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1 KINETIC CHARACTERIZATION AND NEWLY DISCOVERED INHIBITORS FOR VARIOUS CONSTRUCTS OF HUMAN T CELL LEUKEMIA VIRUSI PROTEASE AND INHIBITION EFFECT OF DISCOVERED MOLECULES ON HTLV 1 INFECTED CELLS By AHU DEMIR A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010
2 2010 Ahu Demir
3 To my beautiful mom
4 ACKNOWLEDGMENTS I would like to express my deepest appreciation to my mentor, Professor Ben M. Dunn, for allowing me to work with him on this project and for the independent work environment he provided. I would not be able to finish my dissertation without his guidance and support. I would secondly like to thank my co advisor and committee member Dr. Gail Fanucci. Her support and ideas have lead and helped me a lot in my research. I would also like to thank other committee members Dr. Nicole Horenstein, Dr. Thomas Lyons a nd Dr. Nicolo Omenetto for their guidance and expertise. Their suggestions were invaluable to the progress of this project. Thanks t o Dr. Rob Mc Kenna who was willing to participate in my final defense committee in the last year I would also like t o thank the members of the Dunn laboratory, in particular Dr. Melissa R. Marzhan for her support and invaluable scientific advice and her friendship that has made many days bearable and even enjoyable. Additionally, I would like to thank those that provided technical support during my work including Mr. Alfred Y. Chung. I would also like to thank Dr. David Ostrov and his lab for his help for computational studies, Dr. Alexander Wlodawer and his lab for his great collaboration and for all the help he provided, and Dr. Tomozumi Imamichi and Dr. Raphael Oguariri for their help and support for cell assays. I would specifically l ove to thank my mom who has never stopped believing in me, who has supported me and actually inspired me to accomplish my dreams, who made me a strong woman and made me believe that I can move the world if I try hard enough. I would not have been where I am or who I am w ithout her. I would like to thank my father for his love. I hope he is proud of me; if he is watching as said. I could not have
5 made it without my brother who helped me and supported me throughout. He has always been a good objective supporter. Last, but certa inly not least I want to thank to my friends Meryem Demir and Aysun Altan for listening to all of the complaints, for giving me advice and always being there for me. T here is no way I would have finished without them. I also want to thank Dacia Kwiat kowski painful part of PhD was much more fun with her. Thanks to the many others I cannot finish writing who contributed my beautiful days during my Ph.D. work in Gainesville.
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 LIST OF AMINO ACID ABBREVIATIONS ..................................................................... 11 LIST OF ABBREVIATIONS ........................................................................................... 12 ABSTRACT ................................................................................................................... 14 CHAPTER 1 INTRODUCTION .................................................................................................... 16 Retroviruses ............................................................................................................ 16 HTLV 1 ................................................................................................................... 17 History and Discovery ...................................................................................... 18 Global Implications ........................................................................................... 18 Transmission .................................................................................................... 18 Prevention and Treat ment ................................................................................ 19 Genome and Structure ..................................................................................... 21 Life Cycle .......................................................................................................... 21 Gag and Gag/Pol Processing ........................................................................... 22 HTLV 1 Protease .................................................................................................... 22 Structure ........................................................................................................... 23 Substrate .......................................................................................................... 24 Inhibitors ........................................................................................................... 25 2 MATERIALS AND METHODS ................................................................................ 38 Site Directed Mutagenesis ...................................................................................... 38 Transformation ........................................................................................................ 38 Protein Expression .................................................................................................. 39 Inclusion Bodies Extraction ..................................................................................... 39 Enzyme Purification and Refolding ......................................................................... 40 Kinetic Assays ........................................................................................................ 40 Determination of Ki and Relative Vitality Values ..................................................... 42 Novel Protease Inhibitors ........................................................................................ 43 ELISA and Western Immunoblotting Assays .......................................................... 44 3 EXPRESSION, PURIFICATION AND REFOLDING OF HTLV 1 PR ...................... 46
7 4 KINETIC CHARACTERIZATION AND INHIBITOR DISCOVERIES OF HTLV 1 PR ........................................................................................................................... 59 Truncated Forms of C Terminal Region .................................................................. 59 Kinetic Characterization of Various Constructs ....................................................... 59 Inhibitor Discoveries ................................................................................................ 60 Kinetic Characterization of Various Constructs and Small Molecule Analysis ........ 61 Discussion .............................................................................................................. 62 5 THE EFFECT OF SMALL MOLECULES ON HTLV1 INFECTED CELLS ............. 76 Gag/Pol Processing ................................................................................................ 76 Discussion .............................................................................................................. 77 6 DETERMINING FLAP CONFORMATION OF HTLV 1 PR BY ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPY ............................................. 83 7 CONCLUSIONS AND FUTURE WORK ................................................................. 90 APPENDIX: SEQUENCE .............................................................................................. 94 BIOGRAPHICAL SKETCH .......................................................................................... 111
8 LIST OF TABLES Table page 1 1 Percent prevalence of countries which are highly infected by HTLV 1 PR ......... 27 1 2 Cleavage junction sequence of HTLV 1 PR ( 100). ............................................. 27 4 1 Specificity constants of various constructs of HTLV 1 PR. ................................. 64 4 2 Inhibition constants of 13 inhibitors against L40I and L40I/W98V mutated 116residue HTLV 1 PR.. .......................................................................................... 65 4 3 Ki values of 5 compounds with various constructs of HTLV 1 PR ...................... 67
9 LIST OF FIGURES Figure page 1 1 World map showing HTLV 1 endemic areas.. .................................................... 28 1 2 HTLV 1 genome cartoon picture. ........................................................................ 29 1 3 Retrovirus life cycle ............................................................................................ 31 1 4 HTLV virion. ........................................................................................................ 30 1 5 Mechanism of aspartic acid proteasecatalyzed peptide cleavage ..................... 33 1 6 Cartoon of the crystal structure of 116residue HTLV 1 PR. .............................. 34 1 7 Superposition of seven retroviral PRs shown in ribbon representation. .............. 34 1 8 Sequence Alignment of the Leukemia Retrovirus Proteases with Retroviral Proteases. .......................................................................................................... 35 1 9 Gag Pro Pol sequence of HTLV 1. ................................................................... 32 1 10 Nomenclature of enzyme and substrate subsites ............................................... 36 1 11 Structures of the best inhibitors of HTLV 1 PR. .................................................. 37 3 1 The expression vector pET11a ........................................................................... 50 3 2 DNA gel picture of cloning. ................................................................................. 51 3 3 Alignment of HIV 1 PR and HTLV 1 PR.. ........................................................... 51 3 4 Primers for 121, 122residue and L40I mutation of HTLV 1 PR. ........................ 52 3 5 DNA gel picture of PCR products. ...................................................................... 52 3 6 DNA sequence of HTLV 1 PR vector used in these studies. .............................. 53 3 7 SDS PAGE (18%) gel of expression. .................................................................. 54 3 8 SDS PAGE (18%) gel of inclusion bodies .......................................................... 5 5 3 9 SDS PAGE (18%) gel picture of purification of 121residue HTLV 1 PR. ........... 56 3 11 SDS PAGE (18%) gel of dialysis of HTLV 1 PR ................................................. 57 3 12 Graph of Size Exclusion Chromatography .......................................................... 58
10 4 1 HTLV 1 PR sequence. ........................................................................................ 68 4 2 Kinetic constant determination by Michaelis Menten equation. .......................... 69 4 3 Kinetic constant determination by Lineweaver Burk equation. ........................... 70 4 4 Inhibitor dissociation constant (Ki) Determination. .............................................. 71 4 5 Structures of effective plasmepsin inhibitors. ..................................................... 72 4 6 Possible H bonding distances of PM48, IM37, FS07, IM64 ................................ 73 4 7 Possible H bonding distances of Compound 1 and HTLV 1 PR ......................... 75 5 1 Western Blot of selected inhibitors incubated in MT 2 cells. ............................... 79 5 2 Western Blot of selected inhibitors incubated in MT 2 cells ................................ 80 5 3 Western Blot of selected inhibitors incubated in MT 2 cells. ............................... 81 5 4 ELISA assay graph representation. .................................................................... 82 6 1 Energy diagram of a system with a free electron in the magnetic field. .............. 87 6 2 MTSL label structure .......................................................................................... 87 6 3 Primers for D32N, C90A, C109A and Q64C mutations of H TLV 1 PR. .............. 88 6 4 SDS Page gel of expression of triple mutated HTLV 1 PR. ................................ 88 6 5 SDS Page gel picture of purification of of triple mutated HTLV 1 PR. ............. 89 6 6 of two Cyss of homodimer of HTLV 1 PR. ..................... 89 A 1 DNA sequence of full length HTLV 1 PR with a start methionine with a 5 NdeI site and a 3 BamH1 site for directional cloning into pET 11a. ........................... 94 A 2 Protein sequence of full length HTLV 1 PR. ....................................................... 95
11 LIST O F AMINO ACID ABBREVIATIONS Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic Acid Asp D Cysteine Cys C Glutamic Acid Glu E Glutamine Gln Q Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V
12 LIST OF ABBREVIATIONS BME 2 mercaptoethanol C degrees centigrade CaCl2 calcium chloride ddH2O doubledistilled water DNA deoxyribonucleic acid dNTPs deoxyribonucleotide triphosphate DDT dichlorodiphenyltrichloroethane EDTA ethylenediaminetetraacetic acid Etot total amount of enzyme FPLC Fast Protein Liquid Chromatography g gram(s) HAART Highly Active Antiretroviral Therapy HIV 1 Human Immunodeficiency Virus 1 hr: hour(s) IPTG: isopropyl D 1 thiogalactopyranoside kcat: turnover number (maximum number of enzymatic reactions catalyzed per second) Km Michaelis constant L liter(s) LB Luria Broth LSB Laemmeli sample buffer M molar mg milligram
13 MgCl2 magnesium chloride mL milliliter(s) mM millimolar NaCl sodium chloride NaOH sodium hydroxide ng nanograms(s) nM nanomolar OD optical density PCR polymerase chain reaction PIs Protease Inhibitors BLV Bovine leukemia virus MuLV Mmurine leukemia virus SIV Simian immunodeficiency virus FIV Feline immunodeficiency virus EIAV Equine infectious anemia virus RSV Rous sarcoma virus mwco Molecular weight cut off
14 Abstract of Dissertation Presented to the Graduate School of the University of Flor ida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy KINETIC CHARACTERIZATION AND NEW LY DISCOVERED INHIBITORS FOR VARIOUS CONSTRUCTS OF HUMAN T CELL LEUKEMIA VIRUSI PROTEASE AND INHIBITION EFFECT OF DISCOV ERED MOLECULES ON HTLV 1 INFECTED CELLS By Ahu Demir December 2010 Chair: Gail Fanucci Cochair: Ben Dunn Major: Chemistry Discovered in 1980, HTLV 1 (Human T cell Leukemia Virus 1), was the first identified human retrovirus and is shown to be associated with a variety of diseases including: adult T cell leukemia lymphoma (ATLL), tropical spastic paraparesis/HTLV 1 associated myelopathy (TSP/HAM), chronic arthropathy, uveitis, infective dermatitis, and polymyositis. T he mechanism by which the vi rus causes disease is sti ll unknown. HTLV 1 infection has been reported in many regions of the world but is most prevalent in Southern Japan, the Caribbean basin, Central and West Africa, the Southeastern United States, Melanesia, parts of South Africa, th e Middle East and India. Approximately 30 million people are i nfected by HTLV 1 worldwide, al though only 35% of the infected individuals evolve Adult T cell Leukemia ( ATL ) during their life and the prognosis for those infected is still poor. The retrovira l proteases (PRs) are essential for viral replication because they process viral Gag and Gag (Pro)Pol polyproteins during maturation, much like the PR from Human Immunodeficiency Virus 1 (HIV 1 ) Various antiviral inhibitors are in clinical use and one of the most significant classes is HIV 1 PR inhibitors, which have used for
15 antiretroviral therapy in the treatment of AIDS. HTLV 1 PR and HIV 1 PR are homodimeric aspartic proteases with 125 and 99 residues, respectively. Even though substrate specificities of these two enzymes are different, HTLV 1 PR shares 28% similarity with HIV 1 PR overall and the substrate binding sites have 45% similarity In addition to the 125residue full length HTLV 1 PR, constructs with various C terminal deletions (giving prot eases with lengths of 116, 121, or 122 amino acids) were made in order to elucidate the effect of the residues in the C terminal region. It was suggested that five amino acids in the C terminal region are not necess ary for the enzymatic activity in Hayakaw a et al. 1992. In 2004 Herger et al. had suggested that 10 amino ac ids at the C terminal region are not necessary for catalytic activity A recent paper suggested that C terminal residues are essential; and that c atalytic activity lowers upon truncation, w ith even the last 5 amino acids necessary for full catalytic activity ( 1 ) The m utation L40I has been made to prevent autoproteolysis and the W98V mutation was mad e to make the active site of HTLV 1 PR similar to HIV 1 PR. We have characterized C terminal amino acids of HTLV 1 PR as not being essential for full catalytic activity. We have discovered potential new inhibitors by in silico screening of 116HTLV 1 PR. These small molecules were tested kinetically for various constructs including the 116, 121 and 122amino acid forms of HTLV 1 PR. Inhibitors with the best inhibition constants were used in HTLV 1 infected cells and one of the inhibitors seems to inhibit gag processing.
16 CHAPTER 1 INTRODUCTION Retroviruses Retrovi ruses are a large family of RNA enveloped viruses The mechanism of retroviral transcription differs from other organisms in that RNA is reverse transcribed into DNA which is integrated into the host genome then translated to proteins. Retroviral virions are 100 nm in diameter and packaged in 10 kilo bases double stranded RNA ( 2, 3 ) Retroviruses have recently been classified into two groups; simple and complex viruses, bas ed on their genome organization ( 2 ) Simple viruses include Gag (group antigen), w hich encodes for; the matrix, capsid and nucleocapsid protein, Pol (polymerase), which encodes for reverse transcriptase and integrase and Env (envelope), which encodes for surface and transmembrane proteins such as glycoprotein gp45 and gd20 ( 4 ) Complex viru ses include extra nonstructural genes besides Gag, Pol and Env ( 2 ) Although Murine Leukemia Virus (MLV) is a simple virus, Human Leukemia Virus 1 (HTLV 1) and Human Immunodeficiency Virus (HIV 1) are complex viruses because of their regulatory genes tax and tat, respectively. Beside the recent classification, retroviruses have been combined in three groups as oncoviruses, lentiviruses and spumaviruses according to morphology of the virions as well as the genomic structures. Not all the oncoviruses cause tumor formation and they are classified morphologically into three subgroups a s B C and D type particles ( 5 ) HTLV 1 is an oncovirus ( 2 ) Lentiviruses are associated with slow disease with long latent period such as HIV 1 an d HIV 2 ( 6, 7 ) Spumaviruses, are known as foamy
17 viruses, cause pathogenic changes in the infected cell ( 7 ) Feline foamy virus is an example of spumavirus type ( 6 ) HTLV 1 HTLV 1 is a C type oncovirus, and is classified as a complex virus. According to the The International Committee on Taxonomy of Viruses ( http://www.ncbi.nlm.nih.gov/ICTVdb/ ) it is classified in the Retroviridae group and Deltaretrovirus subgroup with Bovine Leukemia Virus (BLV 1), Simian T lymphotropic Virus 1 2 and 3 (STLV 1, 2, 3) ( 8 ) HIV 1 is in the Lentiretovirus subgroup, and thus differs from HTLV 1. HIV 1 is nononcogenic, while HTLV 1 is oncogenic. HIV 1 has a conical capsid, while HTLV 1 has a spherical capsid ( 9 ) Discovered in 1980, HTLV 1 (Human T cell Leukemia Virus 1), was the first identified human retrovirus and is associated with a variety of diseases including: adult T cell leukemia lymphoma (ATLL) ( 10) tropical spastic paraparesis/HTLV 1 associated myelopathy (TSP/HAM) ( 11) chronic arthropathy ( 12) uveitis ( 13) infective dermatitis ( 14) and polymyositis ( 15, 16) HTLV 1 infection has been reported in many regions of the world but is most prevalent in Southern Japan ( 17) the Caribbean basin ( 18, 19) Central and West Africa ( 2023) the Southeastern United States, Melanesia ( 19) parts of South Africa, the Middle East and India ( 19, 24, 25) ( Figure 11) HTLV 1 subtype A, known as cosmopolitan subtype, is found in many endemic areas like Japan, the United States and Europe ( 26) Subtype B, D and F are found in Central Africa; subtype F in Central and South Africa; and subtype C in Asia ( 26) Approximately 30 million people are infected by HTLV 1 worldwide, and though only 35% of the infected individuals evolve ATL in their life, the prognosis for those infected is still poor ( 27, 28)
18 History and Discovery HTLV 1 was first isolated from a cutaneous lymphoma patient on 1980 by Gallo ( 2932) It was detected in T cells of a pat ient who had mycosis fungoides. The newly recognized ATL was described by Uchiyama on 1977 ( 10) and this research suggested ATL antigen expressed by cell lines from ATL patients was recognized by antibodies in all serum of ATL patients. Anti ATLA (adult T cell leukemia virus as sociated antigen) was detected i n 1982; and another isolation was reported in 1981 ( 33) It was shown that both human T cell leukemia virus (HTLV) and adult T cell leukemia virus (ATLV) isolates were identical based on genespecific probes ( 34) Global Implications Table 11 shows the countries that have the most prevalent HTLV 1 infection. Japan is the country with the highest level of HTLV 1 infection at 10% of the population, followed by the Caribbean with 6%, and subSaharan Africa with 5% HTLV 1 infection ( 19, 25) Most of the data of HTLV 1 prevalence rate is from selected populations, general population information is rare in these studies ( 25) The implications in Europe and United States are due to immig ration or sexual contact with people from endemic areas ( 25 ) T ransmission HTLV 1 is transmitted primarily in three ways: mother infant (mainly through breastfeeding) ( 35) sexual contact ( 36) and infected blood transmission ( 37, 38) Even though there is no case, transmission through saliva might be possible because of presence of proviral DNA and HTLV 1 antibodies ( 39) For all three routes, infected cells must be passed from the infected individual because the living HTLV 1 infected cells are essential for infection that occurs primarily with cell cell contact ( 40, 41) The HTLV 1
19 infected cell and another cell form biological synapses, and viral RNA is transmitted to the target cell. Although HTLV 1 can infect almost any mammalian cell in vitro, it can only infect T cells in vivo for an unknown reason ( 42) The transmission mechanism of HTLV 1 is not clearly understood in vivo or in vitro ( 43) Prevention and Treatment The median survival of adult s wi th T cell leukemia and lymphoma (ATLL) is determined as 1 year despite advances in both che motherapy and supportive care. Cyclophosphamide (inhibits cell division, brand name c ytoxan) adriamycin (doxorubicin/ h ydroxydoxorubicin) vincristine ( o ncovin, inhibits cell division) and p rednisolone drug therapy (CHOP) is one of the methods that has been used for treatment of ATLL patients and results in either complete remission (CR) or partial remission (PR). The more potent chemotherapy, consisting of VCAP (v incristine, cyclophosphamide, and adriamycin which prevents RNA or DNA replication and prednisolone) AMP (doxorubicin, ranimustine [MCNU], and prednisolone), and VECP (vindesine, etoposide, carboplatin, and prednisolone), improves the prognosis of ATL ( 44) However, the overall prognosis of ATL is still poor despi te intensive chemotherapy ( 45) Another combination that had been used is zidovudine plus interferon (ZDV/IFN which is a HIV 1 RT inhibitors ) that gives a better CR result when it is used for the first line therapy ( 46) Zidivudine is a drug used for HIV therapy. There are 6 classes of more than 20 approved antiretroviral drugs used to cure AIDS. Nucleoside/nucleotide reverse transcriptase i nhibitors (NRTIs), nonnucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), fusion inhibitors (FIs), CCR5 antagonists, and integrase strand transfer inhibitors (INSTI) are the 6 different classes of drugs. Two NRTIs plus either one NNRTI or a PI are the most extensively
20 studied combination regimens ( 47) Even though NNRTI PIand INSTI based regimens are equal alternatives according to current guidance, there are selected patients whom PI based regimen seems to show the best result considering toxicity and dosing of the regimens ( 48) Introducing PIs to HIV treatment combinations in 1996 has predominantly lowered the morbidity and mortality due to HIV infection ( 48 ) The addition of an inhibitor of HTLV 1 PR might have the same kind of beneficial effect seen with HAART in HIV therapy. The poor prognosis of HTLV 1 infection is associated with high lac tate dehydrogenase (LDH) level ( 49) Most of the HTLV 1 infected patients are not able to be treated because of drug resistant leukemia cells ( 50, 51) P glycoprotein was found to be overexpressed in various mult idrug resistant cell lines ( 5254 ) Bone marrow transplantation (BMT) has been successfully used to cure ATL ( 55) Allogeneic hematopoietic stem cell transplantation (alloHSCT) provides sustained long term survival for patients with adult T cell leukemia/lymphoma ( 56 ) There is a case report which suggests unrelated cord blood transplantation (UCBT) should be a therapeutic option for ATL patients who do not have suitable donors and those who urgently require treatment ( 57) All of these treatments need to be studied further. There is no specific drug treatment for HTLV 1. Anti HIV or anti cancer regimens have been used as chemotherapic treatment of ATLL ( 51 ) There are studies on discovering drugs targeting HTLV 1 PR, reverse transcriptase (RT), integrase (IN) based on the success of antiretroviral treatment of AIDS and most extensively Tax proteins. Tax is a transcriptional activator viral genes, it transforms and immortalizes the T cells ( 58) Despite the fact that Tax is essential for viral replication, 50% of ATLL patients lose
21 the ability to produce Tax due to mutations ( 59 62) RT and IN exist in low amounts (510 fold less) in the HTLV 1 virion compared to PR; because two frameshifts are nec essary to produce Gag Pro Pol polyprotein while one frameshift is Gag Pro necessary for ( 6366) Therefore HTLV 1 PR is one of the best drug targets for ATLL patients ( 67) Genome and Structure HTLV 1 virus particles (80100 nm in diameter) are enveloped viruses which target CD4 + receptor on the host cell and replicates via a proviral DNA intermediate. The HTLV 1 is a single stranded RNA virus; its genome is approximately 9 kb ( 68) It has Gag, Pol, Pro and env genes and uniquely a pX region at the end of the 3 region. pX encodes for tax and rex which are involved in regulation and synthesis and processing of RNA of the virus as shown in Figure 1 2 ( 68 ) Similar to other retroviruses; Gag encodes for matrix (MA), about 14 kDa, capsid (CA), which provides the core for genomic RNA, is about 24 kDa and nucleocapsid (NC) which is about 12 15 kDa ( 69) Pro encodes for protease (PR) of about 14 kDa that is essential for viral maturation ( 70) ; and pol encodes for reverse trascriptase (RT, 62kDa), which provides the reverse transcription, and integrase (IN, ) which helps the viral DNA int egrate into host genome. Env encodes for surface protein (SU) about a 60 kDa and transmembrane protein (TM) about 21 kDa. Env proteins help virion to go into the cell ( 2, 24) (Figure 1 3 ) After protease cleavage which is necessary for maturation, capsid undergoes a morphological change from circle to pentagonal shape ( 71, 72 ) (Figure 1 3 ) Life Cycle Despite the fact that the mechanism by the virus causes which disease and how is still unknown, steps within the viral replicati on cycle have been shown to be critical for the development of mature, infectious HTLV 1 ( 73)
22 The virus particle attaches to CD4+ receptor with the gp120 and then enters the cell. (Figure 1 4 ) The virus releases its RNA and with the help of reverse transcriptas e (RT), this single stranded RNA is transcribed into double stranded DNA in the cell cytoplasm Then this viral DNA is tran sported into the nucleus of the cell where it is inserted into the host cell DNA with integrase (IN). This form o f retrovirus is called a provirus ( 74) Provirus is transcribed by the host cell RNA polymerase, generating viral RNA The host cell machinery translates the viral RNA into proteins. Proteins and RNA assemble int o virions that eventually bud from the host cell membrane. These new virions mature and continue the cycle of infection ( 9 ) (Figure 1 4) Gag and Gag/P ol Processing The sequence of Gag Pro Pol is shown in Figure 15 Ribosomal frameshifting is a process that uses one mRNA to translate more than one protein by changing the reading frame at a specific site or sites. The genes of the most retroviruses are translated as a single polypeptide by ribosomal frameshifting. This process occurs at the overlapping reading frame. There are two frameshifts for HTLV 1: one is in the 1 direction within the Gag Pro overlap and one is in the ProPol overlap to synthesize Gag, Ga g Pro, and Gag Pro Pol polyproteins ( 65, 75) Gag, Gag Pol processing essential for viral maturation ( 76) HTLV 1 Protease Proteases are enzymes that catalyze the hydrolysis of proteins, belong to hydrolases group of enzymes according to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology They are classified in four groups depending on the type reaction they catalyze, catalytic site residues and relation of the origin of the structure. MEROPS ( http://merops.sanger.ac.uk/ ) is an online
23 database which is a source to get information about proteases (such as name, identifier, gene name, organism and substrates) or their protein inhibitors ( 77 ) Proteases are divided further in three categories based on peptidase, family based on their sequences and clan according to their evolut ionary origin. The most common classification is based on their catalytic residues which are combined in five subgroups: aspartic acid, serine, cysteine, metalloproteases and unknown or mixed active site proteases. HTLV 1 protease belongs to the aspart ic acid protease family; which catalyzes an acidbase mechanism ( 78) An enzyme bound water molecule attacks the amide bond to form a tetrahedral intermediate; then the conjugate base aspartate attacks to the intermediate to take the hydrogen so the amide nitrogen is expelled as the leaving group ( 78) (Figure 1 6 ) The retroviral proteases (PRs) are essential for viral replication because they process viral Gag and Gag (Pro)Pol polyproteins during maturation, much like the PR from HIV 1 (Human Immunodeficiency Virus 1 ) ( 62, 79, 80) Various antiviral inhibitors are in clinical use and one of the most significant classes are HIV 1 PR inhibitors, which have proved to be an invaluable component of antiretroviral therapy in the treatment of AIDS ( 81 ) HTLV 1 PR and HIV 1 PR are homodimeric aspartic proteases with 125 and 99 residues, respectively in the monomers Even though substrate specificities of these two enzymes are different, HTLV 1 PR shares 28% homology with HIV 1 PR overall and the substrate binding sites have 45% homology ( 82) Structure HTLV 1 PR (116 residues) has been crystallized by Mi Li in 2005 ( 27, 83) (Figure 1 7 ) The pr otease was cocrystallized with Ac AlanineProline Glycine Val ine Sta tin Val ine Met hionineHis tidine Pro line inhibitor and the structure was refined at 2.5
24 resolution. It had three homodimeric molecules per unit. When the structure was superimposed with 7 other retroviral proteases, it was seen that the flap, active site and the dimerization regions were conserved; while the elbow region of the proteases were divergent ( 8388) (Figure 1 8 ) One of the different features of HTLV 1 PR compared to the other retroviruses is the presence of the two water molecule between the tips of the flaps ( 83) There are extra additional amino acids at the C terminal region of HTLV 1 protease compared to other aspartic acid proteases; it is only similar to Bovine Leukemia Vi rus 1 (BLV 1) PR ( 83 89) (Figure 1 9 ) Substrate Natural substrate cleavage sites are shown in Table 12 ( 90) Even though natural cleavage sites of HTLV 1 PR and HIV 1 PR are similar, their substrate specificities are different ( 82) Based on cross reactivity of PRs using a vaccinia virus, it was determined that HTLV 1 PR was able process BLV Gag protein, but not HIV 1 PR Gag protein ( 91) This result shows there are other effects on PR cleavage beside the primary amino acid sequence. There is a nomenclature for naming the subsites of the subst rate and the enzyme ( 92) (Figure 1 10) The cleavage bond is called the scissile bond and the amino acid next to it on the left is called P1 amino acid and to the right is called the P1 amino acid; the numbers increase getting further away from the bond. The same procedure is applied for enzyme subsites that interact with each amino acid of a substrate or an inhibitor as S1 and S1 ( 27) All retroviral PRs prefer hydrophobic large residues at the P1 subsite; and HTLV 1 PR prefers a hydrophobic P4 amino acid. S1/S1 The Trp98 has a drastic effect on the H bonding and binding of inhibitor because of its big side chain. Four residues are
25 identical, and two are different in the S1 and S1 pockets in 7 retroviral proteases. This subsite is large and hydrophobic ( 27, 68, 73) S2/S2 Beside Met37, all the other residues are the same or similar. This subsite is large and hydrophobic. In subsites S3/S3 3 amino acids are different out of 6 amino acids. Trp98 has a big effect based on its side chain. Subsites S4/S4 are hydrophobic and subsites S5/S5 are hydrophilic ( 27, 68, 73) Inhibitors Even though, HTLV 1 PR is significantly similar to HIV 1 PR, they have different inhibitor specificity ( 73, 93, 94) A ccording to the literature and our experiments; it was determined that clinically used HIV 1 PR inhibitors have no or little inhibition effect on HTLV 1 PR ( 73) Based on the crystal structure of 116residue HTLV 1 PR; the steric effect of Trp98 and Leu57 side chains might prevent possible inhibitor protease interactions ( 27) Although HTLV 1 PR is an aspartic acid protease, pepst atin; which is an aspartic acid protease inhibitor has a low inhibition effect on HTLV 1 PR (Ki 100 ) ( 95) The best inhibitor for HTLV 1 PR is JG 365, a HIV 1 PR inhibitor, with Ki of 6.0 nM ( 93) The second most efficient inhibitor is a peptide inhibitor with Ki of 38 nM, followed by a nonpeptide inhibitor MES13099 with Ki of 243 nM ( 93, 96) The cleavage products of (30C18 colu mn by eluting with a linear gradient of 3045% acetonitrile (0.1% TFA) and was monitored at 210 nm for these two inhibitors ( 93) Buffer including 100 mM sodium citrate, 5 mM EDTA, 1 mM DTT, and 1 M NaCl, pH 5.3) was used and the reactions were incubated at 37 C for 5 min ( 93 )
26 Th ere are a couple of novel inhibitors (KNI 10729 and KNI 10516 that give 79% and 86 % inhibition at 50 nM concentration, respectively) found by Dr. Wlodawers lab recently ( 83) The hydrolysis of the (0.2 mM ) substrate fluorescent substrate( H Lys ([7 methoxycoumarin4 yl]acetyl) APQVL (p nitrophenylalanine) VMHPL protease was determined in 0.2 M citrate buffer (pH 5.3), 1 mM dithiothreitol, 1 M NaCl, 5 mM EDTA, 6% v/v glycerol, and 2% v/v DMSO solution at different inhibitor concentrations, and the reaction proceeded at 37C for 30 min, then the reaction was terminated with 20% aqueous trichloroacetic acid (15 lL). IC50 value was calculated by measuring the hydrolyzed substrate fragments and/or non hydrolyzed substrate by probit model HPLC using a YMC Pack Pro C18 column (linear gradient: 5 35% CH3CN in 0.1% aqueous TFA; 1.0 mL/min; 215 nm; 40C), and the calculated from standard curves of the areas under the peaks at 6, 10, and 13 min by probit model ( 97 ) The structures of the inhi bitors are shown in Figure 11 1
27 Table 11. Percent prevalence of countries which are highly infected by HTLV 1 PR Country Preval e nce Japan ( 98 ) 10% Caribbean ( 14 ) 6% sub Saharan Africa ( 99 ) 5% Iran and Melanesia ( 100 ) < 5% Europe and USA ( 101 ) 0.01 0.03% Table 1 2 Cleavage junction sequence of HTLV 1 PR Cleavage junction HTLV 1 amino acid sequence MA/CA PQVL/PVMH CA/NC TKVL/VVQP Gag/PR ASIL/PVIP PR/Pol PVIL/PIQA Pro/RT PAVL/GLEL RT RH/IN VLQL/SPAD
28 Figure 11. World map showing HTLV 1 endemic areas. Countries prevalence between 15% are shown in dark brown, less than 1% in orange (adapted from Proietti, F. A et al. 2005). Papua New Guinea Japan South America Iran Sub saharan Africa
29 Figure 12. HTLV 1 genome cartoon picture. Gag, Pro, Pol, Env and Px open reading frames are shown in various color and they are flanked by long terminal repeats shown in red (adapted from Shuker, S. B. et al. 2003).
30 Figure 13 HTLV virion. A) Immature B) Mature. Lipid bilayer is shown in yellow, matrix (MA) is shown in red, capsid (CA) in black, NC in green. The electromicrograph of the mature and immature form of HTLV 1 is shown under the cartoon representation (adapted from Jiang, F. et al. 2000, and Briggs, J. A. 2004). Capsid Capsid B A
31 Figure 14 Retrovirus life cycle (adapted from Coffin, J. M. H. et al. 1997 ).
32 MGQIFSRSASPIPRPPRGLAAHHWLNFLQAAYRLEPGPSSYDFHQLKKFLKIALETPVWICPINY SLLASLLPKGYPGRVNEILHILIQTQAQIPSRPAPPPPSSSTHDPPDSDPQIPPPYVEPTAPQVL PVMHPHGAPPNHRPWQMKDLQAIKQEVSQAAPGSPQFMQTIRLAVQQFDPTAKDLQDLLQYLCSS LVASLHHQQLDSLISEAETRGITGYNPLAGPLRVQANNPQQQGLRREYQQLWLAAFAALPGSAKD PSWASILQGLEEPYHAFVERLNIALDNGLPEGTPKDPILRSLAYSNANKECQKLLQARGHTNSPL GDMLRACQAWTPKDKTKVLVVQPKKPPPNQPCFRCGKAGHWSRDCTQPRPPPGPCPLCQDPTHWK RDCPRLKPTIPEPEPEEDALLLDLPADIPHPKNLHRGGGLTSPPTLQQVLPNQDPTSILPVIPLD PARRPVIKAQIDTQTSHPKTIEALLDTGADMTVLPIALFSSNTPLKNTSVLGAGGQTQDHFKLTS LPVLIRLPFRTTPIVLTSCLVDTKNNWAIIGRDALQQCQGVLYLPEAKRPPVILPIQAPAVLGLE HLPRPPEISQFPLNPERLQALQHLVRKALEAGHIEPYTGPGNNPVFPVKKANGTWRFIHDLRATN SLTIDLSSSSPGPPDLSSLPTTLAHLQTIDLKDAFFQIPLPKQFQPYFAFTVPQQCNYGPGTRYA WRVLPQGFKNSPTLFEMQLAHILQPIRQAFPQCTILQYMDDILLASPSHADLQLLSEATMASLIS HGLPVSENKTQQTPGTIKFLGQIISPNHLTYDAVPKVPIRSRWALPELQALLGEIQWVSKGTPTL RQPLHSLYCALQRHTDPRDQIYLNPSQVQSLVQLRQALSQNCRSRLVQTLPLLGAIMLTLTGTTT VVFQSKQQWPLVWLHAPLPHTSQCPWGQLLASAVLLLDKYTLQSYGLLCQTIHHNISTQTFNQFI QTSDHPSVPILLHHSHRFKNLGAQTGELWNTFLKTTAPLAPVKALMPVFTLSPVIINTAPCLFSD GSTSQAAYILWDKHILSQRSFPLPPPHKSAQRAELLGLLHGLSSARSWRCLNIFLDSKYLYHYLR TLALGTFQGRSSQAPFQALLPRLLSRKVVYLHHVRSHTNLPDPISRLNALTDALLITPVLQLSPA DLHSFTHCGQTALTLQGATTTEASNILRSCHACRKNNPQHQMPQGHIRRGLLPNHIWQGDITHFK YKNTLYRLHVWVDTFSGAISATQKRKETSSEAISSLLQAIAYLGKPSYINTDNGPAYISQDFLNM CTSLAIRHTTHVPYNPTSSGLVERSNGILKTLLYKYFTDKPDLPMDNALSIALWTINHLNVLTNC HKTRWQLHHSPRLQPIPETHSLSNKQTHWYYFKLPGLNSRQWKGPQEALQEAAGAALIPVSASSA QWIPWRLLKRAACPRPVGGPADPKEKDHQHHG Figure 15 Gag Pro Pol sequence of HTLV 1. Matrix is shown in dark blue (130 amino acids) capsid is shown in green (214 amino acids) nucleocapsid is shown in light blue (105 amino acids) protease is shown in red (125 amino acids) reverse transcriptase is shown in blue (563 amino acids) and integrase is shown in orange (300 amino acids)
33 Figure 16 Mechanism of aspartic acid proteasecatalyzed peptide cleavage (adapted from Liu, H. et al. 1996 ). O
34 Figure 17 Cartoon of the crystal structure of 116residue HTLV 1 PR (PDB#2B7F). Aspartic acid residues are shown in orange sticks (adapted from Satoh, T. et al. 2010). Figure 18 Superposition of seven retroviral PRs shown in ribbon representation.HTLV 1 PR is colored blue (PDB#3LIY); HIV 1 PR (PDB#2FLE), green; HIV 2 PR (PDB#3EBZ), dark blue; SIV PR (PDB#2SAM), gray; RSV PR (PDB#2RSP), magenta; EIAV PR (PDB#2FMB), yellow; and FIV PR (PDB#4FIV), red.
35 Figure 1 9 Sequence Alignment of the Leukemia Retrovirus Proteases with Retroviral Proteases of Known Structure (besides BLV). The alignment for HIV 1, HIV 2, SIV, FIV, EIAV, and RSV proteases was generated based on the reported structures (PDB IDs: HIV 1, 2FLE; HIV 2, 3EBZ; SIV, 2SAM; FIV, 4FIV; EIAV, 2FMB; and RSV, 2RSP). Hydrophobic residues are indicat ed in blue hydrophilic residues in yellow
36 Figure 110. Nomenclature of enzyme and substrate subsites (adapted from Schechter, I. et al. 1967)
37 A B R1: Ac Ser LeuAsn R2: IleVal OMe R1: H Pro Val Ile R2: CH2C6H4I C D E Figure 111. Structures of the best inhibitors of HTLV 1 PR. A.JG 365, B.Compound 31, C. MES13 099, D. KNI 10729, E.KNI 10516
38 CHAPTER 2 MATERIALS AND METHODS Site Directed Mutagenesis The HTLV 1 PR gene, which was obtained from NIH, was cloned into a pET11a vector (Novagen) using the restriction sites NdeI (NEB) and BamHI (NEB) To make the truncated forms of HTLV 1 PR, a stop codon has been introduced after the appropriate amino acid coding sequence. Complementary primers (Invitr o ge n) were designed according to the c oding and noncoding DNA strand. The reactions were carried out using the Site Directed Mutagenesis Protocol (Stratagene) with 18 cycles of amplification using melting step at 98C for 3 min followed by an annealing step at 55C for 1 min, and extension at 72C for 7 min, the n the reaction temperature was dropped to 4C. To remove template DNA, 1 mL of the restriction enzyme Dpn1 (10 L / mL) was added to the PCR reaction and the mixture was incubated at 37C for 1 hr. Suc cessful mutagenesis was confirmed by sequencing (ICBR Core, University of Florida, Gainesville, Florida). Transformation Transformation was done with using chemically component cells and One Shot Top 10 (Invitrogen) protocol with some modifications. 2.5 L of the DNA (32.5 ng/ L) of interest was mixed with 25 L of cell stock and the mixtur e was kept on ice for 30 min The reaction mixture was then heat shocked in a 42C water bath for 45 s The reaction mixture was replaced on the ice immediately for 15 mi n. Next, (2.0% Tryptone, 0.5% Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, and 20 mM glucose, pH 7) were added to the reaction tube and placed in an incubator rotating at 250 rpm at 37C for an hour. 75 L of the cell culture were spread onto LB plates
39 containing ampicillin (50 g/mL). Plates were incubated at 37C overnight to promote colony growth. A colony was randomly picked the next day and grown overnight (16 hours ) in 10 mL of LB with ampicil l in (50 g/mL). The plasmid DNA was checked by sequencing to verify the correct amino acid sequence and mutations. Transformation into BL21(DE3) Rosetta pLysS cells (Invitrogen) was done with the same method as above with addition of chloramphenicol (34 g/mL) as well as ampicil l in (50 g/mL). Protein Expression LB medium (10 g Bacto tryptone, 5 g yeast extract, 10 g NaCl in 1L water at pH 7.5 ) was used for expression. The expressions were initiated with a 4% inoculation from overnight cultures of cells grown in LB media supplemented with 50 g/mL of ampici l lin for BL21 Star DE3 cells or 50 g/mL of ampici l lin and 34 g/mL of chloramphenicol for BL21 Star DE3 Rosetta pLysS cells. Expression cultures were grown overnight for ~16 hr. When t he optical density reached 0.8, protein expression was induced with final concentration of 1 mM isopropyl bD thiogalactopyranoside. The cells were allowed to grow for an additional 3 4 h and then pelleted by centrifugation at 10000 x g for 10 min ( 102) Cell pellets were stored at 20 C ( 95) Inclusion Bodies Extraction Cell pellets stored at 20 C overnight were thawed and resuspended in buffer (10 mM Tris pH 7.5, 5 mM EDTA ) and DNase I was added to a final concentration of 0.1 mg/mL. SLMAminco French Pressure Cell at 1000 psi was used to break cells. After lysing the cells, urea was added to solution to 0.5 M final concentration and stirred for 30 min. Ce lls were centrifuged at 5000 x g for 10 min. The procedure was repeated until
40 a clear supernatant was obtained ( 27) Pelleted i nclusion bodies (IB) were stored at 80 C. Samples of I B were run on SDS PAGE gels Enzyme Purification and Refolding The inclusi on bodies were solubilized in buffer B (8 M urea, 10 mM Tris HCl pH 7.5, 5 mM EDTA). All urea solutions were deionized by ion exchange to remove cyanates. Solubilized inclusion bodies were loaded onto a Q Sepharose Fast Flow column (Pharmacia) equilibrat ed with buffer C (6 M urea, 10 mM Tris HCl pH 7.5, 5 mM EDTA). As the pI of HTLV 1 PR is predicted to be 8.89, it did not bind to the Q Column. The flow through from the column was collected and adjusted to pH 4.0 with acetic acid. Various pHs, as pH 3, 4, and 5, have been tried, pH 4.0 resulted the best yield. (data not shown) The pH adjusted solution was then loaded onto a Sepharose Fast Flow SP column (Pharmacia) equilibrated in buffer D (6 M urea, 20 mM sodium acetate pH 4.0, 5 mM EDTA) ( 102 ) The PR that bound to the column was eluted with 0.4 M NaCl. Rapid dilution with excess of 10 mM formic acid was used to refold the purified HTLV 1 PR. S ize excl usion chromatography was used to determine purification and folding success. Purification of HTLV 1 PR wa s determined by 18% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS). Kinetic Assays The enzyme concentrations for HTLV 1 PR preparations were determined by Bradford assay (BioRad) and optical density (OD) reading at 280 nm of the stock solutions after concentrating the purified PR using Amicon ultrafiltration devices (Amicon Ultra 15, Mi llipore). The chromogenic substrate A P Q V L*NphV M H P L, which mimics the natural Gag/P ol MA/CA cleavage site, was synthesized by ICBR Core Facility at UF. This substrate was used to assay enzyme activity in 2 X incubation buffer
41 (0.5 mM potassium phosphate pH 5.6, containing 10% glycerol, 2 mM EDTA, 10 mM dithiothreitol, 2 M NaCl) at 37 C ( 103 ) Various NaCl concentrations were used to determine the optimum ionic strength and 2 M was selected based on the highest protease activity. (D ata not shown) The enzyme mixture and the substrate w ere incubated separately at 37 C for 3 minutes before mixing them and monitoring cleavage of the substrate at 302 nm using a Cary 50 Bio Varian spectrophotometer equipped with an 18cell multitransport system. The initial rate data for each assay was plotted versus substrate concentration to obtain the characteristic Michael is Menten curve. The experimental curve was then fit to the following equation by using Sigma Plot : ) ( *maxS K S V vm (3 1) A non linear Marquardt analysis was used to determine Km and Vmax for the substrate. ( 104 ) In the above Vmax is the maximum velocity, S is the subs trate concentration, and Km is the Michaelis Menten constant which has the unit of molarity. Every enzyme has a distinct Km value for a certain substrate. The Km value of an enzyme represents the substrate concentration required to reach the half of the maximum velocity ( Vmax), is a measure of the concentration of the substrate required for an effective catalysis. The rate of the reaction was derived from the measured change of absorbance per unit time ( The conversion factor was determined by using the exact concentrations of substrate, as determined by amino acid analysis. The initial values of the absorbance (i.e., before substrate cleavage at time zero), and after the completion of substrate cleavage (i.e., after 2 hours) were measured by UV Spectrometry four different substrate
42 concentrations. A graph was then plotted using the absorbance change versus substrate concentrations. The conversion factor is derived from the slope of the graph that convert s the determined by Dr. Roxana Coman. The kcat values were determined using the following equation: tot catE V kmax (3 2) kcat is the turnover number of the enzyme which is a meas ure of its maximal catalytic activity. The reciprocal kcat is the time required by an enzyme molecule to turn over one substrate molecule. Etot is the total enzyme concentration in this formula, it was calculated by OD reading at 280 nm; the absorptivity c oefficient is 14,000 L/mol.cm ( 90) Determination of Ki and Relative Vitality Values Various inhibitors are used to provide information about the active site of the protein. Inhibition was measured as a decrease in the rate of substrate cleavage in the presence of inhibitor over time. After fitting the Michaelis Menten curve in the absence of inhibitor, the assay is repeated twice in the presence of two different concentrations of inhibit or. The curves are then simultaneously fit to the following equation: ) 1 ( ) 1 ( maxi mK I S K V v (3 3) to determine Ki values of classical (nontight b inding) competitive inhibitors. In the above equation, is the rate of product formation, Vmax is the maximum velocity Km is the Michaelis Menten constant S is the concentration of the substrate, I is the
43 concentration of inhibitor and Ki is the inhibition constant which is expressed in units of molarity. The graphs were fit in the cases of uncompetitiv e and noncompetitive inhibitor type of equations, and the best fits were obtained with the competitive inhibitor fit. Km and Kcat were determined for each inhibitor: in order to check the precision of the assay, they were repeated under the same conditions and found to be reproducible. Although no numerical values of the error bars were determined in these assays, but the Km values were very close to each other for each enzyme preparations. Km value was determined and reproduced in the same range even after freezing and thawing. While determining the Ki values for each inhibitor, a consistent Km value was reproduced. Novel Protease Inhibitors In silico screening of over 140, 000 compounds available from the National Cancer Institute Developmental Therapeutics Program was done by docking these small molecules into the active site of the HTLV 1 PR based on the crystal structure of the 116residue HTLV 1 PR available in the Protein Data Bank (PDB 2B7F) using DOCKv5.2. ( 105) The small molecules are available on the NCI websit e. ( http://dtp.nci.nih.gov/index.html ) ( 106) After water molecules were removed from the structure each compound was docked as a rigid body. The interactions between molecules are estimated by calculating approximate molecular mechanics interactio n energies as implemented by the force field function in the DOCK program and compounds were ranked based upon predicted van der Walls and electrostatic interactions. A van der Waals surface representation of the model was generated using the program dms a nd the method of Richards ( 107) Spheres characterizing the potential target sites of the protein were generated using the program sphgen. Subset selection of spheres was performed using sphere select to within 6 of the target site.
44 Electrostatic grid files and bump filters were generated around the target site using the p rogram grid with 10 buffers. The AMBER force field was used for vDW calculations. Docking w as performed using DOCK6.0 ( 108) and all software programs referenced are distributed with the package. A database of 250,251 small molecules derived from the National Cancer I nstitute (NCI) Developmental Therapeuti cs Program (DTP) plated set ( 109 ) was used for the docking calculations. A maximum of 5000 orientations was searched for eac h small molecule in the lig and database. All docking calculations were performed on the University of Florida High Performance Computing cluster. The best 40 compounds were selected and obtained from the National Cancer Institute. The stock solution was o btained by dissolving in 100% DMSO to a final concentration 50 mM and stored at room temperature. ELISA and Western Immunoblotting Assays MT 2 cells were obtained from the AIDS Research and Reference Reagent Program ( 110, 111) National Institute of Allergy and Infectious Disease (Rockville, MD) and maintained in complete RPMI 1640 medium (Invitrogen) as previously described ( 112, 113) MT 2 cells were seeded at 4 x 105 cells per ml and cultured for 4 or 24 hrs at 37oC in the presence or absence of 5, 10 or 50 M of selected inhibitors Total cell lysates were obtained using RIPA lysis buffer ( 25 mM Tris HCl pH 7.6, 150 mM NaCl, 1% NP 40, 1% sodium deoxycholate and 0.1 % SDS) containing protease inhibitor (Sigma Ald rich) and phosphate inhibitor (Thermo Scientific, Rockford, IL) Total cellular protein amount was measured with the BCA Protein Assay Kit (Thermo Scientific) Western blotting was performed as previously described ( 112, 113) Briefly, samples containing a total of 30 g of total cellular protein were loaded onto a 412% SDS Bis Tris Gels (Invitrogen) and subsequently transferred onto a 0.45 M nitrocellulose
45 membrane (Invitrogen). Membranes were probed with anti HTLV 1 p19 monoclonal antibody (Zeptometrix, Buffalo, NY). The primary ant ibody was detected with horseradish peroxidase (HRP) conjugated anti mouse IgG (GE HealthCare, Piscataway, NJ, USA). The membranes were stripped using restore W estern stripping buffer (Thermo Scientific,) and reprobed with monoclonal anti actin (Santa C ruz Biotechnology, Santa Cruz, CA, USA) as internal control. Signals were detected using the enhanced chemilumnescence W estern blotting detection reagent (GE HealthCare). MT 2 cells were seeded at 4 x 105 cells per ml and incubated in the presence or absence of 5 or 50 M of selected inhibitors for 1 or 2 days. Levels of HTLV 1 p19 production in culture supernatants were quantified using enzymelinked immunosorbent assay kits for p19 (Zeptometrix, Buffalo, NY) according to the manufacturer's instructions. Cell growth was estimated by counting the cells using a hemocytometer or a machine counter. Cell viability was determined by counting the viable cells by staining with try pan blue. All the cell assays were done at NCI Frederick facility in Dr. Tomozumi Imamichis laboratory.
46 CHAPTER 3 EXPRESSION, PURIFICATION AND REFOLDING OF HTLV 1 PR The optimum methods and conditions for expression, purification and refolding of HTLV 1 PR have been investigated such as the most efficient E coli cell line, method to lyse the bacterial cells, purification method determined to be ion exchange chromatography, pH for ion exchange chromatography, NaCl concentration for the most active proteas e and the refolding method determined to be rapid dilution. The most efficient E. coli cell line was selected as Rosetta ( DE3 ) pLysS Competent Cells that enhance the expression of eukaryotic proteins that contain codons rarely used in E. coli The pLysS plasmid encodes T7 phage lysozyme, DE3, which contains the T7 bacteriophage gene I. Because p LysS strains express T7 lysozyme, which further suppresses basal expression of T7 RNA polymerase before the induction, they stabilize pET recombinants encoding target proteins that affect cell growth and viability. T7 lysozyme lowers the background expression level of target genes under the control of the T7 promoter but does not interfere with the level of expression achieved after IPTG induction. Rosetta ( DE3 ) pLysS cell strains supply tRNA genes for AGG, AGA, AUA, CUA, CCC, GGA which are the rare codons used in E. coli on a Col E1 compa tible chloramphenicol resistant plasmid. This cell strain has yielded higher expression efficiency of HTLV 1 PR, therefore the assays have continued by this cell strain selection. The pET 11a vector was selected as an expression vector. (Figure 31) Various constructs were cloned into pET11a by using Nde1 and BamH1 restriction enzymes. Digestion products from pET 11a vector and insert HTLV 1 PR are shown in Figure 32,
47 and cut vector and insert were ligated for 1 h, and then transformed into TOP 10 cells. ( See Chapter 2) The 116 residue L40I and W98V amino acids substituted HTLV 1 PR in pET 11a vector and 125residue L40I amino acids substituted HTLV 1 PR in pET 19b vector were provided from NIH. The L40I substitution was used to prevent autoproteolysis and the W98V substitution was introduced to make the active site region of HTLV 1 PR similar to HIV 1 PR (Figure 3 3). The 125residue HTLV 1 PR was cloned in pET 11a vector of E. coli Stop codons were introduced at the 121 and 122 residues to get various construct lengths. The primers are shown in Figure 34. The PCR reactions were conducted on as mentioned in the experimental procedure. (See Chapter 2) Various concentrations of DNA from 0.1 to 0.5 ng were used as template. A gradient was used for anneali ng temperature between 4360C. After the PCR reaction was finished, a DNA gel was run to determine PCR products. (Figure 35) PCR products which have the correct band were transformed in Rosetta ( DE3 ) pLysS E. coli cells. After cloning and site directed mutagenesis (SDM), DNA sequences were checked for correct frame locations of ribosome binding sites and restriction sites for protein translation. (Figure 36) The concentration of 1 mM IPTG was picked as the optimum concentration to induce expression of the gene based on literature ( 114 ) Different expression times were tried as shown in Figure 36. Because there was no significant change between 3 h and 6 h, 3 h of expression was selected. The French Pressure Cell, Bug buster (Novagen) and sonication methods were used to break cells. Resuspension buffer ( 10 mM Tris pH 7.5, 5 mM EDTA 0.5 M
48 urea) was added twice to wash the pellet and the samples centrifuged at 8000 rpm for 10 min. (Figure 3 7) French pressure cell treatment gave the best yield as 2 g of inclusion bodies (IBs) were obtained compared to 1g of IBs obtained with Bug buster and sonication. After solubilizing IBs in buffer B (8 M urea, 10 mM Tris HCl pH 7.5, 5 mM EDTA) Q Sepharose Fast Flow column (Pharmacia) equilibrated with buffer C (6 M urea, 10 mM Tris HCl pH 7.5, 5 mM EDTA) was used to purify the protease from proteins that have lower pIs. The Q column flow through was adjusted to pHs 3, 4, and 5 as well as buffer D (6 M u rea, 20 mM sodium acetate 5 mM EDTA) to apply to a SP column (Pharmacia). pH 4 gave the best purification for HTLV 1 PR. Various concentrations (0.3, 0.4, 0.5 M) of NaCl was used to elute the protease of the SP column. 0.4 M NaC l gave the best elution yield. The SDS PAGE gel of the purification of one of the constructs was shown in Figure 38. Many conditions have been tried for refolding of HTLV 1 PR. For the first condition the purified protease was dialyzed against 20 mM PIP ES, pH7.0, containing 2 mM DTT, 1 mM EDTA, 150 mM NaCl and 10% glycerol at 25 C for 3, 5 ,8 h and 16 h. (Figure 39) None of the time points gave any active protein, so folding was not accomplished. Overnight dialysis resulted in precipitation of the prot ease. No active protease was obtained. Dialysis against 50 mM sodium acetate buffer (pH 5.0) containing 100 mM NaCl at 0C for 3, 5, 9 and 24 h was used in an attempt to get folded protease, but this method failed as well. (Figure 310) As mentioned in Kadas et al.; subsequent dialysis against 25 mM formic acid (pH 2.8) at 0C for 6 h followed by dialysis against 50 mM sodium acetate buffer (pH 5.0) containing 100 mM NaCl at 0C overnight was tried; also
49 yielded inactive protease. After testing them by Size Exclusion Chromatography and kinetic assays; none of these conditions provided correctly folded, active HTLV 1 PR. Only 10x rapid dilution of purified protein in 10 mM formic acid gave active, correctly folded HTLV 1 PR. This result was confirmed by s ize exclusion chromatography in Figure 311.
50 Figure 31. The expression vector pET11a (Novagen)
51 Figure 32. DNA gel picture of cloning. Lane A represents the cut insert of 121residues HTLV 1 PR, lane B the cut vector of pET 11a and the lane C the molecular weight marker (1 kb DNA Ladder (NEB). Figure 33 Alignment of HIV 1 PR and HTLV 1 PR. W98V amino aci d substitution is shown in pink spheres, HTLV 1 PR is shown in pink and HIV 1 PR in blue colors. A B C
52 L40I Upper GCAGACATGACAGTCATTCCGATAGCCTTGTTC Lower GAACAAGGCTATCGGAATGACTGTCATGTCTGC 121 HTLV 1 PR Upper GGCAAAAGGGCCGTAAGTAA TCTTG Lower CCGTTTTCCCGGCATTCATTAGAAC 122 HTLV 1 PR Upper GGCAAAAGGGCCGCCTTAAA TCTTG Lower CCGTTTTCCCGGCGGAATTTAGAAC Figure 34 Primers for 121, 122residue and L40I mutation of HTLV 1 PR. 1 2 3 4 5 Figure 35 DNA gel picture of PCR products. Lane 1 is 0.5 ng 121 residue HTLV 1 PR elongation temperature at 52C, Lane 2 100 pg 121residue HTLV 1 PR elongation temperature at 52C, Lane 3 is 0.5 ng 121residue HTLV 1 PR elongation temperature at 43C, Lane 4 is 100 pg 121residue HTLV 1 PR elongation temperature at 43C. Lane 6 is 0.5 ng 122residue HTLV 1 PR elongation temperature at 52C, Lane 7 100 pg 122residue HTLV 1 PR elongation temperature at 52C, Lane 8 is 0.5 ng 122residue HTLV 1 PR elongation temperature at 43C, Lane 9 is 100 pg 122residue HTLV 1 PR elongation temperature at 43C. Lane 5 and Lane 10 is 1 kb DNA ladder (NEB). 6 7 8 9 10
53 Figure 3 6 DNA sequence of HTLV 1 PR vector used in these studies.
54 A B C D A B C D Figure 37. SDS PAGE (18%) gel of expression. 1. 116residue HTLV 1 PR. 2. 121 residue HTLV 1 PR. Lane A represents the Precision Plus Ladder (Biorad), lane B before IPTG induction, lane C 3 h after IPTG induction and lane D 6 h after IPTG induction. 250 50 37 25 20 15 10 HTLV 1 PR 1 2 250 50 37 25 20 15 10 HTLV 1 PR
55 A B C D Figure 38. SDS PAGE (18%) gel of inclusion bodies 1. Lane A represents the Precision Plus Ladder (Biorad) lane B the first wash of IBs, lane C the second wash of IBs, lane D MW ladder and lane E IBs 121residue HTLV 1 PR. 2. Lane A represents the Precision Plus Ladder (Biorad), lane B before IPTG induction, lane C 3 h after IPTG induction and lane D. IBs 122residue HTLV 1 PR. HTLV 1 PR 250 50 37 25 20 15 10 2 1 250 50 37 25 20 15 10 HTLV 1 PR
56 Figure 39. SDS PAGE (18%) gel picture of purification of 121residue HTLV 1 PR. Lane A is the Precision Plus Ladder (Biorad), lane B is the Q column flow through, lane C is the Q column elution by 1 M NaCl, lane D is the SP column flow through, lane E is the SP column elution by 0.4 M NaCl, lane F is the SP column elution by 1 M NaCl, and the lane G the Precision Plus Ladder ( Biorad). Figure 310. SDS PAGE (18%) gel of dialysis of HTLV 1 PR against 20 mM PIPES, pH 7.0, containing 2 mM DTT, 1 mM EDTA, 150 mM NaCl and 10% glycerol at 25 C. Lane A is the Precision Plus Ladder (Biorad), lane B dialysis after 3 h, lane C dialysis after 5 h, lane D dialysis after 8 h, lane E dialysis overnight, and F is the precipitation after dialysis. 250 50 37 25 20 15 10 HTLV 1 PR 250 50 37 25 20 15 10 HTLV 1 PR
57 Figure 311. SDS PAGE ( 18%) gel of dialysis of HTLV 1 PR against dialysis buffer 1 ( 50 mM sodium acetate buffer (pH 3.0) containing 100 mM NaCl ) at 25 C and dialysis buffer 2 ( 50 mM sodium acetate buffer (pH 4.0) containing 100 mM NaCl) Lane A is the Precision Plus Ladder (Biorad), lane B is dialysis 1 after 3 h, lane C is dialysis 1 after 5 h, lane D is dialysis 1 after 8 h, lane E is dialysis 1 overnight; lane F is dialysis 1 after 3 h, lane G is dialysis 1 after 5 h, lane H is dialysis 1 after 8 h, lane J is dialysis 1 overnight. HTLV 1 PR 250 50 37 25 20 15 10
58 A B Fi gure 312. A. Graph of Size Exclusion Chromatography (Each fraction has 2 mL) B Calibration curve of Size Exclusion Column
59 CHAPTER 4 KINETIC CHARACTERIZATION AND INHIBITOR DISCOVERIES OF HTLV 1 PR Truncated Forms of C Terminal Region There are additional amino acids at the C terminal region of HTLV 1 protease when compared to other aspartic acid proteases such as HIV 1 PR. HTLV 1 PR is most similar to BLV 1 PR. (Figure 17) The function of this extra loop and its effect on enzyme activ ity is still unclear. In the literature, it is controversial as well. An in vivo study by Hayakawa et al. shows the last 5 amino acids at the C terminal region are necessary for protease activity, although 5 amino acids prior to these 5 are not ( 115 ) These results might come from the cell components or other proteases in the cell, since the HTLV 1 P R was not isolated, purified and characterized. Herger et al. showed that 10 residues at the C terminal region are not essential for full catalytic activity, since the full length and 10 residue have the same specificity constant ( 116) Controversially, a 60% decrease in the catalytic act ivity was determined by Li et al. comparing 116residue and full length (125residue) HTLV 1 PR ( 27) A recent study by Kadas et al. shows that 120residue HTLV 1 PR has 3% percent activity, while 116residue has only residual activity ( 1 ) The dimerization tendency and catalytic activity inc rease upon getting closer to full length ( 1 ) We have investigated the effect of C terminal residues at catalytic activity. We have tried to a find construct with full catalytic activity and a crystallizable form of HTLV 1 PR. Kinetic Characterization of Various Constructs 116, 121, and 122residues were used for kinetic characterization of HTLV 1 PR. (Figure 4 1) All the constructs were expressed and purified as described in Materials
60 and Methods. (Chapter 2) Final purity of each protease was verified by 18% SDS PAGE (Figure 3 7 ). The purified construct s were concentrated with polysulfone membrane spin columns ( mwco 10,000; GE Healthcare) to concentrations between 108 0 M and were used for further enzyme characterization. Turnover number of each construct was determined by using various substrate conc entrations from 1575 M. Each substrate concentration has a different rate of reaction plotted and calculated by Sigma Plot software (Systat Software Inc.). (Figure 42) Kinetic constant s determination by Lineweaver Burk equation is shown in Figure 43 E ven though no error bars exists in the figure; Km value was reproduced many times in the same range. The coefficient of determination (R2 ) is the proportion of variability in a data set. Larger values for R2 (close to 1) indicate that the data set fits into the equation. For each kinetic measurement, R2 was kept equal or greater than 0.98 for accuracy of the data. We have found that each construct has a distinct catalytic activity (Table 41). The substrate specificity constant s ( Kcat/ Km) are similar for various lengths of HTLV 1 PR, i ndicating that the C terminal amino acids are not essential for full protease activity. HTLV 1 PR in full length has not been refolded properly in our research; therefore there is no kinetic data for this construct. Each cons truct was utilized in further inhibitor studies. Inhibitor Discoveries Even though HIV 1 PR has been extensively studied, HTLV 1 PR which shares many similarities has not been as well characterized ( 81, 84, 117123 ) Despite the similarities of both retroviral proteases, their substrate and inhibitor specificity are very different from each other ( 82, 94, 103)
61 Different substrate concentrations (1575 M) were used to determine the inhibition constants of each inhibitor. Two different concentrations of inhibitor were used to determine the decrease in the rate of reaction for each substrate concentration and plotted by Sigma Plot software (Systat Software, Inc.). (Figure 4 4) Km value was reproduced and R2 was greater than 0.98. Vari ous aspartic acid protease inhibitors were tested to determine their inhibition effect on HTLV 1 PR. None of the clinically used HIV 1 PR inhibitors (obtained from NIAID) had any inhibition on HTLV 1 PR. (Ki> 100 M ) 22 inhibitors from the laboratory of Anders Hallberg at Uppsala University, 18 compounds from the laboratory of Sergio Romeo at University of Milan, and 19 compounds from other collaborators were tested against HTLV 1 PR. All of these compounds were desi gned against plasmepsins. Only f our of the Swedish inhibitors (available in Dunn lab) have shown inhibition of HTLV_1 PR with Ki values lower than 2 M. Their structures and Ki values are shown in Figure 4 5 and possible H bonding between the inhibitors an d the active site were determined by Chimera software (UCSF) as shown in Figure 46 ( 124, 125) Kinetic Characterization of Various Constructs and Small Molecule Analysis By using the DOCK program (UCSF, San Diego), various inhibitors were docked in the active site of the HTLV 1 PR. 140, 000 compounds available from the National Cancer Institute Developmental Therapeutics Program were used in docking experiments. These compounds obey Lipinski Rules of Five which are rules for druglikeness of a compound ( 126, 127) According to Lipinski absorption or perm eation of a drug is higher when there are less than 5 H bond donors, 10 H bond acceptors, the molecular weight (MWT) is lower than 500 and the calculated Log P (CLogP) is smaller than 5 (or MlogP.4.15) Energy binding values of the inhibitors were determined and
62 ranked by the DOCK program (UCSF, San Diego) ( 108) Top ranked inhibitors were purchased and tested to determine the inhibition on HTLV 1 PR as mentioned in the Method section. Some of the top ranked small molecules identified by in silico screening were tested in inhibition assays and several have shown an inhibition effect on the protease. The inhibition constants range between 178 M for the 116residue HTLV 1 PR (Table 4 3 ). In general, the L40I mutant has lower inhibition constants (Ki), i.e., better binding, when compared to the double m utant ( W98V /L40I) of HTLV 1 PR The best inhibitor is Compound 1 with Ki = 0.8 0.1 M The possible H bonding between Compound 1 and HTLV 1 PR is shown in Figure 47. F ive inhibitors were selected to determine inhibition constants for each construct All of them have significantly better inhibition against the 116residue HTLV 1 PR compared to any of the longer constructs. (Table 4 4 ) D iscussion New therapies are needed to treat patients infected with HTLV 1. The viral target for most of the current treat ments is unknown and most have many side effects. Due to the success seen with targeting the protease from HIV 1, we have focused our studies upon the protease from HTLV 1 which belongs to the same class of enzymes and shares structural and functional characteristics with HIV 1 PR. The specificity constant s stay identical for various lengths of HTLV 1 PR, which indicates that the C terminal amino acids are not essential for full protease activity In an effort to look at the structural differences that may be present in this region and identify specific interactions between the active site residues and the inhibitor c rystallization trials are currently underway, both with and without inhibitors.
63 The compounds selected by the DOCK program have shown inhibit ion both in kinetic and cellular assays. Within the top ra nked inhibitors, 13 of them gave inhibition constants ranging from 1 M to 78 M for the116residue L40I HTLV 1 PR. 15, 3, 5, 7 tetraazatricyclo [126.96.36.199~3, 7~] decane is the most common group in the structure of small molecules. Molecules with an electronegative element (Cl, Br or I) attached to it seem to give better inhibition as in Compounds 2, 4, and 7. Alkenes attached to the 15, 3, 5, 7tetraazatricyclo [188.8.131.52 ~3, 7~] decane molecule have lower inhibition. (Compound 3 and 8) Alcohol group has lower effect on inhibition compared to halogens comparing Compound 4, 7 and 8. Attaching a halogen decreases the inhibition constant for 35 fold, while attaching an alcohol group decreases for 4 fold. (Table 42) The inhibition constants for these small molecules increase for the longer protease constructs. Higher inhibition constant for small molecules means small molecules have lower inhibition for longer constructs. Only Compound 1 has low inhibitions constants for each of the 3 constructs tested here. The selection was made based on the inhibition constants values of 116residue HTLV 1 PR. The different inhibition constant might be based on the extra residues interactions with the inhibitors in the active site of HTLV 1 PR.
64 Table 41. Specificity constants of various constructs of HTLV 1 PR. Residues K m (M ) k cat (s 1 ) k cat /K m (s 1 M 1 ) 116 31 3.6 7.5x10 4 2 x10 5 24 3 121 47 5.2 9.0x10 4 1 x10 5 19 2 122 32.2 4.6 6.5x10 4 3 x10 5 20 3 R2>0.98
65 Table 42. Inhibition constants of 13 inhibitors against L40I and L40I/W98V mutated 116residue HTLV 1 PR. The best of these, marked by asterisk ( ), were used in subsequent experiments. Inhibitor Rankings Numbers Structures L40I K i (M ) W98V/L40I K i (M ) 667746 5,7 1 0.8 0.1 5.1 0.4 168615 6 2 1.1 0.1 8.5 0.4 10408 10 3 27 2 9.3 0.5 172855 12 4 1.1 0.1 5.6 0.5 30049 13 5 4.5 0.4 13 1 35024 14 6 1.1 0.1 5.7 0.5
66 Table 42. continued Inhibitor Rankings Number s Structures L40I K i (M ) W98V/L40I K i (M ) 177977 19 7 8.4 0.5 6.2 0.4 5062 25 8 35 4 .0 45 3 .0 21235 26 9 35 3 .0 58 5 .0 348978 37 10 18 1 .0 35 3 .0 4436 41 11 9.3 0.7 77 9 .0 23429 42 12 38 2 .0 38 3 .0 362403 44 13 3.6 0.2 5.7 0.5
67 Table 4 3 Ki values of 5 compounds with various constructs of HTLV 1 PR Inhibition Constants K i (M ) Compound 116 L40I 121 L40I 122 L40I 1 0.8 0.1 23 2 .0 11 1 .0 2 1.1 0.1 23 1 .0 >100 4 1.0 0.1 >100 >100 7 8.4 0.5 >100 89 10 13 3.6 0.2 14 1 .0 >100 R2>0.98
68 P V I P L D P A R R P V I K A Q V D T Q T S H P K T I E A L L D T G A D M T V L P I A L F S S N T P L K N T S V L G A G G Q T Q D H F K L T S L P V L I R L P F R T T P I V L T S C L V D T K N N W A I I G R D A L Q Q C Q G V L Y L P E A K G P P V I L Figure 41. HTLV 1 PR sequence. R esidue 116 is shown in blue, residue 121 in orange and residue 122 in green.
69 [Substrate] (M) 0 10 20 30 40 50 60 70Rate (M/sec) 0.000 0.005 0.010 0.015 0.020 0.025 0.030 [Substrate] (M) 0 20 40 60 80Rate (M/sec) 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 [Substrate] (M) 0 20 40 60 80 100Rate (M/sec) 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 Figure 42. Kinetic constant determination by Michaelis Menten equation. A.116 B.121 C.122 ( R2>0.98) Km= 31.4 3.6 M Km= 47.4 5.2 M Km= 32.2 4.6 M
70 1/[Substrate] (M) .04 -0.02 0.00 0.02 0.04 0.06 0.081/Rate (M/sec) 20 40 60 80 1/[Substrate] (M) .04 -0.02 0.00 0.02 0.04 0.06 0.081/Rate (M/sec) 10000 20000 30000 40000 50000 60000 70000 1/[Substrate] (M) .04 -0.02 0.00 0.02 0.04 0.06 0.081/Rate (M/sec) 20 40 60 80 Figure 43 Kinetic constant determination by Lineweaver Burk equation. A.116 B.121 C.122 ( R2>0.98)
71 Figure 44 Inhibitor dissociation constant (Ki) Determination. Michaelis Menten curve fit of rate ( mol/min/mg) versus substrate concentration ( R2>0.98
72 Figure 45 Structures of effective plasmepsin inhibitors.
73 Figure 46 Possible H bonding distances of PM48, IM37, FS07, IM64 in the active site of HTLV 1 PR determined by Pymol (UCSF) with the help of Dr. David Ostrov laboratory at University of Florida, Gainesville.
74 Figure 46. continued.
75 Figure 47 Possible H bonding distances of Compound 1 and HTLV 1 PR determined by Pymol (UCSF) with the help of Dr. David Ostrov laboratory at University of Florida, Gainesville.
76 CHAPTER 5 THE EFFECT OF SMALL MOLECULES ON HTLV1 INFECTED CELLS Gag /Pol Processing Gag and Gag/Pol processing take place after HTLV 1 virus buds out of the cell. This process is essential for viral maturation and cell to cell spread ( 70) PR is the functional necessary viral component for this processing; therefore blocking PR is an effective way to inhibit Gag/Pol processing ( 70 ) PR is encoded by the Pro gene which is an open reading frame (ORF) overlaps with both Gag and Pol ORFs ( 69, 70, 128) Pro region does not exist in all the retroviruses like HIV 1 and RSV; but it exist in HTLV 1, HTLV 2, Mouse Mammary Tumor Virus (MMTV) and Bovine Leukemia Virus (BLV) ( 63, 65, 129132 ) Ribosomal shifting in the 1 direction is essential in all the retroviruses to align the various open reading frames ( 133 135) Two frameshifts are necessary for Gag, Pro, and Pol polyprotein synthesis of HTLV 1 ( 75, 136 ) Targeting HTLV 1 PR, the inhibitors were expected to inhibit Gag processing of HTLV 1 ( 111, 137) MT 2 cells were used to test the inhibition effect of HTLV 1 protease inhibitors on HTLV 1 infected ce lls. MT 2 cell line was used for efficient HTLV 1 production ( 138 ) It is derived by co culturing bone marrow CD4+ T lymphocytes of a healthy donor with leukemia cells of an ATL patient ( 139, 140) The selected five best compounds were also used at various concent rations at multiple incubation times to test their antiviral activity. Gag protein precursor, a 53 k Da protein, yields MA (p19, 19 kDa), CA (p24, 24 kDa) and NC (p12, 12 kDa) proteins after proteolytic cleavage. The p19 (MA) antigen has been used for Western Blotting assays as explained in Chapter 2. In Figures 5 1, 5 2 and 5 3 the p53 (Gag) bands are at 53 kDa, p2 8 (MA precursor) are at 28 kDa, and p19 (MA) bands are at 19 kDa. All these
77 bands are present in each sample. The p28 protein, that is found to be linked with kinase activity of protein, is a combination of p19 and part of p24 ( 141 ) In each figure the thick band around 45 kDa appears after incubation of the cells with Compound 1 inhibitor. Beta actin protein was used as a loading control for each sample shown in Figures 5 1, 5 2 and 5 3. None of the i nhibitors show toxic effects wi thin the cells at 4 h, 2 4 h, 2 days, or 6 days post addition of t he inhibitor. The amount of p19 produced is shown to be reduced with the addition of inhibitor and the longer incubation times in ELISA assays. (Figure 54) In Figure 54B the percent inhibition decreases within 48 hours of adding the inhibitor but there is still an inhibition effect on G ag processing. Even though no effect has been seen for C7 by Western Blotting, ELISA assay has shown some inhibition effect on the cells after 24 hours incubation. D iscussion After determining inhibition constants of the computationally top ranked compounds, only five of them were tested in the in vitro cell assays. Their effect on Gag processing was observed. Out of five compounds, the best compound (Compound 1) that was determined to have the lowest inhibition constant (Ki) was the only compound that also showed distinctive bands in the Western Blot and ELISA assays. Even after 4 h of incubation resulted in MA CA uncleaved product, this means the inhibitor effect starts before 4 h. (Figure 52) Reproducing the data at 24 h confirms inhibition of Gag processing. (Figure 51, Figure 52). 2 days and 6 days incubation results the same gene products at 45 kDa (MA CA). (Figure 5 3) A similar e ffect was seen for HIV 1 PR with novel amino acid insertion; partially cleaved Gag products were seen in the Western Blotting ( 142) The ELISA P19 Antigen assay s, which utilized the two best
78 inhibitor compounds identified by Western Blotting and kinetic analysis, support the Western Blotting data as shown in Figure 5 4 The amount of p19 protein was lower in the presence of Compoun d 1 compared to control cells (absence of any inhibitor) in the first or second day. (Figure 54A) The percent inhibition of Compound 1 at the first day is higher than at the second day. (Figure 54B) This result could mean in the case of drug usage, Compound 1 must be taken daily for efficient inhibition effect. Compound 1, which was selected from the kinetic analysis for further testing has also shown an inhibition effect on HTLV 1 infected cells, seeming to stop or slow down MA CA cleavage. Inhibitor screening will continue to identify better compounds and crystal structures will be employed to develop possible drugs for HTLV 1.
79 A Figure 51 Western Blot of selected inhibitors incubated in MT 2 cells. First lane show s the molecular weight marker s, second lane shows control cells without any compound. The rest of the lanes are 50 M of Compound 1 Compound 4 Compound 7 Compound 1 3, Compound 2 control cells without any compound, 5 M of Compound 1 and Compound 7 incubated in MT 2 cells for 24 hours, respectively. 20 g protein has been loaded for 24 hours of incubation cells.
80 Figure 52 Western Blot of selected inhibitors incubated in MT 2 cells for 24 h. (A ) First lane show s the molecular weight marker s, second lane show s control cells without any compound. The rest of the lanes are 10 and 50 M of Compound 1 Compound 7 50 M incubated in MT 2 cells for 4 hours and 24 hours of incubation, respectively. 10 g of protein has been loaded for 4 hours and 20 g protein has been loaded for 24 hours of incubation cells B) Beta actin control loads of MT 2 cells. First lane shows molecular weight marker s, second lane shows control cells without any compound. The rest of the lanes are 10 and 50 M of Compound 1 Compound 7 50 M incubated in MT 2 cells for 4 hours, 24 hours of incubation, respectively. 10 g of protein has been loaded for 4 hours and 20 g protein has been loaded for 24 hours of incubation cells. B A
81 Figure 53 Western Blot of selected inhibitors incubated in MT 2 cells. (A ) First lane show s the molecular weight marker s, second lane show s control cells without any compound. The rest of the lanes are 10 and 50 M of Compound 1 Compound 7 50 M incubated in MT 2 cells for 2 and 6 days of incubation, respectively. B) Beta actin control loads of MT 2 cells. First lane shows the molecular weight marker s, second lane shows control cells without any compound. The rest of the lanes are 10 and 50 M of Compound 1 Compound 7 50 M incubated in MT 2 cells for 2 and 6 days of incubation, respectively. 10 g of protein has been loaded. A B
82 A B Figure 54 A. ELISA assay graph representation. P19 (MA) protein concentration in the cells with 1 and 2 days of incubation with Compound 110 and 50 M, Compound 7 50 M Control cells are shown as quadrangle, 10 M Compound 1 incubated cells are shown as open squares, 10 M Compound 1 incubated cells are shown as squares and 50 M Compound 7 incubated cells are shown as open triangles. B. ELISA assay bar representation. Percent P19 (MA) protein concentration in the cells with 1 and 2 days of incubation with Compound 1 ,10 and 50 M, Compound 7 50 M (No error bars were indicated, because only one set of data was obtained.) 0 200 400 600 800 1000 1200 1400 1600 1800 2000 0.00 0.50 1.00 1.50 2.00 2.50 Time (days) HTLV-1 p19 antigen (ng/ml)
83 CHAPTER 6 DETERMINING FLAP CONFORMATION OF HTLV 1 PR BY ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPY There are many methods that can be used to determine protein structure including X ray crystallography, Nuclear Magnetic Resonance (NMR) Spect roscopy, and Electron Paramagnetic Resonance (EPR) Spectroscopy Because HTLV 1 PR has flexible flap conformations, we have decided to use EPR to determine the flap confirmation of HTLV 1 PR beside the crystallography trials in our research. EPR, is a sens itive method for biological samples; examines the effects of the motion and polarity on the structure and has high accuracy. EPR is a very sensitive method, it can measure fast dynamic changes of a molecule ( 143) EPR was picked for our research based on the quantitative analysis of the flap distances. Using NMR with EPR can provide broad information about the structure of a biomolecule. EPR, is also known as electron spin resonance (ESR) spectroscopy, measure the absorption of a paramagnetic substance when an external magnetic field applied in microwave radiation. Zeeman Effect is the interaction between the substance and the magnetic field. Zeeman effect splits these two unpaired electron in the presence of the magnetic field, these two spin states have degenerate magnetic moments ( ms= 1/2 and ms= +1/2). (Figure 61) The energy difference between these two spin states is calculated by Zeeman equation: E=hv = g B (7 1) Where E i s the energy difference, h is the Plancks constant (6.6260689610 J.s), v is the microwave frequency, g is the splitting factor ( 2 for free electron), is the Bohr magneton (9.27400915 x 10 J T), and B is the applied magnetic field ( 144 ) Enegy diagram is shown in Figure 61.
84 EPR has been used to determine the flap confirmation of HIV 1 P R by Fanucci and her coworkers ( 117, 145, 146) It was found that HIV 1 PR has a broad distri bution of conformation a l change in the absence of the inhibitor, versus a narrow distri bution i n the presence of the inhibitor ( 117, 146) It is assumed HTLV 1 PR acts in a similar manner based on their genomic and sequence similarities The flaps of HIV 1 PR have three conformations: open or semi open forms with no inhibitor, and the closed form with inhibitor bound ( 147 ) This result was supported by NMR results showing large conformational changes at the flap region ( 148 150) Site directed spin labeling (SDSL) is a method to label the specific sites of the macromolecules with spin probes and observe the dynamic changes for macromolecules. The approach to SDSL is introducing a nitroxide site chain at a specific site with using mutagenesis ( 151 ) Nitro xide is the name of the compounds used in the spin labeling. These compounds have R2NO. radicals after removing hy drogen at the hydroxyl groups. There are three commonly used spin labeling probes; the methanethiosulfonate spin label (MTSL), the iodoacetam i do proxyl spin label (IAP) and the maleimido proxyl spin label (MSL). The methanethiosulfonate spin label (MTSL) is commonly used for sitespecific labeling of proteins. It is sensitive to the motions of the protein backbone and secondary structure compared to other labels. The structure of MTSL and its bound structure to free cysteine is shown in Figure 62. The intrinsic motion of spin label, the backbone flexibility in cysteine residue region, and the overall tumbling of the molecule in the solution can be measured by EPR / SDSL. U sing EPR / SDSL, the free electron (ms= 1/2) on the MTSL spin label couples with the nuclear spin from nitrogen (mI =1). Based on 2I+1 rule, three energy transition
85 occur. Because MTSL binds to cysteine amino acid of a protein, any native cysteines are mutated to alanines and a cysteine in a desired region is introduced for analysis. Native cysteines were mutated to alanines at residue positions 90 and 109 for HTLV 1 PR. Based on HIV 1 PR mutations and used methods; three mut ations were made ( 146) The glutamin e at the 64th position, which is equivalent to lysine at 55th position of HIV 1 PR, was mutated to a cysteine for MTSL labeling. K55 was chosen based on the identical activity with the wild type, and the moiety of spin label cooperation ( 146) Mutations were made using the conditions des cribed in the Site Directed Mutagenesis description in Chapter 2. The primers utilized for mutations are shown in Figure 63. It was confirmed that protease with the native cysteines mutated to alanines has the same catalytic properties as the wide type of HTLV 1 PR based on literature ( 82, 103) Protein expression, refolding, and purification methods were identical to those for wild type and purified protein was confirmed by 18% SDS PAGE gel. (Figure 6 4 and 65) The next step is labeling the Cys 64 in the flap region of HTLV 1 P R with MTSL. T he distance between nitroxide spin labels can be measure by two methods. Continuous Wave (CW) method measures the distances based on dipolar interactions and extracts the distance 820 ( 152 ) or Double Electron Electron Resonance (DEER) method ,which is the pulsed EPR, measures the distances by producing a spin echo. DEER capable of measuring distances 2070 The distance between the two alpha carbons of the glutamines at 64th position of HTLV 1 PR were determined by Chimera software (UCSF) to be 19 ( 124) (Figure 6 6) Because the distance between nitroxide spin labels is between 820 CW is a better method to measure the conformational changes of flaps of HTLV 1 PR.
86 Our future work is to determine the molecular dynamics of the HTLV 1 PR flaps in the presence and absence of Compound 1 with using MTSL labeling and EPR spectroscopy.
87 Figure 61. Energy diagram of a sy stem with a free electron in the magnetic field. Figure 62 MTSL label structure a. unbound structure, b. structure of MTSL bound to cystein e a b a
88 >D32N Upper CGAAGCTCTACTAAACACAGGAGCAGACATG Lower CATGTCTGCTCCTGTGTTTAGTAGAGCTTCG > C90A Upper CCTATTGTTTTAACATCTGCGCTAGTTGATACC Lower GGTATCAACTAGCGCAGATGTTAAAACAATAGG > C109A Upper GCCTTACAACAAGCGCAGGGCGTCCTGTACC Lower GGTACAGGACGCCCTGCGCTTGTTGTAAGGC >>Q64C Upper GGGGGCCAAACCTGCGATCA CTTTAAGCTCACC Lower GGTGAGCTTAAAGTGATCGC AGGTTTGGCCCCC Figure 63 Primers for D32N, C90A, C109A and Q64C mutations of HTLV 1 PR. Figure 64. SDS Page gel of expression of triple mutated HTLV 1 PR. Lane A shows the Precision Plus Ladder (Biorad), lane B before IPTG induction, lane C 3 h after IPTG induction and lane D 6 h after IPTG induction and lane E is t he inclusion bodies. A B C D E HTLV 1 PR 250 50 37 25 20 15 10
89 Figure 65. SDS Page gel picture of purification of of triple mutated HTLV 1 PR. Lane A shows the Precision Plus Ladder (Biorad), lane B Inclusion bodies, lane C Q column flow through, lane D Q column elution by 1 M NaCl, lane E SP column flow through, lane F SP column elution by 0.4 M NaCl, lane G SP column elution by 1 M NaCl, and the lane H the Precision Plus Ladder (Biorad). Figure 66 1 PR. A B C D E F G H HTLV 1 PR 250 50 37 25 20 15 10
90 CHAPTER 7 CONCLUSIONS AND FUTURE WORK HTLV 1 was isolated from a cutaneous lymphoma patient in 1980 by Gallo and it was proved to be the causative agent for ATL ( 34, 153) The Centers for Disease Control and Prevention have identified HTLV 1 as emerging pathogen ( 154) HIV 1 and HTLV 1 share many characteristi cs, but HIV 1 is more dangerous than HTLV 1 due to the effect of HIV 1 on the immune system ( 155 ) HTLV 1 infection has been reported in many regions of the world but is most prevalent in Southern Japan, the Caribbean basin, Central and West Africa, the Southeastern United States, Melanesia, parts of South Af rica, the Middl e East and India ( 24 ) Approximately 30 million people are infected by HTLV 1 worldwide, and al though only 35% of the infected individuals evolve ATL in their life, the prognosis for those infected is still poor ( 27) T he overall prognosis of ATL is still poor despi te intensive chemotherapy ( 45) The median survival time of leukemia patients is 7 8 months ( 156 ) There is no specific drug treatment against HTLV 1. Many studies have been focused on drug design against HTLV 1, especially inhibitors for HTLV 1 PR, as PR as a drug target has been successful, especially in the case of AIDS treatment ( 157163) HTLV 1 PR is essential for viral replication and maturation. Therefore, it is a good target for drug design. First, conditions for expression, purification, refolding and kinetic characterization of HTLV 1 PR have been developed as mentioned in the Method chapter. HTLV 1 PR has a loop containing 10 extra amino acids at the C terminal region only similar to BLV PR. It was determined that these 10 amino acids are not necessary for enzymatic activity. Th is research is significant to fully understand the enzymatic activity of HTLV 1 PR and the effects of the C terminal residues on the activity.
91 Recombinant HTLV 1 PR has been used to identify inhibitors against HTLV 1 PR. In silico screening has been used for inhibitor discoveries using the crystal structure of 116residue HTLV 1 PR. 140,000 small molecules were docked in the protease active site, and top ranked molecules were determined by DOCKv5.2 program. Several top ranked small molecules have been assayed in vitro by measuring the cleavage of the substrate A P Q V L*NphV M H P L, which mimics the natural MA/CA cleavage site, and was synthesized by the ICBR Core Facility at UF. Selected inhibitors have been used in in vitro cell culture assays to determ ine the effect in HTLV 1 infected cells. An inhibitor with the lowest inhibition constant has been discovered to inhibit Gag / Pol processing based on Western Blot and ELISA assays. Targeting HTLV 1 PR to treat HTLV 1 related disease is a very promising way based on the issues discussed throughout this diss ertation. These results confirm the inhibition effect of the HTLV 1 PR inhibitors in the cells. In addition to these studies, crystallization trials have been started for the HTLV 1 PR. The conditions fro m the literature and various modifications have been tried. First the conditions that yielded the obtained crystals used to determine the structure of HTLV 1 PR in Li et al. has been employed ( 27 ) Various pHs, salt concentration and precipitate concentrations have been tried, but only needle shaped crystals without any diffractions have been obtained. Hampton Research crystal screening kits and detergent kits have been used; in addition, the purified sample has been sent to Hampton Research to determine optimum conditions for crystal trials. No crystal structure has been obtained yet; new strategies will be tried to obtain a crystal structure while the set up crystal trays might produce crystals in time. Learning X ray structure of
92 the full lengt h HTLV 1 PR might provide a great insight to the inhibitor discoveries and treatment HTLV 1 related disease. EPR studies are underway to determine flap conformation in the presence and absence of inhibitor. Necessary mutations and computational distance m easurements have been made for EPR assays. Labeling and determination of flap conformational studies will continue using information of EPR studies of HIV 1 PR taking consideration of their structural similarities. The discoveries of the flap confirmation of HTLV 1 PR would expand the information about the binding of the inhibitors and their effect on the structures, as well as the effect of the C terminal residues on the structure of the protease. Expressing HTLV 1 PR in a soluble system would prevent fol ding and aggregation problems of HTLV 1 PR. Improving the folding properties by trying new strategies would help understanding the properties of full length of HTLV 1 PR. Obtaining information about the conformation of flaps can give details about the structure and the interactions between inhibitor and the protease. Determining crystal structure by X ray crystallography or NMR would provide detailed information about enzyme active site, enzyme inhibitor interactions.The best inhibitor that found by kinetic characterization can be improved after obtaining crystal structure of inhibitor bound HTLV PR. It can be tried in animal model to observe the effect of the inhibitors. There have been a few animal models identified for HTLV 1 in the listed references ( 164, 165 ) Understanding enzymology and structure of HTLV 1 PR is critical to design an inhibitor. Our in vitro assays help understanding enzymology of HTLV 1 PR, specifically effect of the last residues at the C terminal region. The best inhibitor has effect in the
93 HTLV 1 infected cells. This work can lead a drug targeted at HTLV 1 PR to cure HTLV 1 infected patients.
94 APPENDIX : SEQUENCE C ATATG CCAGTTATACCGTTAGATCCCGCCCGTCGGCCCGTAATTAAAGCCCAGG TTGACACCCAGACCAGCCACCCAAAGACTATCGAAGCTCTACTAGATACAGGAGC AGACATGACAGTCATCCCGATAGCCTTGTTCTCAAGTAATACTCCCCTCAAAAAT ACATCCGTATTAGGGGCAGGGGGCCAAACCCAAGATCACTTTAAGCTCACCTCCC TTCCTGTGCTAATACGCCTCCCTTTCCGGACAACGCCTATTGTTTTAACATCTTG CCTAGTTGATACCAAAAACAACTGGGCCATCATAGGTCGCGATGCCTTACAACAA TGCCAGGGCGTCCTGTACCTCCCTGAGGCAAAAGGGCCGCCTGTAATCTTGGGAT CC Figure A 1. DNA sequence of full length HTLV 1 PR with a start methionine with a 5 NdeI site and a 3 BamH1 site for directi onal cloning into pET 11a.
95 P V I P L D P A R R P V I K A Q V D T Q T S H P K T I E A L L D T G A D M T V L P I A L F S S N T P L K N T S V L G A G G Q T Q D H F K L T S L P V L I R L P F R T T P I V L T S C L V D T K N N W A I I G R D A L Q Q C Q G V L Y L P E A K G P P V I L Figure A 2. Protein sequence of full length HTLV 1 PR.
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111 BIOGRAPHICAL SKETCH Ahu Demir was born in June of 1982 in Ankara, Turkey She completed high school at Cumhuriyet High School in Ankara in June of 200 0 Ahu began her undergraduate work in October of 2000 at the Hacettepe University, Ankara, majoring in C hemistry. Ahu began her research career under the tutelage of Dr. Adil Denizli, studying the folding of tRNAs for three years. Her senior year she work ed for Mining Inc. as a laboratory technician, learning the atmosphere of an industrial work setting. In May 2004 Ahu graduated with a B.S. in C hemistry In January 2005 Ahu joined the Chemistry Department in the College of Life and Science at the University of Florida in G ainesville, Florida. She started working with Dr. Steven Benner on 2005 until his leave. In February 2006 Ahu joined the laboratory of Distinguished Professor Dr. Ben M. Dunn. She spent four years characterizing novel proteins for Human T cell Leukemia Virus 1 Protease, gaining invaluable research and teaching skills. She received her PhD from University of Florida in the fall of 2010.