1 / 45

MOLECULAR MECHANISMS OF POLYMERIZATION, RNase H and DRUG RESISTANCE IN HIV REVERSE TRANSCRIPTASE

MOLECULAR MECHANISMS OF POLYMERIZATION, RNase H and DRUG RESISTANCE IN HIV REVERSE TRANSCRIPTASE. P. N. HIV/AIDS: A GLOBAL HEALTH CRISIS The Culprit . Human immunodeficiency virus (HIV) causes AIDS Untreated HIV infection usually leads to death HIV is a human retrovirus

morse
Download Presentation

MOLECULAR MECHANISMS OF POLYMERIZATION, RNase H and DRUG RESISTANCE IN HIV REVERSE TRANSCRIPTASE

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. MOLECULAR MECHANISMS OF POLYMERIZATION, RNase H and DRUG RESISTANCE IN HIV REVERSE TRANSCRIPTASE P N

  2. HIV/AIDS: A GLOBAL HEALTH CRISIS The Culprit • Human immunodeficiency virus (HIV) causes AIDS • Untreated HIV infection usually leads to death • HIV is a human retrovirus • HIV mutates rapidly: 1 change in 10 kb (+)-strand RNA genome per replication cycle • ~1010 - 1011 virions in an infected individual, ~2 days per replication cycle • “Quasispecies” nature of viral population • => HIV is a moving target, complicating • --drug development (resistance) • --vaccine development (low cross-reactivity)

  3. Life cycle of HIV Bartlett and Moore, Scientific American July 1998 HIV targets and hijacks cells in the immune system HIV programs infected cell to make many copies of itself Billions of new HIV particles each day in infected person Drugs target essential machinery of the virus, including reverse transcriptase and protease HIV-1 Reverse Transcriptase (RT) X-ray crystallography has yielded pictures of HIV-1 reverse transcriptase in atomic detail HIV-1 reverse transcriptase (magnified x 10,000,000) RT is the target of the majority of anti-AIDS drugs: Knowing structure enables design

  4. r pbs u5 A. ppt r u3 • minus strand strong stop synthesis ppt pbs u3 r B. (-) U5 R • minus strand strong stop transfer and extension ppt C. (-) (-) PPT U3 U5 R PBS PPT U3 R U5 PBS • degradation of RNA and plus strand strong stop synthesis D. PBS R U5 U3 (+) PPT R U5 U3 PBS R U5 U3 (+) PPT U3 U5 R PBS • plus strand strong stop transfer and extension The process of reverse transcription • removal of minus strand primer • complete extension of the minus strand 3’ end

  5. thumb RNase H p66 palm Polymerase active site p51 fingers

  6. Bending DNA

  7. Molecular mechanism of nucleic acid polymerization P N

  8. Steps of DNA polymerization RT/DNA RT/DNA/dNTP RT fingers thumb DNAn PPi dNTP E E’/DNAn E’/DNAn+1 E*/DNAn/dNTP E’/DNAn/dNTP E + DNA Conformational change/ catalysis dNTP binding DNA binding Translocation

  9. P site (priming site) Primer dNTP N site (nucleotide binding site) 3’OH a Catalytic carboxylates b g Released as pyrophosphate

  10. Step 1 Step 2 Step 3 Step 4 Step 5 DNA dNTP PPi E’/DNA E+DNA E E#/DNA-dNMP E+/DNA/dNTP E*/DNA/dNTP P P P P PPi N N N N dNTP dNTP Complex N “closed” ternary complex Complex P “open” ternary complex Translocation Step 5’

  11. Molecular mechanism of translocation P N

  12. Structural changes at YMDD motif during the course of polymerization DNA dNTP PPi E’/DNA # E E’/DNA/dNTP E*/DNA/dNTP E+DNA E/DNA+1 Translocation dNTP P site P E 3’OH Y E E’/DNA M E*/DNA/dNTP N N site D D185 E’/DNA D185 II I

  13. 2.5 Å Close contact, charge repulsion destabilize the N complex - -

  14. Structural changes at YMDD motif during the course of translocation Post-translocation complex P P N - “Punched down” pre-translocation complex N 1.5 Å - catalytic complex (E#/DNA+1) D185

  15. Molecular mechanisms of drug resistance P N

  16. HIV-1 Reverse Transcriptase • polymerase and RNase H activities • a major target for inhibitors • extensive drug resistance NRTI NNRTI RNase H Excision-product analogs?

  17. Why triple-drug therapies? Error rate ~10 x e-4 Probability of 3 independent resistance mutations is < 10 x e-12

  18. HIV RT as a drug target • Strategy: a chess match against the virus • 1. Need to think further ahead than the virus: anticipate variation/resistance mutations that can arise • Janssen NNRTIs -- the DAPY compounds • Create inhibitors that require more mutations of the virus than possible in one step: heavy suppression of replication, high activity against common resistant variants -- high genetic barrier • 2. Target conserved portions -- active site residues • RNase H inhibitors: Himmel, Sarafianos, et al. with • Michael Parniak (U of Pittsburgh)

  19. Locations of Drug Binding Sites in HIV-1 RT Structure Fingers Thumb Rnase H Palm Nucleoside inhibitor binding site Non-nucleoside inhibitor binding site

  20. Non-nucleoside RT inhibitors (NNRTIs) • Potent NNRTIs inhibit • HIV-1 RT at nanomolar • concentrations • NNRTIs are chemically • diverse • NNRTIs do not compete • with binding of nucleic acid • or nucleotide substrates: • allosteric inhibitors/low toxicity • Nevirapine (NEV), delavirdine (DLV), efavirenz (EFV), and etravirine (ETV) are approved drugs dNTP binding site NNRTI binding site

  21. Scheme 1 TIBO (R86183) Loviride (R95845) ITU (R100943) DATA (R106168) DATA (R120393)DATA (R129385) DAPY (TMC120-R147681)DAPY (TMC125-165335) DAPY (R185545)

  22. W229 L234 Y188 Y318 F227 Y181 K101 V179 K103 I II III W229 L100 L234 K101 Y188 Y181 K103 V179 8-Cl TIBO a-APA Common binding mode of 1st generation NNRTIs to HIV-1 RT Neviripine W229 L100 L234 K101 F227 Y188 Y181 K103 V179 Ding, Das, et al., Nat. Struct. Biol. (1995) 2:407-415

  23. No pocket present in apo-enzyme (unliganded) HIV-1 RT Aromatic side chains move to create pocket (conformational “breathing”) Must know structure with bound inhibitor for design!

  24. Locations of Drug Resistance Mutation Sites in HIV-1 RT/DNA Structure Nucleoside drug resistance mutation sites Non-nucleoside drug resistance mutation sites

  25. What are the special structural characteristics of 3d generation NNRTIs? What do they teach us in terms of mechanism of action?

  26. Design Considerations in Developing the Ideal Anti-HIV Drug • Potency against a broad range of HIV variants including common drug-resistant viral strains: Don’t allow breakthrough • High oral bioavailability and long elimination half-life, allowing once-daily treatment at low doses: Optimize compliance • Minimal adverse effects • Ease of synthesis and formulation: Global utility

  27. Structure-Based Drug Design • Multi-disciplinary effort • 3DSAR: Information from structures of inhibitor complexes with target enzyme (wild-type and mutant HIV-1 RT) enables design of new inhibitors as drug candidates Das et al., Progress in Biophys. Mol. Biol. (2005) 88:209-231 Janssen et al., J. Med. Chem. (2005) 48:1901-1910

  28. Roadmap: Discovery of DAPY inhibitors a-APAloviride R89439 (1991) ITU R100943 (1993) TIBOtivirapine R86183 (1987) R165335 TMC125 (1999) DAPYR147681 TMC120 (1998) DATA R106168 (1994) R278474 TMC278 (2001) Janssen et al. (2005) J. Med. Chem. 48:1901-1910

  29. Trp229 Leu234 Trp229 Leu234 Leu100 Phe227 Leu100 TMC125- R165335 R185545 Tyr181 Phe227 Tyr181 Tyr188 Val106 Tyr188 Lys101 Val106 Val179 Lys101 Lys103 Val179 Asn103 TMC120-R147681 R120393 Trp229 Trp229 Leu234 Leu234 Leu100 Leu100 Lys101 Tyr181 Phe227 Phe227 Tyr181 Lys101 Tyr188 Tyr188 Val106 Val106 Lys103 Lys103 Val179 Val179 Das et al., J. Med. Chem. (2004)47:2550-2560

  30. Strategic Flexibility Flexible inhibitor Rigid inhibitor Torsional changes (wiggling) Steric hindrance Reorientation and repositioning (jiggling) Das et al., J. Med. Chem. (2004) 47:2550-2560

  31. Dapivirine (TMC120) Vaginal Microbicide Gel Clinical Trial Sponsored by: International Partnership for Microbicides, Inc. Now being expanded to include 10,000+ patients in a long-term prophylaxis study Implications: Prevent heterosexual transmission of HIV-1 Principle has been demonstrated in animal studies TMC120/dapivirine in a microbicidal formulation has been shown to work by binding to and inactivating HIV-1 particles Female-controlled prophylaxis -- important modality considering cultural and traditional barriers >50% efficacy would have dramatic impact on HIV epidemic (better could be expected, but compliance complicated)

  32. Phase II and III trials + approval of TMC125/etravirine/Intelence II TMC125-C223: 24 weeks 400 or 800 mg TMC125 twice daily in patients with documented resistance to NNRTIs and PIs. Saw median 1.04 and 1.18 log drops in viral load in the two groups. (Nadler et al., 10th European AIDS Conference Dublin, abstract LBPS3/7A, Nov. 2005) TMC125 is the first NNRTI that can be used in patients failing available NNRTI therapy. III: TMC125 200 mg (new formulation) twice daily, with TMC114/Ritonavir (600 mg/100 mg twice daily) and two other anti-AIDS drugs. Enrolling 600 patients with documented NNRTI- and PI- resistance mutations. TMC114: protease inhibitor effective against PI-resistant HIV September 2006: Expanded access program for TMC125 September 2007: Paperwork requesting approval sent to US FDA January 2008: FDA: Fast-tracked approval of Intelence/etravirine/TMC125

  33. Roadmap: Discovery of DAPY inhibitors a-APAloviride R89439 (1991) ITU R100943 (1993) TIBOtivirapine R86183 (1987) R165335 TMC125 (1999) DAPYR147681 TMC120 (1998) DATA R106168 (1994) R278474 TMC278 (2001) Janssen et al. (2005) J. Med. Chem. 48:1901

  34. Nevirapine Efavirenz TMC120 TMC125 Wild-type 81 1 1 3 L100I 597 35 11 3 K103N 2,879 28 2 1 Y181C 10,000 2 7 6 Y188L 10,000 78 37 2 G190S 1,000 275 2 3 K103N+ Y181C 10,000 37 54 4 R278474 0.4 0.4 0.3 1.3 2 0.1 1 Activity of R278474 and Reference Compounds Janssen et al. (2005) J. Med. Chem. 48:1901

  35. Clinical Evaluation of TMC278-R278474 (Rilpivirine) Potency: wild-type HIV- EC50=0.5 nM (0.19ng/ml). Selectivity index= 16,000 Half-life: 38 hours Strong binding Conformational flexibility 14th Conf. on Retroviruses and Opportunistic Infections(http://www.retroconference.org/2007/Abstracts/30659.htm) 12th Conf. on Retroviruses and Opportunistic Infections(http://www.natap.org/2005/CROI/croi_11.htm)

  36. Phase II trials of TMC278/rilpivirine IIa: -1.2 log change in HIV viral load after 7 days for 25 mg/day TMC278. Similar effect seen with 50, 100, 150 mg/day doses. No NNRTI-resistance mutations observed. IIb: 48, 96-week study of TMC278 in the U.S. 320 anti-retroviral naïve patients randomized to TMC278 or efavirenz together with two doctor-chosen anti-AIDS drugs. 25 and 75 mg TMC278 once per day comparable to efavirenz at 600 mg/day with very little resistance observed Phase III trial underway with 75 mg TMC278 dose.

  37. Engineered RTs RT2A RT21A RT69A RT22A RT3A RT70A RT23A RT4A RT61A RT51A RT62A RT5A RT24A RT52A Q258C RT63A RT12A RT1A RT6A RT52B RT66A RT13A RT55A RT25A Newtermini RT67A RT26A RT14A RT68A RT7A RT27A RT73A RT8A worse than 5 Å RT28A RT75A worse than 5 Å (larger crystals) RT9A RT29A RT76A 3.5-5 Å RT10A RT30A 3.0-4.0 Å RT71A 2.2-3.0 Å RT31A RT72A better than 2.0 Å RT34A RT35A

  38. Unliganded NNRTI bound

  39. TMC278 and Resistance Mutations Janssen et al. (2005) J. Med. Chem. 48:1901

  40. Locations of mutation sites W229 I II F227 L234 YMDD Y188 Y318 P95 L100 Y181 E138 (p51) OW K103 K101 V179

  41. ~1.5Å 2.1 Å Structure of K103N:Y181C RT/TMC278 Complex YMDD K103N:Y181CRT/ TMC278 (cyan ribbon, yellow side chains & orange TMC278) wt RT/TMC278 (blue ribbon, gray side chains & TMC278). Y183 3.5Å 4.4Å Y/C181 Y188 Loss of aromatic interaction by Y181C mutation is compensated by a new set of interactions between cyanovinyl group and conserved Y183 of YMDD motif. YMDD motif is shifted by ~1.5 Å to facilitate the interaction. K/N103

  42. W229 L234 L/I100 Wiggling ~2 Å Y181 K/N103 F227 Y188 Superposition of wt RT/TMC278 on L100I-K103N RT /TMC278 structure F227 Jiggling Y188 L234 K/N103 Y181 L/I100 K101 2. 9 Å Structure of L100I:K103N RT/ TMC278 Complex TMC278 undergoes structural rearrangement to bind the mutant RT

  43. MOVIE: TMC278 Wiggling and Jiggling in L100I/K103N RT Mutant

More Related