Cuong Ly

Discovery of GluN2A-Selective NMDA Receptor Positive Allosteric Modulators (PAMs): Tuning Deactivation Kinetics via Structure-Based Design.

The N-methyl-D-aspartate receptor (NMDAR) is a Na(+) and Ca(2+) permeable ionotropic glutamate receptor that is activated by the coagonists glycine and glutamate. NMDARs are critical to synaptic signaling and plasticity, and their dysfunction has been implicated in a number of neurological disorders, including schizophrenia, depression, and Alzheimers disease. Herein we describe the discovery of potent GluN2A-selective NMDAR positive allosteric modulators (PAMs) starting from a high-throughput screening hit. Using structure-based design, we sought to increase potency at the GluN2A subtype, while improving selectivity against related α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). The structure-activity relationship of channel deactivation kinetics was studied using a combination of electrophysiology and protein crystallography. Effective incorporation of these strategies resulted in the discovery of GNE-0723 (46), a highly potent and brain penetrant GluN2A-selective NMDAR PAM suitable for in vivo characterization.

Discovery of Dual Leucine Zipper Kinase (DLK, MAP3K12) Inhibitors with Activity in Neurodegeneration Models

Dual leucine zipper kinase (DLK, MAP3K12) was recently identified as an essential regulator of neuronal degeneration in multiple contexts. Here we describe the generation of potent and selective DLK inhibitors starting from a high-throughput screening hit. Using proposed hinge-binding interactions to infer a binding mode and specific design parameters to optimize for CNS druglike molecules, we came to focus on the di(pyridin-2-yl)amines because of their combination of desirable potency and good brain penetration following oral dosing. Our lead inhibitor GNE-3511 (26) displayed concentration-dependent protection of neurons from degeneration in vitro and demonstrated dose-dependent activity in two different animal models of disease. These results suggest that specific pharmacological inhibition of DLK may have therapeutic potential in multiple indications.

Mechanistic and Structural Understanding of Uncompetitive Inhibitors of Caspase-6

Inhibition of caspase-6 is a potential therapeutic strategy for some neurodegenerative diseases, but it has been difficult to develop selective inhibitors against caspases. We report the discovery and characterization of a potent inhibitor of caspase-6 that acts by an uncompetitive binding mode that is an unprecedented mechanism of inhibition against this target class. Biochemical assays demonstrate that, while exquisitely selective for caspase-6 over caspase-3 and -7, the compound’s inhibitory activity is also dependent on the amino acid sequence and P1’ character of the peptide substrate. The crystal structure of the ternary complex of caspase-6, substrate-mimetic and an 11 nM inhibitor reveals the molecular basis of inhibition. The general strategy to develop uncompetitive inhibitors together with the unique mechanism described herein provides a rationale for engineering caspase selectivity.

Potent, Selective, and Orally Bioavailable Inhibitors of the Mammalian Target of Rapamycin Kinase Domain Exhibiting Single Agent Antiproliferative Activity

Selective inhibitors of mammalian target of rapamycin (mTOR) kinase based upon saturated heterocycles fused to a pyrimidine core were designed and synthesized. Each series produced compounds with K(i) < 10 nM for the mTOR kinase and >500-fold selectivity over closely related PI3 kinases. This potency translated into strong pathway inhibition, as measured by phosphorylation of mTOR substrate proteins and antiproliferative activity in cell lines with a constitutively active PI3K pathway. Two compounds exhibiting suitable mouse PK were profiled in in vivo tumor models and were shown to suppress mTORC1 and mTORC2 signaling for over 12 h when dosed orally. Both compounds were additionally shown to suppress tumor growth in vivo in a PC3 prostate cancer model over a 14 day study.

Potent, selective, and orally bioavailable inhibitors of mammalian target of rapamycin (mTOR) kinase based on a quaternary substituted dihydrofuropyrimidine.

A series of inhibitors of mTOR kinase based on a quaternary-substituted dihydrofuropyrimidine was designed and synthesized. The most potent compounds in this series inhibited mTOR kinase with K(i) < 1.0 nM and were highly (>100×) selective for mTOR over the closely related PI3 kinases. Compounds in this series showed inhibition of the pathway and antiproliferative activity in cell-based assays. Furthermore, these compounds had excellent mouse PK, and showed a robust PK-PD relationship in a mouse model of cancer.

Discovery and Biological Profiling of Potent and Selective mTOR Inhibitor GDC-0349.

Aberrant activation of the PI3K-Akt-mTOR signaling pathway has been observed in human tumors and tumor cell lines, indicating that these protein kinases may be attractive therapeutic targets for treating cancer. Optimization of advanced lead 1 culminated in the discovery of clinical development candidate 8h, GDC-0349, a potent and selective ATP-competitive inhibitor of mTOR. GDC-0349 demonstrates pathway modulation and dose-dependent efficacy in mouse xenograft cancer models.

Antagonists of inhibitor of apoptosis proteins based on thiazole amide isosteres.

A series of IAP antagonists based on thiazole or benzothiazole amide isosteres was designed and synthesized. These compounds were tested for binding to the XIAP-BIR3 and ML-IAP BIR using a fluorescence polarization assay. The most potent of these compounds, 19a and 33b, were found to have K(i)s of 20-30 nM against ML-IAP and 50-60 nM against XIAP-BIR3.

Tailoring small molecules for an allosteric site on procaspase-6.

Although they represent attractive therapeutic targets, caspases have so far proven recalcitrant to the development of drugs targeting the active site. Allosteric modulation of caspase activity is an alternate strategy that potentially avoids the need for anionic and electrophilic functionality present in most active‐site inhibitors. Caspase‐6 has been implicated in neurodegenerative disease, including Huntington’s and Alzheimer’s diseases. Herein we describe a fragment‐based lead discovery effort focused on caspase‐6 in its active and zymogen forms. Fragments were identified for procaspase‐6 using surface plasmon resonance methods and subsequently shown by X‐ray crystallography to bind a putative allosteric site at the dimer interface. A fragment‐merging strategy was employed to produce nanomolar‐affinity ligands that contact residues in the L2 loop at the dimer interface, significantly stabilizing procaspase‐6. Because rearrangement of the L2 loop is required for caspase‐6 activation, our results suggest a strategy for the allosteric control of caspase activation with drug‐like small molecules.

GluN2A-Selective Pyridopyrimidinone Series of NMDAR Positive Allosteric Modulators with an Improved in Vivo Profile

The N-methyl-d-aspartate receptor (NMDAR) is an ionotropic glutamate receptor, gated by the endogenous coagonists glutamate and glycine, permeable to Ca2+ and Na+. NMDAR dysfunction is associated with numerous neurological and psychiatric disorders, including schizophrenia, depression, and Alzheimers disease. Recently, we have disclosed GNE-0723 (1), a GluN2A subunit-selective and brain-penetrant positive allosteric modulator (PAM) of NMDARs. This work highlights the discovery of a related pyridopyrimidinone core with distinct structure-activity relationships, despite the structural similarity to GNE-0723. GNE-5729 (13), a pyridopyrimidinone-based NMDAR PAM, was identified with both an improved pharmacokinetic profile and increased selectivity against AMPARs. We also include X-ray structure analysis and modeling to propose hypotheses for the activity and selectivity differences.

Abstract B23: Crucial deubiquitinases in cancer cell survival

Deubiquitinases (DUBs) are enzymes that proteolytically cleave ubiquitin from substrates. Substrates include oncogenes, tumor suppressors and polyubiquitinated proteins marked for degradation by the proteasome. Ubiquitin specific peptidase-7 (USP7) deubiquitinates MDM2 (an oncogene). MDM2 is a ligase that ubiquitinates p53 (a tumor suppressor protein), targeting it for proteosomal degradation. As such, USP7 is a promising cancer target because its inhibition stabilizes p53 and thereby promotes apoptosis and cell cycle arrest, processes that are often deregulated in tumors (Nicholson and Suresh Kumar, 2011). We found that USP7 was selectively druggable following a fragment-based lead discovery effort to obtain USP7 antagonists. Cellular and xenograft studies confirm that inhibiting USP7 activity stabilized p53 levels and p53-downstream target, p21. Additionally, normal primary and p53-null cells were less sensitive than the corresponding p53-WT cancer cells to USP7 inhibition. To investigate whether other DUBs are involved in cancer cell survival, we carried out a drop-out CRISPR screen using a pooled DUB library in HCT116 and A549 cells. Out of the approximately 100 DUBs targeted, nine, including USP7, were found to affect cell viability. These hits were validated using siRNA-mediated knockdown in cancer cell lines (A549, HCT116, MCF7). Three DUBs that robustly decreased cell proliferation were further tested in normal cells (Human Mammary Epithelial Cells and Human Bronchial Epithelial Cells). DUB protein expression levels and activity were also determined. In general, DUB expression levels, activity and knockdown efficiency were higher in cancer cells compared to normal cells. Collectively, our studies support the hypothesis that USP7 inhibition may be an efficacious strategy to promote cancer cell death. Furthermore, there are other DUBs that should be considered as novel cancer targets. Citation Format: Lorna Kategaya, Trinna Cuellar, Ben Haley, Jinfeng Liu, Andy Tran, Yi Cao, David Stokoe, Mark McCleland, Beth Blackwood, Sharon Yee, Joy Drobnick, Jake Drummond, James Ernst, Michael Kwok, Cuong Ly, Richard Pastor, Paola Di Lello, Chudi Ndubaku, Robert Blake, Vickie Tsui, Jeremy Murray, Till Maurer, Ingrid Wertz. Crucial deubiquitinases in cancer cell survival. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B23.