In the Quest for Potent and Selective Malic Enzyme 3 Inhibitors for the Treatment of Pancreatic Ductal Adenocarcinoma.

pubs.acs.org/acsmedchemlett Letter In the Quest for Potent and Selective Malic Enzyme 3 Inhibitors for the Treatment of Pancreatic Ductal Adenocarcinoma Gaurav Sheth, Shailesh R. Shah,* Prabal Sengupta, Tushar Jarag, Sabbirhusen Chimanwala, Kalapatapu V. V. M. Sairam, Vaibhav Jain, Rashmi Talwar, Avinash Dhanave, Mehul Raviya, Soumya Menon, Shivangi Trivedi, and Trinadha Rao Chitturi* Cite This: ACS Med. Chem. Lett. 2023, 14, 41−50 ACCESS Metrics & More Read Online Article Recommendations sı Supporting Information * ABSTRACT: The genome of pancreatic ductal adenocarcinoma (PDAC) is associated with frequent deletion of the tumor suppressor gene SMAD family member 4 (SMAD4) with collateral deletion of its chromosomal neighbor malic enzyme 2 (ME2). In SMAD4−/−/ME2−/− PDAC cells, ME3 takes over the function of the ME2 enzyme, and hence therapeutic targeting of ME3 is expected to arrest tumor growth. Hitherto no selective small molecule inhibitor of ME3 has been reported in the context of PDAC. Based on the molecular docking studies and structure− activity relationships with the reported ME1 inhibitor, several analogues of 6-piperazin-1-ylpyridin-3-ol amides have been synthesized and screened for their ME inhibition activity. Among them, compound 16b is identified as the most potent and selective ME3 inhibitor with an IC50 of 0.15 μM on ME3, and with 15- and 9-fold selectivity over ME1 and ME2, respectively. In the cell viability assay, compound 16b exhibited an IC50 of 3.5 μM on ME2-null PDAC cells, viz., BxPC-3. KEYWORDS: ME3 inhibitors, Anticancer compounds, PDAC, Collateral lethality, Malic enzyme, Molecular Docking P and de novo fatty acid synthesis. Advancement of glycolysis and fatty acid synthesis is supported by bioprecursors like pyruvate and cofactors like nicotinamide adenine dinucleotide (NAD) phosphate NAD(P)+ and NAD(P)H. Expression of enzymes involved in the mitochondrial tricarboxylic acid (TCA) cycle is also upregulated in numerous cancers leading to metabolic alterations to meet the enhanced demand for bioenergy and biomass for chronic proliferation of cancer cells. In this context, malic enzymes (MEs) play a crucial role in catalyzing oxidative decarboxylation of L-malate to form pyruvate and CO2 while simultaneously reducing NAD(P)+ to NAD(P)H.7 Both pyruvate and NADPH have an important role in energy production in cells, for the maintenance of redox balance and for the production of building blocks for biosynthesis. In mammalian context, three isoforms of malic enzyme have been identified. These are classified based on their cofactor specificities and subcellular ancreatic ductal adenocarcinoma (PDAC) is an aggressive pancreatic cancer originating from exocrine cells and accounts for more than 90% of pancreatic tumors.1 PDAC patients remain asymptomatic at early stages, and a majority of them are diagnosed only at unresectable stages.2 Due to its poor prognosis, it is one of the leading causes of cancer-related mortalities with a five year survival rate of less than 11%.3 Chemotherapeutic combinations, viz., gemcitabine and paclitaxel or FOLFIRINOX (a combination of 5-fluorouracil, irinotecan, oxaliplatin, and folinic acid), are the current major standards of care for PDAC. However, there is an unmet clinical need for novel targeted therapies.4 Activating mutations in the Kirsten rat sarcoma virus (KRAS) oncogene is associated with more than 90% of PDAC cases along with subsequent deletion of tumorsuppressor genes like INK4A/ARF, tumor protein 53 (TP53), and SMAD family member 4 (SMAD4).5 In PDAC, oncogenic KRAS mutation (KRasG12D) driven activation of downstream signaling pathways like MAPK and PI3K-mTOR mobilizes uncontrolled proliferation and survival of cancer cells. Rapid proliferation of neoplastic cells requires an adequate supply of energy and biosynthetic precursors as cellular building blocks.6 This enhanced energetic and anabolic requirement is satisfied through metabolically rewired processes which include aerobic glycolysis, glutaminolysis, © 2022 American Chemical Society Received: August 7, 2022 Accepted: December 8, 2022 Published: December 13, 2022 41 https://doi.org/10.1021/acsmedchemlett.2c00369 ACS Med. Chem. Lett. 2023, 14, 41−50 ACS Medicinal Chemistry Letters pubs.acs.org/acsmedchemlett localization: cytosolic NADP+-dependent ME (ME1), mitochondrial NAD(P)+-dependent ME (ME2), and mitochondrial NADP+-dependent ME (ME3).8 These enzymes are essential for the maintenance of mitochondrial health and related energetics. ME1 is a potential oncogene product linked to energy metabolism, redox status, and epithelial to mesenchymal transition (EMT) in squamous cell carcinoma (oral, head, and neck), breast cancer, and gastric cancer.9−11 ME2 is overexpressed in human primary oral squamous cell carcinoma tissue and in lung carcinoma tissue for helping tumor cell proliferation and colony formation.12,13 ME2 is also known to promote cell proliferation, invasion, and migration of glioma cells in glioblastoma.14 ME3, a paralogous isoform of ME2, is localized in mitochondria, and its aberrant expression contributes to the carcinogenesis of pancreatic cancer.15 In nearly one-third of PDAC cases, the tumor-suppressor gene SMAD4 is homozygously deleted, and neighboring gene ME2, which is in the same locus, is codeleted conferring “collateral lethality”. Through systematic in vitro and in vivo genetic experiments, Dey et al. have demonstrated that, in such a situation, the paralogous enzyme ME3 takes over the role of ME2, and depletion of this enzyme creates cancer specific metabolic vulnerability leading to cell death.16 Based on their compelling data, it was conceived that targeting ME3 with selective small molecule pharmacological inhibitors could be an attractive therapeutic strategy for specific PDAC patients having the SMAD4/ME2 deletions. Herein, we report the design and synthesis of small molecules that potently and selectively inhibit ME3. In order to choose appropriate chemotype(s) for the development of novel ME3 inhibitors, the reported inhibitors A−E (Table 1) were synthesized and screened for their inhibitory potency against all three ME isoforms.17−20 Among these, compound A showed superior inhibition on ME3 with an IC50 of 0.15 μM. Hence, compound A was selected as a lead chemotype for the design and optimization of potent and selective ME3 inhibitors. To understand and to analyze the possible binding interactions of compound A with the ME3 enzyme, docking studies needed to be carried out. Since the crystal structure of ME3 either in the apo form or with a substrate/inhibitor and cofactor (NADP+) was not available in the protein data bank (PDB), it was crystallized as an oxalate whose crystal structure was resolved (resolution: 1.8 Å). Using this crystal structure, the in silico binding mode of compound A and its analogues in the malate binding site of the ME3 enzyme was established by the induced fit docking (IFD) (...truncated)