Identification of ESM based

RIPK Inhibitor

FTE based Integrated Discovery Proposal

 HEADER

Biology

Aurigene Pharmaceuticals Services Limited

 RIPK3 Kinase assay Whole blood Tox
Chemical Stability
(Human) Necroptosis assay in Non-GLP 7/ 14 day
PAMPA
RIPK1 Kinase assay hCE1 Tg mice rodents
(Human) PPB & Stability : hu &
Stability in hCE-1 Tg Non-GLP MTD higher
ms plasma
RIPK2 & 4 selectivity mouse plasma species
Work Flow Scheme

Ion channel off target

pMLKL inhibition profiling
Mouse RIPK 3 &
assay in THP1 cells PK in hCE-1 Tg mice
RIPK1 kinase assay Kinase profiling
stimulated with TSZ
CEREP profiling

Necroptosis assay in
THP1 (invivogen, Kinase Panel Acute PD Study
DMPK Lead Profile
THP1-HMGB1-Lucia selectivity TNFα+z-VAD induced
cells) cells post PK in higher species
(in house panel) hypothermia model
stimulus

Ester Hydrolysis:
h Rec. CE 1 & 2 Th. solubility (pH 7.4) in vitro Tox
Rev. CYP Inhibition
m Rec. CE 1 Stability in liver hERG, Mini AMES,
Log D
Stability in human microsomes: hu & ms MNT
liver S9

Acid Accumulation:
Hu T-78 , Wild type Chronic Efficacy
Necroptosis assay in Dose escalation, 7- Study
THP1 cells & THP1 Hu T-78 cells day repeated dose
CES1 CRISPR KO Mice UUO model
cells
In vitro screening for RIPK3 &1 inhibition – Cell-free and cell-based assays

• RIPK3 Kinase assay (Human, IC50 <200 nM for initial hits, IC 50 ADP-Glo assay
<10 nM ? for lead nomination) Ki determination for lead compounds
• RIPK1 kinase assay (Human, IC50 <500 nM for initial hits, IC50
Biochemical
assay <30nM ?for lead nomination)
Hit compounds to be screened
for RIPK2 & RIPK4 selectivity

• pMLKL inhibition assay in THP1 cells stimulated with TSZ

• IC50 <500 nM for initial hits, IC50< 100 nM (?) for lead
Cell based assay
nomination
Target mediation
• To increase the through put, pMLKL will be measured by ELISA

• Necroptosis assay in THP1 (invivogen, THP1-HMGB1-Lucia

cells) cells post stimulus
• EC50 <500 nM for initial hits, EC50< 100 nM (?) for lead
Cell based assay
phenotype nomination)

Aurigene Pharmaceuticals Services Limited

Selectivity for hCE1 expressing cells and preliminary DMPK studies

• Ester Hydrolysis by Human Recombinant Carboxylesterases 1 & 2

• Ester Hydrolysis by mouse Recombinant Carboxylesterase 1 D
hCE1
hydrolysis • Stability in human liver S9

• Accumulation in Hu T-78 , Wild type THP1 cells & THP1 CES1 CRISPR KO cells
Accumulat • Accumulation in THP1 CES1 CRISPR KO cells below LOQ
ion of acid

• Necroptosis assay in Hu T-78 cells ( hCE1 negative cell line)

Cell type • Minimum 10 fold less potent over THP1 cells IC 50
selectivity Only for ESM motif
assay attached compounds

Tier-1 in • Thermodynamic solubility at pH 7.4

vitro • Stability in liver microsomes: human and mice
DMPK

• Selectivity against other kinases of small panel (BRAF, VEGFR2, PDGFRA, PDGFRB &
Selectivity SRC)
against • Minimum 30 fold selectivity
other
kinases

Aurigene Pharmaceuticals Services Limited

Selectivity against small kinase panel & in vitro activity against mouse

• Mouse RIPK 3 & RIPK1 kinase assay, IC 50 < 100 nM (?) for initial hits to
Mouse proceed for in vivo efficacy studies
RIPK assay

• Stability in hCE-1 Tg mouse plasma

• Mouse whole blood Necroptosis assay in hCE1 Tg mice whole blood
Mouse
Phenotype
assay

• Chemical Stability (48h @ 25°C; pH 4.5, pH 7.4, DMSO)

• PAMPA
Tier 2 in- • PPB & Stability : Mouse & human plasma
vitro DMPK

• hCE1 Tg mice PK Bioavailability F >15%, Cl <70% liver blood flow

PK study in (LBF) for initial hits and <30% for lead nomination
mouse

Aurigene Pharmaceuticals Services Limited

Mouse PK studies, acute efficacy and Tier-2 DMPK studies

• TNFα+ z-VAD induced hypothermia model establishment in hCE-1 Tg mice

• Thioglycolate induced macrophage model in hCE-1 Tg mice ( out sourced for
Acute PD lead molecules)
model

• Reversible CYP Inhibition

Tier-3 • Log D
in vitro
DMPK

• Dose escalation PK in hCE-1 Tg mouse

• 7 day repeated dose PK in hCE-1 Tg mouse
Tier-2 PK
studies

• Mice UUO model establishment and validation (In house / out source)
• Demonstration of activity in animal model with dose response and PK/PD in hCE-1 Tg mice
Chronic • Profiling of lead molecules in STZ induced diabetic nephropathy or aneurysmal subarachnoid
efficacy model hemorrhage ( out sourced to relevant CRO)

Aurigene Pharmaceuticals Services Limited

Disease specific efficacy model, advanced selectivity profiling, Tox studies

• hERG
• Mini AMES
Tox invitro • MNT in vitro

• Solubility in different pH
• Microsomal stability, Plasma stability, PPB in additional species
Lead • CYP TDI, CYP Induction, Reactive Metabolites, Met ID (if required)
profiling • Brain Penetration PK, PK in higher species

• Ion channel off target profiling

• Kinase profiling
Lead • CEREP profiling
Profiling

• Non-GLP 7/ 14 day tox in rodents

• Non-GLP MTD study in higher species
Tox

Aurigene Pharmaceuticals Services Limited

Expected Deliverables: first six months

Chemistry: Synthesize four standard compounds (TAK 632, Comp 26, GSK-074 and BMS-9). Optimize the
chemistry for tethering ESM motif on standard compounds. A set of standards (up to 10 compounds) with ESM
linked ready for various profiling. Initiate scheme for optimization for three different novel series. Synthesize at least
3 compound / FTE / month for identifying the first novel hit as per agreed criteria.

CADD: Identify ESM link sites on standards, finalize the compound prioritization of novel design and Initiate
distinctly different back up hit identification via library screening.

Biochemistry: : Establish ATP competitive assay for both RIPK 1 and 3 in Mouse and Human. Identify no HIT
kinases from the agreed upon panel. Initiate compound screening.

Cell based assays: Cell based mechanistic assays in THP1 and Hu T-78 cells line.

DMPK: ESM cleavage assay establishment and screening for active compounds. Tier 1 in vitro ADME and PK
studies.

Pharmacology: Establishment and Validation of TNF + z-VAD-FMK induced hypothermia model and screening of
compounds.
Establishment of UUO model and validation with standard compound (inhouse/ outsourced)

Aurigene Pharmaceuticals Services Limited

 HEADER

Chemistry

Aurigene Pharmaceuticals Services Limited

Step-wise planning

• Synthesis / Procurement of standard compounds (GSK074, TAK632 and Compd. 26 (JMC 2019,
62, 6665−6681; BMS-9)

• One patentable change at a time to develop a distinct compound after two iteration.

• Will take help of SBDD to rank ordering the designs.

• Synthetic feasibility and patentability will be the other criteria to shortlist compounds.

• With help of modeling Identify areas of scaffold where EMS Motif can be tethered.

• Stability of ester at Various pH and RIPK activity of corresponding acids will be considered critical

Aurigene Pharmaceuticals Services Limited

RIPK Sequence analysis: Kinase domain

• Human and mouse RIPK3 kinase domain share low sequence identity compared to other isoforms.
This information may be utilized in biochemical to in-vivo model translation
RIPK human and mouse kinase
domain sequence identity
Sequence 1 Sequence 2 Identity
RIPK3_Human RIPK3_Mouse 72
RIPK1_Human RIPK1_Mouse 78
RIPK2_Human RIPK2_Mouse 93.5
RIPK4_Human RIPK4_Mouse 91.6
Kinase domain sequence
identity and similarity wrt human RIPK3
Sequence Identity Similarity
RIPK3_Mouse_KD 72 79
RIPK1_Human_KD 34.46 43.35
RIPK2_Mouse_KD 34.46 40.34
Kinase domain RIPK4_Mouse_KD 34.34 40.73
sequence alignment RIPK1_Mouse_KD 34.08 42.77
RIPK4_Human_KD 33.96 39.13
• RIPK3 human kinase domain shares low sequence homology with RIPK2_Human_KD 32.96 40.52
other isoform. This may guide compound selectivity
Aurigene Pharmaceuticals Services Limited
RIPK3 Structural insight: Activated kinase structure

• ATP binds to RIPK3 in activated kinase conformation and this can be exploited to design Type-I
inhibitors

PDB ID: 4M69-Mouse RIPK3-ATP complex

Hinge

• RIPK3 human and mouse kinase domain share reasonable sequence similarity 79 % and identity 72 %

Aurigene Pharmaceuticals Services Limited

 RIPK3 Selective Type-I inhibitors from various chemical classes

• Type-I inhibitor occupy kinase back pocket and trajectory directed towards solvent region. Inhibitor core scaffold
makes crucial interaction with hinge region (shown in arrow)

ATP/ GW39’B
Proposed trajectory for ESM motif attachment

ATP/ Dabrafenib ATP/ GSK-843 ATP/ HS-1371 ATP/ GSK-872 ATP/ GSK-840
Note: Docking models overlaid onto ATP X-ray structure
Ref: ACS Med. Chem. Lett. 2020, 11, 266−271 J. Med. Chem. 2019, 62, 6665−6681
Aurigene Pharmaceuticals Services Limited
RIPK3 Structural insight Type-II inhibitor

• Available Type-II inhibitor bound RIPK3 X-ray structure may guide future design
PDB ID: 6OKO-
Mouse RIPK3

Proposed trajectory for ESM motif attachment

Aurigene Pharmaceuticals Services Limited Ref: ACS Med. Chem. Lett. 2020, 11, 266−271
 RIPK3/1 Type-II inhibitors from various chemical class

• GSK-074 and compound 26 is a dual RIPK1/3 inhibitor. Type-II RIPK3 selective inhibitor is known in
literature. Known compounds occupy hinge (anchor) and back pocket region in inactive conformation.

Compd-9-BMS/Cpd-26

Compd-9-BMS/GSK-074

Compd-9-BMS/Sorafenib Compd-9-BMS/Ponatinib
Compd-9-BMS/Compd-18-BMS
Proposed trajectory for ESM motif attachment
Ref: ACS Med. Chem. Lett. 2020, 11, 266−271 J. Med. Chem. 2019, 62, 6665−6681
Aurigene Pharmaceuticals Services Limited
 Scaffold selection decision tree

• Type-I inhibitor hypothesized to inhibit kinase Chemotype • Type-II inhibitor hypothesized to inhibit
activity through activated kinase conformation selection scaffolding and kinase activity by inhibiting
inhibition conformational transition
RIPK3/1 dual
inhibitor

Kinase Kinase Scaffold

Type-I Type-II
or Scaffold
inhibitor inhibitor
function
RIP3 selective
Type-II inhibitor

RIPK1 potency
RIP3 selective Active In-active

optimization
Type-I inhibitor conformation conformation
RIPK1 potency
optimization

Compound-15,18 & 9/BMS,

TAK-632, Compound-
compound-42, GSK-074,
GSK-843, HS-1371, GSK-872, Work around GSK-843, HS-1371, 26, GSK-074 or novel
sorafenib, ponatinib or
GSK-840, GW39’B, GSK-872, GSK-840, GW39’B to in-house design
novel in-house design
dabrafenib or novel in-house bring in RIPK1 potency or novel
design in-house design

Aurigene Pharmaceuticals Services Limited

RIPK3/1 dual inhibitors design strategy

• Design strategy:

• 1-Scaffold morphing based on

• RIPK3/1 dual Type-II inhibitor

• RIPK3/1 dual Type-I inhibitor

• 2-Hybrid design-Scaffold swapping

• 3-Virtual screening: In-house kinase focus library

Note: In-house kinase focus library comprise of hinge, linker, back-pocket, ribose and
solvent region elements will be used to identify novel chemotype

Aurigene Pharmaceuticals Services Limited

RIPK3/1: Structural insight inactive conformation

• RIPK3/1 inactive conformation X-ray structure is available for compound design

PDB ID: 6OKO-Mouse RIPK3, PDB ID: 4NEU-Human RIPK1

Linker
Hinge Back-
Hinge
pocket

DFG/DLG

• Despite RIPK3/RIPK1 low sequence identity, several Type-II reported compounds have dual
potency, hence provide scope for dual Type-II compound development
Aurigene Pharmaceuticals Services Limited
RIPK3/1 Type-II dual inhibitors designs-1

Linker Hydrophobic • Selected Type-II scaffold will be sequentially modified retaining elements
tail fixed keeping novelty in mind
Hinge

Compd-9-BMS/Cpd-26

Compound-26:
Proposed hinge
modification

Ref: ACS Med. Chem. Lett. 2020, 11, 266−271

Aurigene Pharmaceuticals Services Limited
Few specific example of design 1

Aurigene Pharmaceuticals Services Limited

RIPK3/1 Type-II dual inhibitors designs

Linker
Hinge • Selected Type-II scaffold will be sequentially modified retaining elements fixed keeping
novelty in mind

Hydrophobic
Compd-9-BMS/GSK-074 tail

Few specific example of Design 1

GSK-074:
Proposed hinge
modification
Ref: ACS Med. Chem. Lett. 2020, 11, 266−271
Aurigene Pharmaceuticals Services Limited
Optimization of RIPK3 selective Type-II inhibitors for RIPK1

Hinge • Selected RIPK3 Type-II scaffold will be sequentially modified to optimize RIPK1
potency keeping novelty and potency in mind
Linker

Hydrophobic
Compd-9-BMS/Compd-18-BMS tail

SAR

Proposed hinge
modification

Ref: ACS Med. Chem. Lett. 2020, 11, 266−271

Aurigene Pharmaceuticals Services Limited
Optimization of RIPK3 selective Type-I inhibitors for RIPK1

• Selected Type-I scaffold will be sequentially modified retaining elements fixed keeping novelty in mind

Proposed hinge
modification

ATP/ GSK-843 ATP/ HS-1371 ATP/ GW39’B

Ref: ACS Med. Chem. Lett. 2020, 11, 266−271
Aurigene Pharmaceuticals Services Limited
 Compound design process: RIPK3/1 dual inhibitor

Structural Characterization of Selection of

Understanding of Preparation kinase Docking and rank
insight: X-ray active site and patentable
Type-I and Type-II scaffold knowledge ordering of designs in
structure PDB understanding key scaffold for both
inhibitor base for Type-I and both active and inactive
ID: 4M69 & pharmacophore Type-I & II
interaction Type-II inhibitor conformation
6OKO features inhibitors

Kinase focus Development of Type-I & II

Introduction of common pharmacophore
library
Lys359 chemical diversity model
RIPK3/1 active and Relevance of key
Key interaction
inactive interactions and Arg367
between ATP and
conformation series-based SAR
Type-II inhibitors Literature and patent Short-listed designs for
comparison understanding
compound SAR understating further consideration
•MolSoft suite of software

Top ranked designs

for synthesis and
evaluation

Aurigene Pharmaceuticals Services Limited

RIPK1/3 Active site comparison

• Human and mouse RIPK3 active site is mostly conserved except region 1 and 2

• RIPK3 human kinase domain share low sequence homology with RIPK1

RIPK3 homology model based on PDB ID: 6OKO-Mouse Sequence Identity Similarity
PDB ID: 4NEU-Human RIPK1
RIPK1_Human_KD 34.46 43.35
RIPK1_Mouse_KD 34.08 42.77 RIPK3
RIPK1
2

1 2

hRIPK3
mRIPK3
Active site

hRIPK3 hRIPK3
mRIPK3 hRIPK1
Kinase domain sequence identity and similarity wrt human RIPK3
RIPK3 Type-II inhibitors and ESM attachment site

GSK-074
• Human RIPK3 homology model built on mouse
RIPK3 X-ray structure (PDB ID: 6OKO template)

Solvent
exposed
• Earlier proposed RIPK3 binding model in mouse
TAK-632 was reproduced again human RIPK3 kinase
domain with similar interaction

Solvent
exposed Proposed trajectory for ESM motif attachment

Cpd-26

Solvent
exposed
Aurigene Pharmaceuticals Services Limited
RIPK1 Type-II inhibitors and ESM attachment site

GSK-074 • Human RIPK1 X-ray structure PDB ID: 4NEU

was used to dock these compounds

Solvent
exposed • Compound trajectory is similar to proposed
RIPK3 binding model for all three compounds
TAK-632

• A minor conformational change observed for

Solvent TAK-632 and Cpd-26 at hinge region
exposed
Cpd-26

Solvent
exposed Proposed trajectory for ESM motif attachment
Aurigene Pharmaceuticals Services Limited
hCE1: Structural insight and understanding

• Human Carboxylesterase 1 Complexed with the Alzheimer's Drug Tacrine (PDB ID: 1MX1)
X-ray structure Docking model Compound 2
Docking model
Sub-
pocket-1

Solvent
exposed
Solvent
exposed

Sub-
pocket-2 Note: Water molecule 7019 was removed while docking Compound 2

• Docking model of Tacrine and Compound 2 was reproduced to establish protocol

X-ray structure
Docking model

Ref: Med. Chem. Commun., 2012, 3, 1070, Med. Chem. Commun., 2012, 3, 1070, Chemistry & Biology, Vol. 10, 341–349, April, 2003
Aurigene Pharmaceuticals Services Limited
Proposed design prioritization: GSK-074 scaffold

• ESM1 docking model in hCE1, RIPK3 and RIPK1

Sub-pocket-1

RIPK3: Docking
model

Sub-pocket-2 hCE1: Docking

model Solvent
exposed
RIPK1: Docking
• ESM motif is flipped conformation in hCE1, RIPK3 and RIPK1 model
docking model is consistent retaining critical hinge and DF(L)G
interaction

Aurigene Pharmaceuticals Services Limited

Proposed design prioritization: GSK-074 scaffold

• ESM2 docking model in hCE1, RIPK3 and RIPK1

Sub-pocket-1

RIPK3: Docking
model

Sub-pocket-2
hCE1: Docking
model Solvent
exposed
RIPK1: Docking
model
• ESM motif mimic exact orientation in hCE1, RIPK3 and RIPK1
docking model is consistent retaining critical hinge and DF(L)G
interaction

Aurigene Pharmaceuticals Services Limited

Proposed design prioritization: GSK-074 scaffold

• ESM3 docking model in hCE1, RIPK3 and RIPK1

Sub-pocket-1

RIPK3: Docking
model

Sub-pocket-2
hCE1: Docking
model Solvent
exposed
RIPK1: Docking
model
• ESM motif mimic exact orientation in hCE1, RIPK3 and RIPK1
docking model is consistent retaining critical hinge and DF(L)G
interaction

Aurigene Pharmaceuticals Services Limited

Structural Requirements for hCE-1 Hydrolysis

Representation of ESMTM compound in HCE-1

Hydrolysis ESMTM Amino Acid ESMTM atom spacer

Other ESMTM technology observations Ring Position Substituent alcohol
• L-amino acid required, D-isomer typically much slower hydrolysis
• Carbonyl adjacent to amino acid nitrogen introduces
hCE-2 & -3 hydrolysis and consequently reduces macrophage selectivity Rapid para Leu, Phe, tBuSer 1o,2o 2-3
• Therefore alkyl linker preferred
• With phenyl as aromatic spacer hCE-1 hydrolysis is generally good Intermediate meta Val, Phg, cHxg 2o 1-2
• Large substituents detrimental to hCE-1 hydrolysis
• Introduction of heteroatoms good for modulating hydrolysis Slow/None ortho Ser, His, Trp 3o 1 or bond
rate to achieve oral exposure; pyridine present in oral ESM TM HDACi

Aurigene Pharmaceuticals Services Limited

Balancing Intracellular & First Pass Liver Hydrolysis

Aurigene Pharmaceuticals Services Limited

Specific Hybrid compounds based on GSK-074

Aurigene Pharmaceuticals Services Limited

Specific Hybrid compounds based on Compound-26 & TAK-26

Aurigene Pharmaceuticals Services Limited

Specific Hybrid compounds based on BMS-9

Aurigene Pharmaceuticals Services Limited

Thank You