Aurigene-Modulus Collaboration

JSC meeting

Target : c-KIT Allosteric Inhibitor

8th June 2020

CONFIDENTIAL

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Presentation Outline

• Introduction
• Objective and hypothesis
• Present Flow Scheme (Compound triaging funnel)
• Summary of Progress
• Stage 1 criteria
• Status of assays in biology
• Chemistry Hit generation strategies
• Profile of interesting compounds
• Key challenges in the project
• FTE Utilization Summary (Nov’19-Jan 2020)

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Introduction - KIT Kinase

 c-KIT, is a 145 kDa transmembrane glycoprotein, which

belongs to class III of the RTK family. It has three
domains: a hydrophobic transmembrane, an
extracellular ligand-binding domain, and a cytoplasmic
domain with tyrosine kinase activity.

 c-KIT is mainly considered a stem cell factor receptor,

which participates in vital functions of the human body,
such as fertility, homeostasis, and melanogenesis.

 Deregulation of c-KIT, including overexpression and gain

of function mutations, has been detected in several
human cancers and plays a crucial role in
carcinogenesis.
KIT structure and schematic of several KIT interactors and the
 c-KIT activating mutation is found in almost all cases of phosphorylated tyrosine residues to which they bind

gastrointestinal stromal tumor, systemic mastocytosis Babaei et al., Drug Design, Development and Therapy ,2016 :
and other hematopoietic cancers. 10 2443–2459

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Objective and Hypothesis

 Objective
• To develop a first-in-class, selective, orally bioavailable and patentable allosteric KIT inhibitor for
the treatment of cancer
 Hypothesis
• The receptor tyrosine kinase c-KIT is mutated in several types of cancers, notably
gastrointestinal tumours (GIST), systemic mastocytosis and subsets of acute myeloid
leukaemia.
• Oncogenic activating and secondary mutations prevent binding of ATP competitive small
molecule inhibitors to the catalytic domain of KIT.
• This leads to resistance in the cancer stem cells that do not respond to treatment with current
KIT inhibitors.
• Development of allosteric inhibitors that inhibits the activity of KIT will thus allow to overcome
the resistance to approved drugs in the clinic.

Assay set up and Time line has

Hit Identification Feb 2020 been Extended
validation

September 2019
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Flow scheme
Detection of Solubility,
Compound KIT kinase assay phosphorylation of KIT permeability, plasma Hit
design and (FP or ELISA type) (Tyr 719/703) Metabolic stability (M
synthesis ICW/WB R H)

KIT enzymatic assay 3-day proliferation

assay
(ADP glow)

Selectivity against Phosphorylation of

closely related ERK (Thr202/Tyr204)
kinases ICW/WB

Assay can be setup

Outsourced assay
in-house
Routine Assay

Profiling Assay

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Summary of progress
 Biochemical assays including ADP Glo, Fluorescent polarization and TR-FRET
displacement assay, has been standardized and validated with tool compounds. Routine
screening of compounds is ongoing
 Cell based assays including proliferation assay, western blot-In cell western assay for
pKIT and downstream target gene pERK has been standardized and validated with
reference inhibitors in Kasumi-1 and GIST-T1 cell line
 Expression of KIT protein in insect cell system has been demonstrated. Crystals obtained
in the presence of MDG-032. Data collection and analysis is on going
 Potential hits have been identified from virtual screens and De novo designed
compounds by Modulus. Binding mode needs to be authenticated by co-crystal structure
 Further efforts are in progress to improve the potency of the identified hit by classical
medicinal chemistry approach

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Stage 1 Criteria (Hit ID and Assay Setup – 6 months)
Parameter
Establish relevant in vitro
assays
Initiate chemistry design
Structural studies
Hit characterization: In vitro
pharmacology
Criteria for stage transition has not been decided
Deliverables Time line
Establish relevant in vitro assays and validate them with reference inhibitor
• Biochemical assay:
• Binding assay:
• Competitive ELISA-like assay
• Fluorescent polarization assay for c-KIT
• Enzymatic assay: ADP Glo
• Cell based assay
• Inhibition of phosphorylation of KIT (at Tyr719 and/or Tyr703),
ERK (Thr202/Tyr204)
• 3-day proliferation assay
Feb 2020
• Design and synthesis of type III pocket binding compounds based on key
interaction in peptide c-KIT complex co crystal structure
• Determination of co-crystal structures with preliminary hit compounds
(conditions published in literature will be evaluated)
• Begin in vitro testing of analogs.
• Biochemical assay IC50 < nM (TBD)
• Proliferation assay IC50 < nM (TBD)
TBD – To be decided
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Status of Assays in Biology
Parameter Deliverables Status
Establish  Protein production and crystallography  Expression of KIT protein in Ecoli is on hold, as protein
relevant in-  c-KIT expression for crystallization in was found to be aggregating in inclusion bodies
vitro  E.coli regardless of the conditions tested.
assays  Insect cells  Expression of KIT protein was observed in the soluble
fraction when expressed in insect cells. Crystallization
trials are on going. c-kit crystals were obtained in the
presence of MDG-032. Data collection and analysis is on
going
 Biochemical Assay  ADP Glo, FP and TR-FRET displacement assay, has
 ADP Glo assay for c-KIT been standardized and validated. Routine screening of
 Fluorescence polarization based compounds is ongoing
competition assay for c-KIT  Competitive ELISA-like assay is on hold for now
 Competitive ELISA-like assay for c-KIT
 TR-FRET displacement assay
 Cellular assays  Kasumi-1 and GIST-T1 cell lines have been procured
  3d proliferation assay has been standardized and
3 day Proliferation assay
validated with Imatinib/ sunitinib in both cell lines
 Western blot for pKIT (Tyr719 and  Western blot and in cell western for pKIT and pERK has
Tyr703), pERK (Thr202/Tyr204) been standardized and validated with reference
inhibitors in GIST-T1 cell line.
 In vivo pharmacology  Tumour take studies are initiated for GIST T1 cell line in
NOD SCID and Balb/c nude mice

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Chemistry strategies for hit generation
 Virtual Screening hits: ~172 compounds purchased & screened
• VS hit compound (MDG-023) was synthesized and data validated
• Enantiomers of MDG-023 separated, Peak-2 (MDG-032) is the active isomer
• SAR around MDG-032 produced more hits with structural variation
• Few more potent compounds were identified in a follow up commercial compound screen

 De novo designed compounds: ~ 25 compounds synthesized & screened

 MDG-001 and few other analogues of it are potent in the ADP Glo assay
 MDG-115 is the most potent compound with good correlation between assays, submitted to co-
crystallization. SAR is planned around this hit.

 PeptiDreams's Virtual Screen hits: 28 compounds were designed

 17 compounds were synthesized, few micro molar hits were obtained
 Rest of the compounds synthesis is in progress
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SAR around Virtual Screening hit
peak-2 peak-2 H racemic peak-1 racemic F
H H N S S F
N H
S O N S O H N H N H N
O H N N F
Structure N
H N N N N N N N O H
N N NH2 N N H H H N N
H H O O
O H
O O O

MDG Code / MDG-032 MDG-119 MDG-062 MDG-114 MDG-124 MDG-112

AU Code AU-CGO-19599 AU-CGO-XXXXX AU-CGO-19738 AU-CGO-20104 AU-CGO-20331 AU-CGO-20079

MW / ClogP 342.42 / 2.44 344.43 / 2.10 306.33 / 2.21 340.45 / 3.69 340.45 / 3.69 349.31 / 3.40
ADP Glo IC50
0.156 0.1 / 0.13 0.153 / 0.219 0.16 / 0.378 0.05 0.35
(µM)

FP IC50 (µM) 0.574 1.7 / 1.1 4.6 TBD TBD TBD

TR-FRET EC50
7 22 / 11 No displacement 3 TBD TBD
(µM)
GIST-T1 GI50
1 0.362 0.931 0.714 TBD 0.566
(µM)
pKIT Tyr703
1.06 IP 0.718 0.206 TBD 0.398
ICW IC50 (µM)
pKIT Tyr719
64% @ 100µM IP 58% @ 100µM 68% @ 100µM TBD 0.987
ICW IC50 (µM)
Eq. Sol.@
47 32.8 TBD 7.6 TBD 2.8
pH 7.4 (µM)
MiLM % rem.
81 91.67 84.94 14.10 TBD >95
@ 60 min.
MiLM CLH
32.62 18.19 30.95 78.74 TBD NC
(mL/min/kg)

• 50-200 nM activity in ADP Glo assay is achieved with many compounds

• These compounds show 300nM - 1 µM anti proliferative activity in GIST-T1 cell line
• MDG-119 & MDG-124 were submitted for co-crystallization
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Follow up Virtual Screening hits
H
N N O N N
H
N
Structure O O H
N
N F N N
H
O H
O

MDG Code /
Z828307670 Z1286571265
AU Code

MW / ClogP 392.39 / 3.29 347.38 / 2.57

ADP Glo IC50 (µM) 0.054 / 0.06 / 0.057 0.16 / 0.12 / 0.095

FP IC50 (µM) 0.23 / 0.5 / 0.36 0.5 / 0.56 / 1.3

TR-FRET EC50 (µM) 1.5 / 0.4 / 0.35 1 / 2.8 / 1.4

GIST-T1 GI50 (µM) 0.359 1.15

pKIT Tyr703
0.65 1.08
ICW IC50 (µM)
pKIT Tyr719
3.25 5.79
ICW IC50 (µM)
Eq. Sol.@
pH 7.4 (µM)
MiLM % rem.
@ 60 min.
MiLM CLH
(mL/min/kg)

• These compounds have 50-100 nM activity in ADP Glo assay

• Few more compounds were planned to extend this SAR understanding

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PK Profile of AU-CGO-19599 (MDG-032) in male CD1 Mice
In vitro ADME profile MDA-032
Mol Wt;, CLogP; tPSA 342.42; 2.43; 82.59
Equil. Sol. (pH7.4) in µM 46.8
MLM stability
81.14; 32.62
% rem @60 min/ Hep Cl (mL/min/kg)
Caco-2 (E-6 cm/sec) A-B/B-A ; ER 3.983/30.813; 7.73

IV PO
Parameters Units
1 mpk 3 mpk PK profile of AU-CGO-19229 (MDG-032) in male CD1 Mice
AUC0-last h*ng/mL 1149 2217
AUC0-inf 10000
h*ng/mL 1182 2265 IV-1 mg/kg

Plasma conc (ng/ml)

Cl mL/min/kg 14.25 - PO-3 mg/kg
1000
Vdss L/kg 0.83 -
Beta t1/2 hr 0.84 1.30 100
C0/Cmax ng/mL 1130.20 717.18
10
Tmax hr - 0.67
F % - 63.85
1
0 1 2 3 4 5 6 7 12 24

IV: 2% NMP + 30% PEG-200+ Normal Saline QS Time (h)

PO: 0.5% Tween-80 + 0.5% Methyl cellulose in Water QS

• MDG-032 exhibited low clearance, moderate volume of distribution with 64 % oral bioavailability
• Reasonable IVIVC observed
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Confidential
 Profile of interesting compounds from de-novo designs
O
NH2
NH
N

H H F N CF3
Structure N N N
N H H F
N H F N N H F O
O N N N N
N
O
O N N
H H

MDG Code / MDG-001 MDG-018 MDG-085 MDG-115

AU Code AU-CGO-18697 AU-CGO-19412 AU-CGO-19915 AU-CGO-20106
MW / ClogP 352.41 / 4.87 381.46 / 3.82 423.49 / 3.68 410.42 / 4.53

ADP Glo IC50 (µM) 0.55 / 0.72 0.851 0.34 / 0.5 / 0.89 0.116

FP IC50 (µM) 14 / 15.6 2.5 / 2 3.4 0.8/ 0.46 / 0.68

TR-FRET EC50 (µM) 13.5 35 / 23.5 2.3 0.7/ 0.7 / 0.49

GIST-T1 GI50 (µM) 2.46 4.57 1.008 0.189

pKIT Tyr703
15.66 5.52 0.86 1.99
ICW IC50 (µM)
pKIT Tyr719
42.43 62% @ 100µM 68% @ 100µM 78% @ 30µM
ICW IC50 (µM)

Eq. Sol.@pH 7.4 (µM) <2 >180 28.9 52

MiLM % rem. @
8.31 60.35
60 min.

MiLM CLH
78.79 55.0
(mL/min/kg)

• 100 - 500 nM activity in ADP Glo assay is achieved in this series

• MDG-085 & MDG-115 were submitted for co-crystallization
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PeptiDream’s virtual screen hits
F
N
H
N H O O NH N
F N N F HN Me
Structure N F O
O
Me
NH

O N
H

MDG Code / MDG-P01 / MDG-P02 / MDG-P17 /

AU Code AU-CGO-20070 AU-CGO-20071 AU-CGO-20362

MW / ClogP 370.47 / 3.44 357.44 / 5.10 611.76 / 3.33

ADP Glo IC50 (µM) 7.5 38 60

FP IC50 (µM) TBD

TR-FRET EC50 (µM) TBD

GIST-T1 GI50 (µM)

pKIT Tyr703
ICW IC50 (µM)
pKIT Tyr719
ICW IC50 (µM)
Eq. Sol.@pH 7.4 (µM)
MiLM % rem. @
60 min.
MiLM CLH (mL/min/kg)

• MDG-P01 is a good find considering the complete structural diversity

• More data is awaited in this series
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Key challenges in the project and strategies to overcome the challenges

 To conclusively determine if the hit compounds are binding to the

allosteric site of the Kit protein
• To solve the crystal structure of the Kit protein with hit compounds to determine their
binding mode
• To determine if the hit compounds are potent in mutant kit proteins that are insensitive
to inhibitors imatinib and sunitinib

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Confidential
Stage 1: Hit /Assay Validation Activities
Approved FTE’s - Mar-May 2020

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Confidential
Thank You

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