DISCOVERY OF A SERIES OF SMALL MOLECULE SHMT1/2 INHIBITORS

• Synthetized compounds exert potency in SHMT1/2 biochemical assay as well as in

cellular assay (measured by the C13 serine to glycine conversion)

• Tested compounds inhibit cancer cell growth

• Reduced viability could be rescued with formate

SHMT1: SEL302-01612

SHMT2: SEL302-01612

SHMT1: SEL302-00332

SHMT2: SEL302-00332

SHMT1: SEL302-00621

SHMT2: SEL302-00621

Discovery of novel SHMT small molecule inhibitors for cancer treatment

Anna Bartosik, Paweł Guzik, Marta Sowińska, Karolina Gluza, Marcin Król, Anna Wróbel, Agnieszka Dreas, Faustyna Iwańska, Magdalena Zastawna, Urszula Kulesza, Nicolas Boutard, David Schultz,

Justyna Wujkowska Karolina Pyziak, Agnieszka Sroka-Porada, Agnieszka Przybyłowicz, Agnieszka Adamus, Magdalena Sieprawska-Lupa, Przemysław Golik,

Piotr Kowalczyk, Krzysztof Brzózka, Tomasz Rzymski, Mateusz Nowak

Selvita S.A. Krakow, Poland www.selvita.com

Glucose

3-P-GlyceratePHGDH

THF 5,10-CH2-THF 10-formyl-THF

MTHFD1

Formate

DNA pyrimidine methylation

MTHFD1

Redox Proteins

Pyruvate

Serine Glycine

THF 5,10-CH2-THF 10-formyl-THF

Serine Glycine

SHMT2

Formate

MTHFD2/2L MTHFD1L

Redox ATP

SHMT1

Mitochondria

Cytoplasm

Breast (TCGA)

Enrichment Score (ES) 0.76

Normalized Enrichment Score (NES)

2.20

Nominal p-value 0.001

Enrichment Score (ES) 0.57

Normalized Enrichment Score (NES)

1.86

Nominal p-value 0.005

CRC (TCGA) NSCLC (TCGA)

Enrichment Score (ES) 0.54

Normalized Enrichment Score (NES)

1.71

Nominal p-value 0.03

INTRODUCTION

Over-activation of the serine synthesis pathway, upregulation of SHMT2 has beendescribed in over 20% of solid tumors (e.g. breast, lung, colorectal cancers). Such cancercells are highly dependent on serine. Serine hydroxymethyltransferase (SHMT isoform 1and 2) plays a key role in a so-called one-carbon pathway, a group of biochemicalreactions involved in amino acid metabolism. SHMT catalyzes the conversion of serine toglycine and also plays a role in the folate (vitamin B9) cycle. Antagonists of folatemetabolism or antifolates are an established chemotherapy in certain cancers. Folateantagonism disrupts cell division, DNA/RNA synthesis and protein synthesis. Pemetrexed(for non-small cell lung carcinoma, mesothelioma) and methotrexate (for autoimmuneconditions like rheumatoid arthritis and certain cancers) are two well established andeffective antifolates. The main drawback with antifolates in cancer treatment is thedevelopment of resistance. In this study we report development of a series of smallmolecule SHMT1/2 inhibitors. Synthetized compounds exert potency in SHMT1/2biochemical assay as well as in cellular assay (measured by the C13 serine to glycineconversion) in a low nanomolar range. Therapeutic effect of the compounds wasinvestigated in the panel of cancer cell lines with different genetic background as well aswith different SHMT2 levels. We identified several cell lines in which tested compoundsinhibited cancer cells growth with nM GI50 values. Taken together, presented datasupports our rationale for using SHMT1/2 inhibitors as a novel and interesting promisingapproach for the cancer therapy.

SSP/1C highSSP/1C highSSP/1C high

SSP POSITIVE SOLID TUMORS ARE C-MYC POSITIVE AND

SHOW INDUCTION OF AMINOACID INTEGRATED STRESS

RESPONSE (ISR)

PSAT1

CONCLUSIONS

Gene Set Enrichment Analysis for the class of SSP/1C high solid tumors

SSP and 1C pathways in cancer

Project supported by the European Regional Development Funds under the Measure 1.1.1. Operational Program - Smart Growth (POIR.01.01.01-00-0673/15-00)

SHMT INHIBITORS DECREASE VIABILITY OF BREAST AND LUNG

CANCER CELLS BY THE INHIBITION OF 1C PATHWAY

FORMATE SENSITIZES CELL LINES DEFICIENT IN GLYCINE

TRANSPORT TO SHMT1/2 INHIBITORS

• Cancer specific SSP and 1C proteins are attractive targets for pharmacological inhibition in oncology

• SHMT1/2 inhibitors are a novel approach for cancer therapy

• Inhibition of SHMT by small molecules effectively blocks influx of 1C units form serine in cancer cells

• Anticancer effects of compounds are SHMT-dependent as revealed by rescue experiments

• Defective glycine import as well as glycine starvation could be a promising therapeutic strategy for SHMT2

inhibitors in DLBCL

References:

1. Yang and Vousden. Serine and one-carbon metabolism in cancer. Nat. Rev. Can. 2016

2. Jain et al. Metabolite profiling identifies a key role for glycine in rapid cancer cell proliferation. Science. 2012

3. Labuschagne et al. Serine, but not glycine, supports one-carbon metabolism and proliferation of cancer cells. Cell Reports. 2014

4. Newman and Maddocks. One-carbon metabolism in cancer. British Journal of Cancer. 2017.

TOP TABLE

COMPOUND SLV# SEL302-01612 SEL302-00687 SEL302-00745 SEL302-00753

IC50 [µM] SHMT2 0.02 0.03 0.05 0.04

IC50 [µM] SHMT1 0.35 tbd tbd tbd

AD

ME/

DM

PK

Metabolic Stability(mic. mouse) (1h)

0% 80.4% 80.7% 45.8%

Metabolic StabilityMouse S9 fraction (phase I and II)

0% 86.9% tbd 55.6%

hERG Channel Inhibition [uM] 17 tbd tbd >100

CEL

L A

SSA

YS

Ser-Gly Flux InhBreast cancer cell line

IC50 [uM] 0.03 0.34 tbd tbd

Viability 72hBreast cancer cell line

GI50 [uM](efficiency)

0.2 4.1 2.3 2.5 1.1 1.8

Viability 72hLung cancer cell line

GI50 [uM](efficiency)

0.3 >10 >10 9.7 5.5 6.9

Results from combination study indicate that glycine starvation (e.g. in a form of low glycine diet)

can be used to render similar dependency in cancer cell without glycine transport deficiency.

Failure of formate to rescue growth in the DLBCL cell lines is most likely due to a

requirement for both glycine and 1C units made by SHMT in these cells. In the

absence of glycine, formate can enhance the cytotoxicity of SHMT inhibition, by

driving residual SHMT enzymatic function in the glycine-consuming direction.

Human SHMT inhibitors reveal defective glycine import as a targetable metabolic vulnerability of diffuse large B-cell lymphoma. Proc NatlAcad Sci U S A. 2017 Oct 24;114(43):11404-11409.

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SEL302-00332

biochemical assay SHMT1 IC50 (µM) SHMT2 IC50 (µM)

SEL302-01612 0.01 0.02

SEL302-00332 0.22 0.01

SEL302-00621 0.08 0.16

serine flux IC50 (µM)

SEL302-01612 0.03

SEL302-00332 0.05

SEL302-00621 0.4

SEL302-01612 SEL302-00621

SEL302-01612

SEL302-00332

SEL302-00621

FULL MEDIUM 1 mM FORMATE

10X GLYCINE 10x GLYCINE + 1 mM FORMATE

SEL302-01612Breast cancer

cell line (-FA)

Breast cancer

cell line (+FA)

Lung cancer

cell line (-FA)

Lung cancer

cell line (+FA)

GI50 (µM) 0.23 > 10 0.35 > 10LC (µM) 2 > 10 3 > 10

SEL302-00332Breast cancer

cell line (-FA)

Breast cancer

cell line (+FA)

Lung cancer

cell line (-FA)

Lung cancer

cell line (+FA)

GI50 (µM) 0.9 > 10 0.6 > 10LC (µM) > 10 > 10 7 > 10

SEL302-00621Breast cancer

cell line (-FA)

Breast cancer

cell line (+FA)

Lung cancer

cell line (-FA)

Lung cancer

cell line (+FA)

GI50 (µM) 0.89 > 10 3.2 > 10LC (µM) 2.6 > 10 > 10 > 10

Combination study was performed using DLBCL cell line with defective glycine import.