Discovery and optimization of novel small molecule HIV-1 entry inhibitors using field-based virtual screening and bioisosteric replacement

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 infectionsand can be further subdivided into nine distinct genetic subtypes, or clades. Clade Bis prevalent North America, Western Europe, and Australia. Clades A, C, and D aremost prevalent in Latin America, Africa and Asia.

Human Immunodeficiency Virus-1 (HIV-1) Life cycle and Intervention pointsAttachment

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.

HR1HR2

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.

HR1HR2

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

HR1HR2

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.

HR1HR2

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 Luminescence

• Determine IC50of each compound

Results from initial single-round infection screenCompound IC50 YU-2 (µM) IC50 AMLV (µM)

1113.1 ± 1.7 N.A.

1253.5 ± 3.0 N.A.

2833.7 ± 4.5 N.A.

32

79.4 ± 11 N.A.

34153 ± 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 andpotency of compoundsidentified within thisstudy with specificactivity against HIV-1YU-2. N.A. = not activeover concentrationrange 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. Thediscovery 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 SC12IC50 = 80nM AP Tanimoto = 0.58

O

O

N

NO

NNN HN

HN

O

SC45IC50 = 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 NN

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