Transcript Document 7228023

HIV-1 Integrase Inhibitors:
Past, Present and Future?
Daria Hazuda and the Merck
Integrase Discovery Team
1
The HIV Life Cycle: Multiple Targets for Intervention
Most inhibitors in
development fall into
the three known classes:
reverse transcriptase or
protease.
Integrase is the third
enzyme; required for stable
maintainance of the viral
genome and efficient
viral transcription.
Integrase Structure/Function
integration/reverse transcription/virus architecture
Zinc Finger
Catalytic Core
HH CC
1
D64
DNA Binding
SH3
D116-(35)-E152
50
212
288
“DDE” residues coordinate the essential
active site metals(s).
Conserved in all integrases and
transposases.
Bujacz, G. et al. (1997) J. Biol. Chem. 272, 18161-18168
Integrase Structure/Function
integration/reverse transcription/virus architecture
Zinc Finger
Catalytic Core
HH CC
1
D64
50
DNA Binding
SH3
D116-(35)-E152
212
288
Class 1 mutants:
•no effects on viral DNA synthesis
•inhibit integration, increase 2LTR circles
•may affect interaction with host factors (BAF, LEDGF)
Class 2 mutants:
•pleiotropic affects, reverse transcription and/or viral assembly
Integration presents multiple potential
points for intervention
Integrase Presents Multiple Potential
Binding Sites for Inhibitors
adapted from Parillo, Current Med Chem (2003)
Novel Integrase Inhibitors Selectively Inhibit
Strand Transfer in vitro and in Cells
(Hazuda et. al Science 2000; 287, 646.)
O
OH
N
O
F
L-731988
OH
Blocking Integrase Strand Transfer Shifts
the Metabolic Fate of HIV-1 DNA
*cellular recombination
Integrase binds to viral DNA
& catalytically processes 3’ ends
and repair processes
Li et. al (2001), EMBO.
PIC
LTRs
Integrase joins viral
and cellular DNA
Inhibitors block
strand transfer
Integration
*Gap repair/ligation
Note: *cellular functions
1 & 2 LTR
circles
Biochemical and genetic evidence has validated
Integrase Strand Transfer Inhibitors (InSTIs) as
bona fide integration inhibitors in cells

The biochemical profile of these inhibitors is
recapitulated in HIV-1 infected cells

Mutations that confer resistance map to the
integrase catalytic core
.
Integrase strand transfer inhibitors define a
functionally distinct mechanistic class
Biochemical mechanism: Espeseth, (2000) PNAS USA 97, 11244.
No
effect on assembly w/DNA, 3’ processing or disintegration
High
affinity binding requires assembly with (viral) donor DNA
Inhibitors
compete with the (host cell) target DNA.
Molecular mechanism: Grobler, (2002) PNAS USA 99, 6661 .
Inhibitors
bind and sequester divalent metal ions in
integrase the active via the dka motif.
Specificity/affinity
derived from pendant substituents.
Diketo acids Sequester the Active Site
Metals in Integrase
O
HN
N
O
N
N
OH
O
N
N
O
O
N
O
O
F
F
Binding (Kd, nM)
no metal
>10,000
Mn
15
Mg
30
Activity (IC50, nM)
Mn
50
Mg
54
F
>10,000
110
3100
60
470
>10,000
320
590
>100,000
>100,000
•binding requires metal
•isosteric replacements exhibit distinct metal dependence
•neutral replacements bind but do not inhibit enzymatic activity
O
A model for the interaction of the DKA
with two active site metals in integrase

Integrase inhibitors sequester
active site metals
– Inhibitor binding requires
metal
–
Pendant substituents (R)
confer specificity and
affinity
2.0
Mg2+ 3.6
Mg2+
O 2.7 O
2.0
2.9
R
O
OH

DKA analogs can inhibit other phosphoryltransfer enzymes
with homologous 2-metal active site architecture
(nucleases, e.g., HIV-1 RNase H; polymerases, e.g., HCV polymerase)
Naphthyridine carboxamides are bioisosteric with
2,4-diketobutanoic acids
2,4-Diketobutanoic acid strand transfer inhibitor
(L-731,988)
8-Hydroxy-1,6-naphthyridine-7-carboxamide
( L-870,810 )
O
Fluoroaromatic ring
S
O
O
O
OH
N
H
N
OH
N
..
O
F
Lone Pair Electrons
Hydroxyl Group
Carbonyl Oxygen
F
N
OH
N
..
Naphthyridines establish POC for the
efficacy of InSTIs in retroviral infection
Biochemical & biological mechanism of action = diketo acids
Retain activity in human serum, oral bioavailable, good PK
SHIV (SIV) infection (Hazuda et. al Science 2004; 305, 528)
L-870812 monotherapy
•Sustained a 1 to > 4 log decrease in vRNA for 75 (180) days
•Incomplete suppression selected N155H in integrase
•The N155H virus has impaired RC (Fransen #725 CROI ‘05)
HIV-1 infection (Little, et. al #161 CROI ‘05)
L-870810 monotherapy
•After10 days, avg 1.77 log decrease in vRNA
in both treatment naive and experienced patients
•Development on hold due to findings in preclinical toxicology
Beyond L-870810: MK-0518
•
•
•
•
•
Potent InSTI (IC50 ~ 10 nM in strand transfer)
In vitro activity: IC95 ~ 33  23 nM (50% NHS)
Synergistic with currently licensed ARTs in vitro
Active against HIV isolates resistant to licensed ARTs
Preclinical safety studies: no issues
12 month studies in rats and dogs
No genotoxicity; in vitro and in vivo
• Phase I results
Excellent tolerability
Suitable pharmacokinetics (BID)
Low potential for drug interactions
MK-0518 Clinical Study Design
Randomized, double-blind, placebocontrolled study of MK-0518 vs Placebo in
HIV infected ART-naïve patients

Study regimen: 10 day monotherapy
– 100mg, 200mg, 400mg, 600mg p.o. or placebo BID

Endpoints:
– HIV RNA and CD4 counts
–

Adverse experiences
Inclusion Criteria (selected):
– HIV RNA ≥ 5000 copies/mL
–
CD4 count ≥ 100 cells/uL
MK-0518 Clinical Data Summary
Efficacy
–
HIV RNA declined by 1.7-2.2 logs (all MK-0518 doses)
–
HIV RNA < 400 cp/mL in > 50% patients ;
approx. 20% patients achieved < 50 cp/mL
Tolerability
–
No significant AEs
–
No food effect
–
No drug interactions requiring dose adjustment
Phase 2 naive and salvage studies ongoing
Phase 3 to initiate in early 2006
Novel Inhibitors of Integrase Strand Transfer
2004
2003
2002
DKA patents
non-DKA patents
2001
2000
diketo acid POC in cells
1999
0
5
10
15
# of new patents
20
Examples of Structurally Diverse InSTIs
R2
R4 N
N
R1
O
N
R3
O
OH
IRBM WO 2003035076
WO 2003035077
WO 2004058756
WO 2004058757
OPh
N
N
R1
N
N
OH
Shionogi WO 2003047564
N
O
OH
O
R2
O
R1
OR
N
Merck WO 2004047725
OH
O
R1
R2
N
O
OH
R3
N
O
R2
OH
BMS WO 2004004657
R2
N
R1
R1
OH
O
N
N OH
N
O
Japan Tobacco WO 2004046115
O
Pfizer WO 2004039803
WO 2004067531
Diverse inhibitors are structurally and
functionally homologous
Retain metal binding motif of the diketo acids
Selectively inhibit strand transfer in vitro
Inhibit integration and increase 2 LTR circles
in HIV-1 infected cells
Potential for InSTI cross resistance?
Distinct mutations selected in cell culture by
DKA and naphthyridine carboxamide analogs
Compound
observed mutations in integrase
Diketo acids
L-708906, L-731988
T66I + S153Y or M154I or V151I
Diketo acid ‘411
S153Y (4 months)
T66I + S153Y (13 months)
S153Y + N155S (26 months)
COOH
O
O
Naphthyridine
L-870810
O
F121Y + T125K (6 months)
V72I + F121Y + T125K (7 months)
V72I + F121Y + T125K + V151I (9 months)
S
N
O
N
H
N
N
O
F
OH
Limited Cross Resistance Between
Two Structurally Diverse InSTIs
IC50 Ratio of Mutants versus WT HIV in Infectivity Assay
30
411
Single cycle
810
infection assay
20
using site-directed
mutants
10
in HIV HXB2
0
T66I/S153Y
N155S
DKA mutants
N155H
F121Y
Naphthyridine Mutants
Resistance maps to conserved
residues in the integrase active site
Zinc Finger
HH CC
1
Catalytic Core
D64
SH3
D116-(35)-E152
50
Resistance
Mutations:
DNA Binding
212
T66I
V151I
F121Y
T125K
S153Y
M154I
N155S/N155H
Residues associated with DKA, Naphthyridine
resistance are proximal to the “DDE” residues that
coordinate the essential active site metals(s).
288
DKA and naphthyridine mutations map to distinct
regions, suggesting unique ligand interactions
green=DKAR
red=napthyridineR
yellow=“DDE”
Hazuda et. al, (2004.) A novel naphthyridine carboxamide
provides evidence for discordant resistance between
mechanistically identical inhibitors of HIV-1 integrase.
Proc. Natl. Acad Sci. USA
10
The critical elements of the DKA and
naphthyridine carboxamide pharmacophores
can be aligned in either orientation
Forward mode
Reverse mode
Modeling the two inhibitors in the reverse
orientation is consistent with a conserved
mechanism and distinct resistance profile
pink=DKA
green=napthyridine
blue=“DDE”
Different mutations at N155 have distinct
affects on DKA and naphthyridine carboxamide
activity
green=DKAR
red=napthyridineR
yellow=“DDE”
N155S = naphthryridineR and DKAR
N155H = naphthryridineR but DKAS
10
The discordant resistant profiles of
the diketo acids and naphthyridine
carboxamides suggest
the potential for at least two putative ligand binding
surfaces in the integrase active site and for
developing InSTIs with limited cross-resistance
Integrase Phenosense Assay:
(Virologic/Monogram Sciences)
Resistance Test Vector
DNA (pol
(pol or RHIN)
A-MLV env
DNA
+
Transfection
Infection
IN
Inhibition
60
12
10
MK-0518
50
8
40
6
30
4
20
2
10
0
MK-CCR5 inhibitor
0
0.01 0.03 0.06 0.16 0.4
'612 FC
1
2.5 6.3 10
0.01 0.03 0.06 0.16 0.4
CCR5 FC
1
2.5 6.3 10
MK-0588 maintains potency across diverse clinical isolates.
Specific infectivity of HIV-1 integrase mutants
selected with structurally diverse inhibitors
100
Single cycle
80
infection assay
60
using site
directed
40
mutants
20
in HIV HXB2
0
WT
T66I/
M154I
T66I/
N155S N155H F121Y
M153Y
411 mutants
810/812mutants
%
Integrase inhibitors: past, present & future
1) Complex biology and biochemistry present several
opportunities for potential intervention.
2) Integrase strand transfer inhibitors (InSTIs) are a
distinct mechanistic class.
3) Efficacy of InSTIs has been established
4) Compounds are advancing in the clinic
(MK-0518 phase 3 to initiate in 2006)
Integrase inhibitors: past, present & future
1) Complex biology and biochemistry present several
opportunities for potential intervention.
2) Integrase strand transfer inhibitors (InSTIs) are a
distinct mechanistic class.
3) Efficacy of InSTIs has been established
4) Compounds are advancing in the clinic
(MK-0518 phase 3 to initiate in 2006)
5) It is possible to identify InSTIs with discordant
resistance profiles.
6) Some InSTI resistant mutants exhibit
replication defects, could increase the barrier to
the development of resistance in vivo.
The HIV-1 Integrase Inhibitor
Discovery Team
Medicinal Chemistry
Antiviral & Vaccine Research
John Wai, Thor Fisher, Linghang Zhuang,
Mark Embrey, Melissa Egbertson, Linda Payne,
Jeff Melamed, Marie Langford, Michelle Kuo,
Neville Anthony, Bob Gomez, Sam Jolly,
Jim Guare, Terrence Hamill, Joe Vacca,
Joel Huff, Steve Young
Vincenzo Summa, Michael Rowley, Paola Pace,
Cristina Gardelli
Daria Hazuda, Pete Felock, Abigail Wolfe,
Kara Stillmock, Marc Witmer, Mike Miller,
Carol Blau, Amy Espeseth, Jim Cole, Jay Grobler,
Robert Danovich, Bill Schleif, Greg Moyer,
Lori Gabryelski, John Shiver, Wendy Trigona,
Emilio Emini
Virologics Inc.
Drug Metabolism & Animal Resources
Lixia Jin, Patrice Ciecko, Juinn Lin, Jim Yergey,
Brad Wong, I-Wu Chen, Paul Pearson
Hilton Klein, Larry Handt
Safety Assessment
Ramon Kemp, Hugo Vargas, Pete Siegl,
Patty Bunting, Sheila Galloway
Chris Petropolous, Gabrielle Heilock,
Neil Parkin
NMR & Mass Spectroscopy
Steve Pitzenberger, Sandor Varga, Joan Murphy,
Mike Bogusky, Tom Kalisker, Harri Ramjit,
Chuck Ross, Art Coddington
Protocol 004 Study Team
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J. Morales-Ramirez
C. Kovacs
R. Steigbigel
D. Cooper
R. Liporace
R. Schwartz
C. Tsoukas
M. Markowitz
J. Galpin
S. Brown
M. Miller
D. Hazuda
J. Vacca
M. Rowley
V. Summa
M. Iwamoto
Clinical Res. Puerto Rico, Inc., San Juan, PR
Canadian Immun. Res. Collaborative, Inc., Toronto, CAN
State Univ. of New York, Stony Brook, NY
St. Vincent’s Hosp., Darlinghurst, Australia
Albany Med. College, Albany, NY
Associates In Research, Fort Myers, FL
Montreal General Hospital, Montreal, Canada
Aaron Diamond AIDS Research Ctr, New York, NY
Shared Medical Research Foundation, Tarzana, CA
AIDS Research Alliance, West Hollywood, CA
Merck Research Labs, West Point, PA
Merck Research Labs, West Point, PA
Merck Research Labs, West Point, PA
Merck Research Labs, IRBM, Italy
Merck Research Labs, IRBM, Italy
Merck Research Labs, Rahway, NJ