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Discovery and optimization of novel small molecule HIV-1 entry inhibitors
using field-based virtual screening and bioisosteric replacement
Simon Cocklin, Ph.D.
HIV-1 Global Burden
Group M viruses are the most widespread, accounting for ~99% of global infections and can be further subdivided into nine distinct genetic subtypes, or clades. Clade B is prevalent North America, Western Europe, and Australia. Clades A, C, and D are most prevalent in Latin America, Africa and Asia.
Human Immunodeficiency Virus-1 (HIV-1) Life cycle and Intervention points
Attachment
e.g., sCD4
Fusion
e.g., T-20
Reverse Transcription
e.g., NRTIsIntegration
Translation
Transcription
Budding and Maturation
Viral Assembly
e.g., integrase inhibitors
e.g., protease inhibitors
Attachment of virus to cells
Fusion
Reverse Transcription
Integration
Transcription
Translation
Virus assembly
Budding and Maturation
In development – disrupts viral attachment to cellular receptors
In clinical use – disrupts the processes involved in viral and cell membrane fusion
In clinical use – disrupts the process of changing the RNA genome to DNA
In clinical use– disrupts the process of changing the RNA genome to DNA
In clinical use – disrupts the HIV-1 protease maturation of viral gene products
A. The envelope protein complex is a heterotrimer of three gp41 molecules and three gp120 molecules, embedded in the viral envelope.
HR1
HR2
Entry of HIV-1 is a multistep process mediated by the Envelope complex
Schematic
Viral membrane
PDB code: 4NCO
Julien et al. (2013) Science. Dec. 20 (342(6165): 1477-83
gp120
gp41
A. The envelope protein complex is a heterotrimer of three gp41 molecules and three gp120 molecules, embedded in the viral envelope
B. The first specific high affinity interaction occurs between CD4 and the gp120 subunit of the envelope complex.
HR1
HR2
Entry of HIV-1 is a multistep process mediated by the Envelope complex
A. The envelope protein complex is a heterotrimer of three gp41 molecules and three gp120 molecules, embedded in the viral envelope
B. The first specific high affinity interaction occurs between CD4 and the gp120 subunit of the envelope complex.
C. This interactions promotes a subsequent interaction with another cell surface receptor, which is usually a G-protein coupled receptor, and usually either CXCR4 or CCR5
HR1
HR2
A. The envelope protein complex is a heterotrimer of three gp41 molecules and three gp120 molecules, embedded in the viral envelope
B. The first specific high affinity interaction occurs between CD4 and the gp120 subunit of the envelope complex.
C. This interactions promotes a subsequent interaction with another cell surface receptor, which is usually a G-protein coupled receptor, and most predominantly either CXCR4 or CCR5
D. This interaction causes rearrangement in the gp41 subunit, such that the fusion peptide is inserted into the membrane, and the helical regions (HR1 and HR2) fold back and interact with each other, facilitating the bringing together of the two membranes and eventual fusion.
HR1
HR2
The piperazine derivatives developed by BMS are the most potent entry inhibitors to date
N
N
O
ON
N
OO
N
N
O
ON
NO
O
O
NN
ON
N
N
N
NO
O
O
BMS-378806
BMS-488043
BMS-626529
EC50 1.47 nMCC50 >300 µM
EC50 0.88 nMCC50 >300 µM
EC50 0.4 nMCC50 >300 µM
Lin et al. 2003. PNAS. 100(19):11013
Wang et al. 2009. JMedChem. 52:7778-7787
Nowicka-Sans. 2012. AAC. 56(7):3498-3507
• The BMS compounds have a broad therapeutic spectrum indicating their binding site is conserved and available
• They are specific to HIV-1
• Limited by their low solubility and low bioavailability
Therefore, we proposed to find other chemotypes that bind to the BMS binding site on Env but that potentially have improved drug-like properties
As there is no information regarding BMS binding site, we templated BMS-377806, BMS-488043 and BMS-626529 to develop a Field Point pharmacophore.
This was used to probe the Cresset in silico compound library using Blaze.
Blaze analysis resulted in the ranked identification of 1000 potential hit compounds
50 compounds were individually chosen for antiviral testing using the single-round infection assay
Pseudo-virus
• Transfect producer cells (293T) with:• Env (AMLV, YU-2) Plasmid• Rev plasmid• Backbone Plasmid• (NL4-3; Luc Reporter gene)
Co-incubation
• Incubate CD4+ CCR5+ cells with:• Pseudo-virus• Compound of interest
Measure Luminescenc
e
• Determine IC50 of each compound
Results from initial single-round infection screenCompound IC50 YU-2 (µM) IC50 AMLV (µM)
11 13.1 ± 1.7
N.A.
12 53.5 ± 3.0
N.A.
28 33.7 ± 4.5
N.A.
32
79.4 ± 11
N.A.
34 153 ± 44
N.A.
ON
NO
N
N
O
O
O
O
S
N
NS
N
N O
S
O
N
N
OO
N
N
N
N S
O
O
Table 1: Structure and potency of compounds identified within this study with specific activity against HIV-1 YU-2. N.A. = not active over concentration range tested.
N
N
O
O
N
N
O
O
ON
NOPiperazine is core scaffold
of BMS compounds
SC03 BMS-378806
Improved novelty - search for piperazine bioisosteres
Mowbray, C.E., et al. Challenges of drug discovery in novel target space. The discovery and evaluation of PF-3893787: a novel histamine H4 receptor antagonist. Bioorganic & medicinal chemistry letters 21, 6596-6602 (2011)
NN
2-methyloctahydropyrrolo[3,4-c]pyrrole
BIF% with piperazine = 57%
~7-fold reduction in potency
ON
NO
N
NO
O
SC03 SC04
IC50 = 15µM IC50 = 100µM
N
NO
O
SC04 has a new central scaffold and is specific for HIV-1
But acenanapthene can be genotoxic and
Very low potency
O
N
Cl
N
O
O
Chose “headgroups” from BMS patent literature and checked BIF% to acenapthene ring
O O
N
N
OO
O
BIF% = 100 BIF% = 57 BIF% = 64
O
N
Cl
NH
O
O
N
N
ON
O
NO
NHN
O
OO
SC07 SC08
IC50 = 16µM
N
NO
O
SC04
IC50 = 100µM IC50 = 0.19µM
N
N
O
ON
NO
O
O
NN
ON
N
N
N
NO
O
O
BMS-488043
BMS-626529
EC50 0.88 nMCC50 >300 µM
EC50 0.4 nMCC50 >300 µM
BMS improved compound by substituting methoxy for a methyltriazole
N
O
NO
NHN
O
OO
SC08
IC50 = 0.19µM
N
O
NO
NHN
OO
N
N
N
SC11
IC50 = 1 nM
SC11 has poor drug-like score, just like BMS compounds. Spark experiment on phenyl group and assessment using StarDrop. Chose compound for synthesis based upon BIF% and drug-like score.
N
O
NO
NHN
OO
N
N
N
SC11
IC50 = 1 nM
N
O
NO
NHN
OO
N
N
N
SC26
IC50 = 0.6nM
Drug Score = 0.3815 ± 0.2323 Drug Score = 0.0179 ± 0.026
De novo and Spark-inspired chemotype diversification
N
N
O
NHN
N
N
N
O
OO
BMS-626529
Drug Score = 0.02 ± 0.027
N
O
NO
NHN
OO
N
N
N
SC26
IC50 = 0.6nM
Drug Score = 0.3815 ± 0.2323
IC50 = 0.4nM
SC15
O
O
NHN
OO
N
N
N
N
IC50 = 3nM
AP Tanimoto = 0.69
AP Tanimoto = 0.47
O
O
N
NN
NH
N
O
N
N N SC12
IC50 = 80nM AP Tanimoto = 0.58
O
O
N
NO
NNN HN
HN
O
SC45
IC50 = 100nM
AP Tanimoto* = 0.55
piperazine dipyrrolidine azetidine
pyrrolo-pyrazole
tetrahydropyridine
Potency improvement on the pyrrolo-pyrazole scaffold
O
O
N
NN
NH
N
O
N
N N
SC12
IC50 = 80nM AP Tanimoto = 0.58
O
O
N N
N
HN
N
NN
N
F
SC38
IC50 = 8nM AP Tanimoto = 0.43
TI (CC50/IC50) = 81,000
Summary
• Successfully used Fieldscreen to identify new entry inhibitors
• Improved novelty of hit using a combination of literature searching coupled with Spark analysis
• Improved potency using patent literature coupled with Spark analysis
• Used Spark coupled to StarDrop to improve predicted drug-like properties of dipyrrolidine lead.
• Identified a total of 4 new “core” chemotypes with ≤100nM potency
Acknowledgements
Drexel University College of Medicine
Marina TuyishimeMathew Danish
DFCI/Harvard Medical School
Navid MadaniAmy PrinciottoJoseph Sodroski
Cresset Group
Rae Lawrence
Compounds were synthesized by outsourcing to WuXi Apptec and HD Biosciences (China) and AsisChem (USA). Special thanks to Drs. Henry-Georges Lombart and Joel Berniac from AsisChem.
Funding – NIH and W. W. Smith Charitable Trust