docResistance Pathways for Potent and Broadly Active HIV
download 
Report Transcription
Resistance Pathways for Potent and Broadly Active HIV
Resistance Pathways for Potent and Broadly Active HIV-1 Maturation Inhibitors Emiko Urano Eric Freed Lab HIV Drug Resistance Program National Cancer Institute, Frederick HIV-1 Virion Maturation Immature PR Mature Keller et al., JVI. 2010 Balasubramaniam & Freed, Physiology 2011 Bevirimat: First-in-class HIV-1 Maturation Inhibitor Bevirimat (BVM, PA-457, DSB) (dimethylsuccinyl betulinic acid) H H O H OOC C OOH H O H Gag Is Processed in a Highly Ordered Cascade MA CA NC SP1 MA CA NC SP1 MA CA p6 SP2 NC CA p6 SP2 SP1 MA NC SP1 Gag p6 SP2 p6 SP2 Li et al., PNAS, 2003, Zhou et al., JVI, 2004 Bevirimat: First-in-class HIV-1 Maturation Inhibitor Bevirimat (BVM, PA-457, DSB) (dimethylsuccinyl betulinic acid) H H O H OOC C OOH H O H Mechanism: Inhibits CA-SP1 processing virus BVM CA NC SP1 MA CA NC SP1 MA CA Gag p6 SP2 p6 SP2 SP1 CA NC SP1 p6 SP2 + 80 60 40 20 0 MA - 100 p6 SP2 NC + CA-SP1 CA % CA-SP1 MA - BVM Li et al., PNAS, 2003, Zhou et al., JVI, 2004 Bevirimat: First-in-class HIV-1 Maturation Inhibitor Bevirimat (BVM, PA-457, DSB) (dimethylsuccinyl betulinic acid) H H O H OOC C OOH H O H Mechanism: Inhibits CA-SP1 processing virus BVM CA NC SP1 MA CA NC SP1 MA CA Gag p6 SP2 p6 SP2 SP1 CA NC SP1 p6 SP2 + 80 60 40 20 0 MA - 100 p6 SP2 NC + CA-SP1 CA % CA-SP1 MA - BVM Li et al., PNAS, 2003, Zhou et al., JVI, 2004 Phase II BVM Clinical Trials (Panacos Pharmaceuticals; ca. 2007) • ~50% of patients responded with a drop in viral load of 12 logs • ~50% of patients did not respond • Polymorphisms in SP1 (e.g., V7A) were associated with lack of response both in vivo (Baelen et al., AAC 2009, Margot et al., AAC 2010) and in vitro (Adamson et al., Retrovirology 2010) Clinical development of BVM put on hold Polymorphisms in SP1 (e.g., V7A) Reduced HIV-1 Susceptibility to BVM and Were Associated with a Lack of Response in Patients CA >M GROUP CONSENSUS >CONSENSUS_A1 >CONSENSUS_A2 >CONSENSUS_B >CONSENSUS_C >CONSENSUS_D >CONSENSUS_F1 >CONSENSUS_G >CONSENSUS_H >CONSENSUS_K GGP - - - - - - - - - - - - - - - - - - - S HK G - - - G - - - - - G - - - - - G - - SP1 ARVL - - - - - - - - - - - - - - - - - - - - - - - - - -I - AE - - - - - - - - - - AMSQV - - - - - - - - - - - - - - - -A - - - -A - - - -A - - - -A - - - - - - - - - T Q Q N S - N H G - Goal: to develop BVM analogs that more effectively inhibit HIV-1 replication and display broader activity against viral strains harboring SP1 polymorphisms Second-Generation Maturation Inhibitor Program to Overcome SP1-V7A Restriction C -1 9 A n a l o g s : A lc o h o l s , A m i n e s , K e to n e s C H2 H 3C 19 H C H3 H 3C C H3 O H 3 HO C H3 H C - 2 8 A n al o g s : A c i d s , A c i d Is o s te r e s , A l c o h o l s , A m i d e s , A m i n e s a n d D e r i v a ti v e s , E s te r s , E th e r s C O 2H 28 C H3 O O H 3C H C H3 A l te r n a te T e m p l a te s C -3 A n a l o g s : A c id s , A c i d Is o s te re s , A l c o h o l s , A m i n e s , A m i d e s , E s te r s Chemistry effort led by Panacos and DFH Pharma; Synthesis effort assisted by the CCR Chemical Biology Laboratory ~500 Analogs Synthesized Screening: Identification of Second-Generation Maturation Inhibitors CA-SP1 accumulation : 100 nM Compound 7t 7s 7r 0 7q 0 7p 10 7o 10 7n 20 7l 20 7k 30 BVM 30 DMSO 40 7t 40 7s 50 7r 50 7q 60 7p 60 7o 70 7n 70 7m 80 7l 80 7k 90 BVM 90 DMSO % CA-SP1 SP1-V7A 100 7m WT 100 Second-generation BVM Analogs Are Active Against SP1-V7A Compound BVM 7k 7l 7m 7n 7o 7p 7q 7r 7s 7t WT IC50 136 ± 30 nM 3±1 14 ± 7 3 ± 0.3 7±3 9±3 3 ± 0.4 5±1 5±3 21 ± 19 2 ± 0.5 SP1-V7A IC50 >500 145 ± 29 201 ± 44 9±2 14 ± 4 22 ± 5 26 ± 6 22 ± 7 10 ± 3 8±2 19 ± 6 The IC50 values represent the mean ± SEM from four independent experiments. Second-Generation BVM Analogs Are Active Against Primary Isolates in PBMC IC50: PBMC BVM Compound 7r NL4-3 Subtype B 5 nM 5 nM 93IN101 Subtype C 2,651 67 99UGA07412MI Subtype D 15 15 CMU02 Subtype E 3,327 96 93BR029 Subtype F 7 13 JV1083 Subtype G 4,188 221 YBF30 Group N 1,489 43 BCF02 Group O 444 7 Identification of BVM-Resistant Mutants CA NC HIV-2 …… SP1 231 1 Wild-Type CA-H226Y CA-L231F CA-L231M SP1-A1V SP1-A3T SP1-A3V 225 CA Gag p6 …………………… 14 MA GHKARVLAEAMSQVTNPATIM… -Y-------------------… ------F--------------… ------M--------------… -------V-------------… ---------T-----------… ---------V-----------… -Q---LM---LKE... Adamson et al., JVl. 2006 Second-Generation BVM Analogs Are Active Against Most BVM Resistant Viruses CA-SP1 accumulation : 100 nM 90 DMSO DMSO BVM BVM GTP03-16 7m GTP03-21 7r 80 % CA-SP1 70 60 50 40 30 20 10 0 WT CA H226Y CA-H226Y CA L231F CA-L231F CA L231M CA-L231M SP1 A1V SP1-A1V Identification of PF96-Resistant Mutants PF-46396 (PF96) BVM H H O H OOC C OOH H O H Blair et al., AAC, 2009 • We observed that resistance to PF-96 resulted from mutations near the CA-SP1 cleavage site and far upstream in CA, including the major homology region (MHR) • The MHR mutants were markedly compound-dependent Waki et al., PLoS Pathog 2012 Second-Generation BVM Analogs Potently Inhibit HIV-1 Replication Compound 7m R: H R’: -CH2CH2N(CH3)2 Compound 7r BVM R: H R’: 9000 Mock MOCK WT 8000 RT-activity (cpm/ul) GTP-03-16 7m (2 nM)(2nM) 7000 GTP-03-21 7r (2 nM) (2nM) DMSO DMSO 6000 5000 4000 3000 2000 1000 0 1 3 5 7 9 11 13 15 17 Day Post-transfection 19 21 23 25 Mutations in CA and SP1 Were Acquired During Selection in Second-Generation BVM Analogs CA NTD (1-145) Pr55Gag SP1 MA 157 MHR ILDIRQGPKEPFRDYVDRFYKTLRAE A CTD (146-231) NC SP2 CA/SP1 1 GPGHKARVLAEAMSQVT V p6 Mutations in CA and SP1 Were Acquired During Selection in Second-Generation BVM Analogs CA NTD (1-145) Pr55Gag CTD (146-231) SP1 MA 157 MHR ILDIRQGPKEPFRDYVDRFYKTLRAE A P157: 99.94% A157: 0.01% (~30,000 seq) Courtesy Wei Shau NC SP2 CA/SP1 1 GPGHKARVLAEAMSQVT V p6 Mutations in CA and SP1 Were Acquired During Selection in Second-Generation BVM Analogs CA NTD (1-145) Pr55Gag SP1 MA 157 MHR ILDIRQGPKEPFRDYVDRFYKTLRAE A CTD (146-231) NC SP2 CA/SP1 1 GPGHKARVLAEAMSQVT V A1: 99.63% V1: 0.017% (~30,000 seq) Courtesy Wei Shau p6 The P157A Mutation in the CA MHR Confers Resistance to BVM Analogs CA-SP1 accumulation: 100 nM 90 90 % CA-SP1 WT CA-P157A 80 80 70 70 60 60 50 50 40 40 30 30 20 20 10 10 0 0 DMSO BVM 7m 7r DMSO Compound BVM 7m 7r Replication Kinetics of Resistant Mutants With BVM Analogs WT 9000 SP1-A1V 12000 1 BVM BVM 2525 nM nM 8000 7000 10000 6000 8000 2 BVM BVM 100 nM nM 100 9 7r GTP03-021 5 nM 5 nM 10 10 nM 7rGTP03-021 10 nM 11 25 nM 7rGTP03-021 25 nM 5000 RT-activity (cpm/ul) 12 50 nM 7rGTP03-021 50 nM 6000 4000 3000 17 DMSO DMSO 4000 18 MOCK w/ DMSO MOCK 2000 2000 1000 0 0 0 10 20 30 40 50 60 70 CA-V230I/SP1-V7A 10000 0 8000 8000 7000 7000 20 30 BVM 25 nM BVM 25 nM BVM 100 100 nM nM BVM GTP03-021 nM 7r 5 5nM GTP03-021 nM 7r 1010nM GTP03-021 nM 7r 2525nM GTP03-021 nM 7r 5050nM DMSO DMSO MOCK w/ DMSO MOCK CA-P157A 9000 9000 10 6000 6000 5000 5000 4000 4000 3000 3000 2000 2000 1000 1000 0 0 0 10 20 30 40 0 5 Days Post-transfection 10 15 The CA-P157A and CA-V230I/SP1-V7A Mutants Display WT CA-SP1 Processing Kinetics Pulse-chase CA-SP1 accumulation: without Compounds 90 WT WT 90 WT WT 80 SP1-A1V A1V 80 SP1-V7A V7A 70 CA-V230I/SP1-V7A V230I/V7A CA-P157A P157A % CA-SP1 70 60 60 50 50 40 40 30 30 20 20 10 10 0 0 0 30 60 120 0 Chase (min) 30 60 120 Conclusions A set of C-28 alkyl amine derivatives of BVM have been identified that exhibit low-nM activity across major clades of HIV-1; these compounds are active against SP1 polymorphic viruses that display reduced susceptibility to BVM These second-generation maturation inhibitors also retain some activity against HIV-1 isolates selected for resistance to BVM Selection for resistance to the second-generation BVM analogs gave rise to a mutation in a highly conserved position in the CA major homology region (MHR) The level of CA-SP1 accumulation was significantly reduced in the presence of BVM and BVM analogs against CA-P157A mutant, however, the extent of CA-SP1 processing of CA-P157A was similar to that of WT The CA-P157A mutant was able to replicate in the Jurkat T-cell line, and conferred partial resistance to all BVM analogs. Efforts are underway to define the structure of the maturation inhibitor binding pocket to facilitate the rational design of additional maturation inhibitors and to better understand the function of CA-SP1 region of Gag in virus maturation The Structure of the MI Binding Pocket Remains Unresolved CA-NTD Schur et al. & Briggs Nature 2015 CA-CTD SP1 Acknowledgments NCI-HIV DRP Virus-Cell Interaction Section Sherimay Ablan Nishani Kuruppu Justin Kaplan Melissa Fernandez Maria Novikova Maya Swiderski Rachel Van Duyne Abdul Waheed DFH Pharma, Inc. David E. Martin T. J. Nitz Carl T. Wild NIAMS-NIH Alasdair Steven Juan Fontana South Asian University Ritu Gaur Rebecca Mandt Kayoko Waki Eric Freed Hetero Research Foundation NCI-Chemical Biology Lab Gary Pauly Dina Sigano Joel Schneider NCI-HIV DRP Retrovirus assembly Section Siddhartha Datta Alan Rein University of Delaware Tatyana Polenova Southern Research Institute University of Missouri Anna T Gres Karen A Kirby Stefan G Sarafianos Model for Maturation Inhibitor Binding WT Gag + PF-46396 + BVM CA-CTD PR X PR X PR SP1 Waki et al., PLoS Pathog 2012 Model for Maturation Inhibitor Binding Mutations in MHR + PF-46396 CA201 MHR-Mut PR PR Multimerization Defect WT-like Structure • The distinct but partially overlapping panel of resistant mutants suggests that PF-46396 and BVM may share portions of a binding pocket. • The structure function relationship between the CA-CTD and SP1 is suggested. Waki et al., PLoS Pathog 2012 The Structure of the MI Binding Pocket Remains Unresolved The binding between BVM and HIV-1 Gag • BVM is able to block PR-mediated CA-SP1 processing in the context of in vitro-assembled Gag but does not block processing in non-assembledmonomeric Gag (Li F et al. Proc Natl Acad Sci U S A. 2005, Sakalian et al. J Virol. 2006) • Direct binding of 3H-BVM to immature but not mature HIV-1 particles has been detected (Zhou et al. J Biol Chem. 2005, Zhou et al. J Virol. 2006) • A recent study using photoactivatable BVM derivatives observed direct crosslinking of the compound to residues in the vicinity of the CA-SP1 cleavage site (Nguyen et al. Retrovirology. 2011) CA-NTD CA-CTD SP1 Schur et al. & Briggs Nature 2015 PF-46396 antagonizes the activity of BVM when cells are treated simultaneously with both compounds 70 Virus CA-SP1 % 60 50 40 30 20 10 0 PF-46396 (mM) BVM (mM) 1- 0.1 2 0.5 3 41 0.5 5 0.5 6 - - - - 0.01 0.05 0.5 7 0.5 8 0.1 0.5 -9 10 0.01 0.05 -11 12 0.1 0.5 CA-SP1 CA SP1-T8I Phenocopies the Effect of MIs by Blocking CA-SP1 Processing WT PF-46396 - P157S + - + P157S/ G225D/ G225D T8I T8I T8I - + - + - + - + 100 Cell p55- % CA-SP1 75 p41- CA-SP1CA- 50 25 PF-46396 0 + WT - + P157S - + P157S/T8I - + G225D - + G225D/T8I - + T8I Virus CA-SP1CA- WT - + T8I - + PF-46396 CA Assembly CA Monomer NTD Ganser-Pornillos COSB 2008 CTD MHR CA Hexamer Mature Capsid Pornillos Cell 2009 Pornillos Nature 2011 The MHR Mutants were markedly Compound-dependent CA NTD (1-145) MA Pr55Gag CTD (146-231) SP1 NC SP2 p6 160 156 157 MHR DIRQGPKEPFRDYVDRFYKTLRA ES L CA G156E 0mM WT 25000 0uM 5uM 20000 CA P157S 18000 12000 15000 10000 6000 5000 0 0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 5mM Blocking CA-SP1 Cleavage Inhibits Virion Maturation Immature Non-Infectious Mature Infectious BVMTreated Poorly-Infectious Keller et al., JVI. 2010 Second-Generation BVM Analogs Are Active Against Primary Isolates in PBMC IC50: PBMC BVM GTP03-021 NL4-3 Subtype B 4.8 nM 5.2 nM 93IN101 Subtype C 2,651.0 66.6 99UGA07412MI Subtype D 15.4 14.5 CMU02 Subtype E 3,327.0 95.6 93BR029 Subtype F 6.9 12.9 JV1083 Subtype G 4,188.0 221.0 YBF30 Group N 1,489.0 43.4 BCF02 Group O 444.0 6.9 The CA P157A Mutation Confers Partial Resistance to All BMV Analogs in Replication Assay WT 9000 8000 1 BVM 25 nM 2 BVM 100 nM 5 GTP03-016 5 nM 6 GTP03-016 10 nM 7 GTP03-016 25 nM 8 GTP03-016 50 nM 17 DMSO 18 MOCK w/ DMSO 10000 7000 6000 8000 5000 6000 4000 RT-activity (cpm/ul) SP1 A1V 12000 3000 4000 2000 2000 1000 0 0 0 8000 10 20 30 40 50 60 70 0 12000 CA V230I/SP1 V7A 7000 10 20 CA P157A BVM 25 nM BVM 100 nM GTP03-016 5 nM GTP03-016 10 nM GTP03-016 25 nM GTP03-016 50 nM DMSO MOCK w/ DMSO 10000 6000 8000 5000 6000 4000 3000 30 4000 2000 2000 1000 0 0 0 10 20 30 40 0 Day Post-transfection 5 10 15 Future Directions To develop more potent maturation inhibitors • Continue screening to identify maturation inhibitor candidates. more • Evaluate the stability and inhibitory compounds in vivo (animal model). second-generation potency of these • Evaluate the breadth of antiviral activity against diverse HIV-1 isolates. To define the structure of the maturation inhibitor binding pocket • Characterize resistance mutations that arise during propagation of HIV-1 in the presence of second-generation maturation inhibitors. • Measure the binding activity of maturation inhibitors to HIV-1 Gag (Mass spec, SPR, 3H-labeling, cryo-electron tomography). • Solve the structure of assembled Gag bound to maturation inhibitors (ssNMR). The Double Mutations CA V230I/SP1 V7A Confers Resistance to All BVM Analogs NL4-3 WT 92UG031 Subtype A CA SP1 HKARVLAEAMSQVTN--PATIM ****I*******AQH**T-N** CA-SP1 accumulation : 100 nM 90 DMSO BVM GTP03-16 GTP03-21 80 70 % CA-SP1 60 50 40 30 20 10 0 WT V7A V230I V230I/V7A The CA P157A Mutation Confers Partial Resistance to BVM Analogs in Replication Assays 9000 12000 WT 8000 7000 12000 SP1 A1V BVM 25 uM nM BVM 100 nM CA P157A 10000 10000 8000 8000 6000 6000 GTP03-016 50 nM DMSO 4000 4000 MOCK w/ DMSO 2000 2000 6000 GTP03-016 5 nM GTP03-016 10 nM GTP03-016 25 nM 5000 RT-activity (cpm/ul) 4000 3000 2000 1000 0 0 0 10 20 30 40 50 60 9000 70 0 10 20 30 12000 8000 10000 7000 6000 8000 5000 6000 4000 3000 4000 2000 2000 1000 0 0 5 10 15 9000 BVM 25 uM nM 8000 BVM 100 nM 7000 GTP03-021 5 nM 6000 GTP03-021 10 nM 5000 GTP03-021 25 nM 4000 GTP03-021 50 nM 3000 DMSO 2000 MOCK w/ DMSO 1000 0 0 0 10 20 30 40 50 60 70 0 0 5 10 15 20 25 Day Post-transfection 0 5 10 15 Structural Proximity of PF96-Resistance Mutations CA CTD (146-231) SP1 SP2 NC DIRQGPKEPFRDYVDRFYKTLRA ES L p6 NANPDCKTILKA V 225 226 CA/SP1 201 160 MHR 156 157 Pr55Gag NTD (1-145) MA 3 GPGHKARVLAEAMSQVT DY V T Structural Proximity of PF96-Resistance Mutations CA NTD (1-145) MA Pr55Gag CTD (146-231) SP1 SP2 NC DIRQGPKEPFRDYVDRFYKTLRA ES NANPDCKTILKA V L ES 225 226 201 CA/SP1 160 156 157 MHR p6 L 3 GPGHKARVLAEAMSQVT DY V T CA-NTD CA-NTD CA-CTD CA-CTD G156 P157 P160 von Schwedler et al., JVI. 2003 I201 SP1 Schur et al. & Briggs Nature 2015 The MHR Mutants Are Markedly Compound-dependent CA CTD (146-231) SP1 NC MHR 160 156 157 Pr55Gag NTD (1-145) MA DIRQGPKEPFRDYVDRFYKTLRA ES L WT 25000 0uM 5uM 20000 CA P157S 18000 12000 15000 10000 6000 5000 0 0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 SP2 p6
