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
•
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)
Life cycle of HIV
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
Bartlett and Moore, Scientific American July 1998
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
The process of reverse transcription
A.
r
u5
R
U5
pbs
(-)
r
ppt u3
r
•minus strand strong stop synthesis
pbs
B.
ppt u3
(-)
PBS
PPT U3 R U5
•minus strand strong stop transfer and extension
ppt
C.
U3 R U5 PBS
(-)
PBS
D.
U3 R U5 PBS
PBS
PPT U3 R U5
•degradation of RNA and plus strand strong stop synthesis
•removal of minus strand primer
PPT U3 R U5
(+)
PPT U3 R U5
•plus strand strong stop transfer and extension
•complete extension of the minus strand 3’ end
(+)
thumb
p66
Polymerase
RNase H
palm
active site
p51
fingers
Bending DNA
Molecular mechanism of nucleic acid polymerization
P
N
Steps of DNA polymerization
RT/DNA
RT
RT/DNA/dNTP
fingers
thumb
DNAn
E
PPi
dNTP
E’/DNAn
DNA binding
E’/DNAn/dNTP
dNTP binding
E*/DNAn/dNTP
Conformational change/
catalysis
E’/DNAn+1
Translocation
E + DNA
P site
Primer
(priming site)
dNTP
3’OH
N site
(nucleotide binding site)
a
Catalytic
carboxylates
b
g
Released
as
pyrophosphate
Step 1
Step 2
DNA
E
Step 3
Step 4
dNTP
E’/DNA
Step 5
PPi
E+/DNA/dNTP
P
P
N
E*/DNA/dNTP
P
N
E#/DNA-dNMP
PPi
P
N
N
dNTP
dNTP
Complex P
“open”
ternary complex
Step 5’
Translocation
“closed”
ternary complex
Complex N
E+DNA
Molecular mechanism of translocation
P
N
Structural changes at YMDD motif during the course of polymerization
DNA
dNTP
E’/DNA
E
PPi
E’/DNA/dNTP
#
E*/DNA/dNTP
E /DNA+1
E+DNA
Translocation
dNTP
P site
E
Y
M
I
3’OH
N
N site
D
E’/DNA
P
E
E’/DNA
E*/DNA/dNTP
D185
D185
II
Close contact, charge repulsion destabilize the N complex
- 2.5 Å
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
D185
catalytic complex
(E#/DNA+1)
Molecular mechanisms of drug resistance
P
N
HIV-1 Reverse Transcriptase
polymerase and RNase H activities
 a major target for inhibitors

NRTI
RNase H

NNRTI
Excision-product
analogs?
extensive drug resistance
Why triple-drug therapies?
Error rate ~10 x e-4
Probability
of 3 independent resistance
mutations is < 10 x e-12
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)
Locations of Drug Binding Sites in
HIV-1 RT Structure
Fingers
Thumb
Rnase H
Palm
Nucleoside inhibitor binding site
Non-nucleoside inhibitor binding site
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
dNTP binding site
NNRTI binding site
• Nevirapine (NEV), delavirdine (DLV), efavirenz
(EFV), and etravirine (ETV) are approved drugs
Scheme 1
N
Br
O
Cl
H
N
NH
N
Br
NH2
O
S
Cl
H
N
Cl
NH
TIBO (R86183)
Loviride (R95845)
N
NH
S
ITU (R100943)
N
H
N
Cl
Cl
N
Cl
N
N
NH
N
N
NH2
Cl
Cl
O
N
N
N
HN
NH
N
NH2
NH2
N
DATA (R106168)
HN
N
DATA (R120393)
N
N
NH
O
N
Br
N
N
NH
N
NH2
DAPY (TMC120-R147681)
DAPY (TMC125-165335)
DATA (R129385)
N
N
O
Br
N
NH
N
OH
DAPY (R185545)
W229
W229
L234
L100
Y188
Y318
L234
F227
Y188
Y181
Y181
Cl
N
V179
NH
N
N
Cl
NH2
V179
a-APA
K103
Common binding mode
of 1st generation
NNRTIs to HIV-1 RT
I
II
Neviripine
N
W229
L234
K103
H
N
O
8-Cl TIBO
O
Cl
O
K1
01
S
HN
K101
L100
K101
F227
Y181 Y188
V179
K103
III
Ding, Das, et al., Nat. Struct. Biol. (1995) 2:407-415
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!
Locations of Drug Resistance Mutation
Sites in HIV-1 RT/DNA Structure
Nucleoside drug resistance mutation sites
Non-nucleoside drug resistance mutation sites
What are the special structural characteristics
of 3d generation NNRTIs?
What do they teach us in terms of
mechanism of action?
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
Structure-Based Drug Design
Synthesis
of molecules
Antiviral
screening
Best
compounds
Metabolic
screening
Selected
compounds
• Multi-disciplinary effort
Molecular
Modeling
Crystallographic
studies in complex
with HIV-1 RT
Clinical trials
• 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
Roadmap: Discovery of DAPY inhibitors
N
N
Cl
Cl
O
H
N
N
Cl
H2N
NH
S
TIBO
tivirapine
R86183
(1987)
O
N
a-APA
loviride
R89439
(1991)
Cl
Cl
H
N
Cl
Cl
H
N
NH
NH
S
ITU
R100943
(1993)
NH
N
NH
N
N
N
R165335
TMC125
(1999)
N
O
N
NH
N
N
Cl
Cl
N
N
Br
NH2
HN
N
NH
N
N
HN
R278474
TMC278
(2001)
N
NH
N
DAPY
R147681
TMC120
(1998)
NH
N
NH2
DATA
R106168
(1994)
Janssen et al. (2005) J. Med. Chem. 48:1901-1910
N
TMC120-R147681
H
N
Trp229
R120393
Leu234
N
N
Leu100
Tyr181
Trp229
Cl
HN
Leu234
NH2
N
Leu100
N
NH
N
Lys101
HN
Phe227
N
Lys101
Phe227
Tyr181
Tyr188
Tyr188
Val106
Val106
Lys103
Val179
Lys103
Val179
N
N
Trp229
N
N
Leu234
O
Phe227
N
NH
Trp229
Leu234
NH2
TMC125R165335
NH
Br
Leu100
Br
Tyr181
N
N
N
Leu100
O
OH
Tyr181
Phe227
R185545
Tyr188
Val106
Tyr188
Val106
Lys101
Lys101
Val179
Lys103
Val179
Asn103
Das et al., J. Med. Chem. (2004) 47:2550-2560
Strategic Flexibility
Rigid
inhibitor
Steric hindrance
Flexible
inhibitor
Torsional changes
(wiggling)
Reorientation and
repositioning (jiggling)
Das et al., J. Med. Chem. (2004) 47:2550-2560
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)
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
Roadmap: Discovery of DAPY inhibitors
N
N
Cl
Cl
O
H
N
N
Cl
H2N
NH
S
TIBO
tivirapine
R86183
(1987)
O
N
a-APA
loviride
R89439
(1991)
Cl
Cl
H
N
Cl
Cl
H
N
NH
NH
S
ITU
R100943
(1993)
NH
N
NH
N
N
N
R165335
TMC125
(1999)
N
O
N
NH
N
N
Cl
Cl
N
N
Br
NH2
HN
N
NH
N
N
HN
R278474
TMC278
(2001)
N
NH
N
DAPY
R147681
TMC120
(1998)
NH
N
NH2
DATA
R106168
(1994)
Janssen et al. (2005) J. Med. Chem. 48:1901
Nevirapine Efavirenz
TMC120
TMC125
R278474
81
1
1
3
0.4
597
35
11
3
0.4
K103N
2,879
28
2
1
0.3
Y181C
10,000
2
7
6
1.3
Y188L
10,000
78
37
2
2
G190S
1,000
275
2
3
0.1
10,000
37
54
4
Wild-type
L100I
K103N+
Y181C
1
Activity of R278474 and Reference Compounds
Janssen et al. (2005) J. Med. Chem. 48:1901
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)
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 doctorchosen 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.
Engineered RTs
RT2A
RT21A
RT69A
RT22A
RT3A
RT70A
RT23A
RT4A
RT61A
RT51A
RT62A
RT5A
RT24A
Q258C
RT1A
RT6A
RT52A
RT63A
RT12A
RT52B
RT66A
Newtermini
RT13A
RT14A
RT25A
RT26A
RT7A
RT27A
worse than 5 Å
worse than 5 Å (larger crystals)
3.5-5 Å
3.0-4.0 Å
2.2-3.0 Å
better than 2.0 Å
RT8A
RT28A
RT55A
RT67A
RT68A
RT73A
RT75A
RT9A
RT29A
RT10A
RT30A
RT31A
RT34A
RT35A
RT76A
RT71A
RT72A
Unliganded
NNRTI bound
TMC278 and Resistance Mutations
1,000.0
R278474
R165335
efavirenz
10.0
L100I+K103N
F227C
K101E
Y188L
Y181C
K103N+Y181C
F227L+V106A
V179D
E138K
K101E+K103N
wild-type
L100I
V179E
L234I
P225H
K103N
G190A
F227L
P236L
V108I
V106A
Y188H
0.1
G190S
1.0
Y188C
EC50 (nM)
100.0
HIV-1 wild-type (IIIB) and mutant strains
Janssen et al. (2005) J. Med. Chem. 48:1901
Locations of mutation sites
N
N
W229
I
F227
HN
II
N
NH
L234
YMDD
N
Y188
Y318
P95
L100
Y181
E138 (p51)
OW
K103
V179
K101
2.1 Å Structure of K103N:Y181C
RT/TMC278 Complex
~1.5Å
YMDD
Y183
4.4Å 3.5Å
K103N:Y181C RT/ TMC278 (cyan ribbon, yellow
side chains & orange TMC278)
wt RT/TMC278 (blue ribbon, gray side chains &
TMC278).
Y/C181
Y188
1. Loss of aromatic interaction by Y181C
mutation is compensated by a new set
of interactions between cyanovinyl
group and conserved Y183 of YMDD
motif.
2. YMDD motif is shifted by ~1.5 Å to
facilitate the interaction.
K/N103
W229
2. 9 Å Structure of
L100I:K103N RT/
TMC278 Complex
L234
~2 Å
L/I100
Wiggling
Y181
K/N103
F227
Y188
Superposition of wt RT/TMC278 on
L100I-K103N RT /TMC278 structure
Jiggling
F227
TMC278 undergoes
structural rearrangement
to bind the mutant RT
Y188
L234
K/N103
Y181
L/I100
K101
MOVIE:
TMC278 Wiggling and Jiggling
in L100I/K103N RT Mutant
QuickTime™ and a
Sorenson Video 3 decompressor
are needed to see this picture.