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Structure-based design was utilized to optimize 6,6-diaryl substituted dihydropyrone and hydroxylactam to obtain inhibitors of lactate dehydrogenase (LDH) with low nanomolar biochemical and single-digit micromolar cellular potencies. Surprisingly the replacement of a phenyl with a pyridyl moiety in the chemical structure revealed a new binding mode for the inhibitors with subtle conformational change of the LDHA active site. This led to the identification of a potent, cell-active hydroxylactam inhibitor exhibiting an in vivo pharmacokinetic profile suitable for mouse tumor xenograft study.

Activating mutations in PIK3CA are among the most significant oncogenic events across all cancers, making it an important target for drug development. Yet the application of PI3K inhibitors in the clinic has been limited by the difficulty of achieving an adequate therapeutic window, due to the critical role that PI3K signaling plays in normal physiologic processes, such as glucose homeostasis. In theory, the therapeutic window could be improved if it were possible to design mutant selective inhibitors, as has been demonstrated with other oncogenes such as EGFR. However, unlike EGFR, the most predominant PIK3CA activating mutations do not reside in the kinase active site, presenting a major challenge for rational structure-based design. Nevertheless, it was recently shown that the PI3K inhibitor taselisib is able to achieve modest levels of mutant selectivity both across cancer lines as well as in cell lines that were engineered to express mutant or wild-type PIK3CA. Taselisib was also shown to selectively induce degradation of mutant versus wild-type PIK3CA, leading to the speculation that this degradation may be responsible for the observed selectivity. In order to better understand the origins of mutant selectivity for taselisib and several other PIK3CA inhibitors, we assessed these inhibitors in a variety of biophysical and biochemical assays under conditions designed to mimic physiologic settings. In parallel, we also investigated the mechanistic basis of this selectivity in our engineered cell lines. Our results are consistent with the hypothesis that selective degradation of mutant PIK3CA is the predominant mechanism underlying mutant selectivity for this class of PIK3CA active site inhibitors. This abstract is also being presented as Poster B03. Citation Format: Lan Nguyen, Kyle Edgar, Kyung Song, Stephen Schmidt, Victorai Schutz, Noriko Ishisoko, Eric Torres, Akash Das, Divya Murali, Steve Sideris, Timothy Wendorff, Matt Saabye, Hans Purkey, Jawahar Sudhamsu, Steven Staben, Emily Hanan, Georgia Hatzivassiliou, Lori Friedman, Nicholas F. Endres. Selective degradation of mutant PIK3CA promotes increased mutant specificity in a subset of PI3K ATP-competitive inhibitors [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr PR03.

Optimization of 5-(2,6-dichlorophenyl)-3-hydroxy-2-mercaptocyclohex-2-enone using structure-based design strategies resulted in inhibitors with considerable improvement in biochemical potency against human lactate dehydrogenase A (LDHA). These potent inhibitors were typically selective for LDHA over LDHB isoform (4–10 fold) and other structurally related malate dehydrogenases, MDH1 and MDH2 (>500 fold). An X-ray crystal structure of enzymatically most potent molecule bound to LDHA revealed two additional interactions associated with enhanced biochemical potency.

Activating mutations within the epidermal growth factor receptor (EGFR) kinase domain, commonly L858R or deletions within exon 19, increase EGFR-driven cell proliferation and survival and are correlated with impressive responses to the EGFR inhibitors erlotinib and gefitinib in nonsmall cell lung cancer patients. Approximately 60% of acquired resistance to these agents is driven by a single secondary mutation within the EGFR kinase domain, specifically substitution of the gatekeeper residue threonine-790 with methionine (T790M). Due to dose-limiting toxicities associated with inhibition of wild-type EGFR (wtEGFR), we sought inhibitors of T790M-containing EGFR mutants with selectivity over wtEGFR. We describe the evolution of HTS hits derived from Jak2/Tyk2 inhibitors into selective EGFR inhibitors. X-ray crystal structures revealed two distinct binding modes and enabled the design of a selective series of novel diaminopyrimidine-based inhibitors with good potency against T790M-containing mutants of EGFR, high selectivity over wtEGFR, broad kinase selectivity, and desirable physicochemical properties.

A novel class of 3-hydroxy-2-mercaptocyclohex-2-enone-containing inhibitors of human lactate dehydrogenase (LDH) was identified through a high-throughput screening approach. Biochemical and surface plasmon resonance experiments performed with a screening hit (LDHA IC50 = 1.7 μM) indicated that the compound specifically associated with human LDHA in a manner that required simultaneous binding of the NADH co-factor. Structural variation of this screening hit resulted in significant improvements in LDHA biochemical inhibition activity (best IC50 = 0.18 μM). Two crystal structures of optimized compounds bound to human LDHA were obtained and explained many of the observed structure–activity relationships. In addition, an optimized inhibitor exhibited good pharmacokinetic properties after oral administration to rats (F = 45%).

A novel 2-thio-6-oxo-1,6-dihydropyrimidine-containing inhibitor of human lactate dehydrogenase (LDH) was identified by high-throughput screening (IC50=8.1 μM). Biochemical, surface plasmon resonance, and saturation transfer difference NMR experiments indicated that the compound specifically associated with human LDHA in a manner that required simultaneous binding of the NADH co-factor. Structural variation of the screening hit resulted in significant improvements in LDHA biochemical inhibition activity (best IC50=0.48 μM). A crystal structure of an optimized compound bound to human LDHA was obtained and explained many of the observed structure-activity relationships.

Lowering of intra-ocular pressure is the primary pharmacologic approach for the treatment of glaucoma and a number of distinct mechanisms of action have been clinically validated. Targeting of multiple mechanisms in combination therapies has proven effective both clinically and commercially although potential improvements with regards to efficacy, tolerability and dosing frequency remain. Application of Theravance's multivalent approach to drug discovery towards linked dual-pharmacology prostaglandin F receptor (FP) agonist/carbonic anhydrase (CA)-II inhibitor compounds is described. Compound 29 exhibits weak potency (pEC(50)=5.7, IA>1.0) as an FP agonist with high binding affinity (pK(i)=8.1) to the CA-II enzyme, and has comparable corneal permeability to the CA-II inhibitor dorzolamide.

Inhibitors targeting the activating mutants of the epidermal growth factor receptor (EGFR) have found success in the treatment of EGFR mutant positive non-small-cell lung cancer. A secondary point mutation (T790M) in the inhibitor binding site has been linked to the acquired resistance against those first generation therapeutics. Herein, we describe the lead optimization of a series of reversible, pan-mutant (L858R, del746–750, T790M/L858R, and T790M/del746–750) EGFR inhibitors. By use of a noncovalent double mutant (T790M/L858R and T790M/del746–750) selective EGFR inhibitor (2) as a starting point, activities against the single mutants (L858R and del746–750) were introduced through a series of structure-guided modifications. The in vitro ADME-PK properties of the lead molecules were further optimized through a number of rational structural changes. The resulting inhibitor (21) exhibited excellent cellular activity against both the single and double mutants of EGFR, demonstrating target engagement in vivo...

This Letter describes the discovery and key structure-activity relationship (SAR) of a series of 2-aminobenzimidazoles as potent Aurora kinase inhibitors. 2-Aminobenzimidazole serves as a bioisostere of the biaryl urea residue of SNS-314 (1c), which is a potent Aurora kinase inhibitor and entered clinical testing in patients with solid tumors. Compared to SNS-314, this series of compounds offers better aqueous solubility while retaining comparable in vitro potency in biochemical and cell-based assays; in particular, 6m has also demonstrated a comparable mouse iv PK profile to SNS-314.

The treatment of epidermal growth factor receptor (EGFR)-driven non-small cell lung cancers with the T790M resistance mutation remains a significant unmet medical need. We report the identification of 4-aminoindazolyl-dihydrofuro[3,4-d]pyrimidines as non-covalent inhibitors of EGFR, with excellent activity against the T790M resistance double mutants and initial single activating mutants. Using an optimization strategy focused on structure-based design and improving PK properties through metabolite identification, we obtained advanced leads with high oral exposure.

Metabolic reprogramming in tumors represents a potential therapeutic target. Herein we used shRNA depletion and a novel lactate dehydrogenase (LDHA) inhibitor, GNE-140, to probe the role of LDHA in tumor growth in vitro and in vivo. In MIA PaCa-2 human pancreatic cells, LDHA inhibition rapidly affected global metabolism, although cell death only occurred after 2 d of continuous LDHA inhibition. Pancreatic cell lines that utilize oxidative phosphorylation (OXPHOS) rather than glycolysis were inherently resistant to GNE-140, but could be resensitized to GNE-140 with the OXPHOS inhibitor phenformin. Acquired resistance to GNE-140 was driven by activation of the AMPK-mTOR-S6K signaling pathway, which led to increased OXPHOS, and inhibitors targeting this pathway could prevent resistance. Thus, combining an LDHA inhibitor with compounds targeting the mitochondrial or AMPK-S6K signaling axis may not only broaden the clinical utility of LDHA inhibitors beyond glycolytically dependent tumors but also reduce the emergence of resistance to LDHA inhibition.

SummaryPolychlorinated biphenyls (PCBs) and their hydroxylated metabolites (OH-PCBs) are known to bind to transthyretin (TTR) in vitro, possibly explaining their bioaccumulation, rodent toxicity, and presumed human toxicity. Herein, we show that several OH-PCBs bind selectively to TTR in blood plasma; however, only one of the PCBs tested binds TTR in plasma. Some of the OH-PCBs displace thyroid hormone (T4) from TTR, rationalizing the toxicity observed in rodents, where TTR is the major T4 transporter. Thyroid binding globulin and albumin are the major T4 carriers in humans, making it unlikely that enough T4 could be displaced from TTR to be toxic. OH-PCBs are excellent TTR amyloidogenesis inhibitors in vitro because they bind to the TTR tetramer, imparting kinetic stability under amyloidogenic denaturing conditions. Four OH-PCB/TTR cocrystal structures provide further insight into inhibitor binding interactions.

Tetrameric transthyretin is involved in transport of thyroxine and, through its interactions with retinol binding protein, vitamin A. Dissociation of these structures is widely accepted as the first step in the formation of transthyretin amyloid fibrils. Using a mass spectrometric approach, we have examined a series of 18 ligands proposed as inhibitors of this process. The ligands were evaluated for their ability to bind to and stabilize the tetrameric structure, their cooperativity in binding, and their ability to compete with the natural ligand thyroxine. The observation of a novel ten-component complex containing six protein subunits, two vitamin molecules, and two synthetic ligands allows us to conclude that ligand binding does not inhibit association of transthyretin with holo retinol binding protein.

A series of 3,6-disubstituted dihydropyrones were identified as inhibitors of human lactate dehydrogenase (LDH)-A. Structure activity relationships were explored and a series of 6,6-spiro analogs led to improvements in LDHA potency (IC50

The development of 1,3,4,4a,5,10a-hexahydropyrano[3,4-b]chromene analogs as BACE1 inhibitors is described. Introduction of the spirocyclic pyranochromene scaffold yielded several advantages over previous generation cores, including increased potency, reduced efflux, and reduced CYP2D6 inhibition. Compound 13 (BACE1 IC50 = 110 nM) demonstrated a reduction in CSF Aβ in wild type rats after a single dose.

Eight small molecules were synthesized to evaluate the structure activity relationships (SAR) of N-substituted anthranilic acids. The molecules were synthesized by benzylation or arylation of methyl anthranilate. A light scattering-based amyloid fibril formation assay was used to evaluate potential inhibitors of transthyretin (TTR) amyloid fibril formation in vitro. The m-carboxyphenylated and o-trifluoromethylphenylated anthranilic acids are potent inhibitors that will be subjected to further SAR and structural analysis.

A pyridazin-4-one fragment 4 (hCatS IC50 = 170 μM) discovered through Tethering was modeled into cathepsin S and predicted to overlap in S2 with the tetrahydropyridinepyrazole core of a previously disclosed series of CatS inhibitors. This fragment served as a template to design pyridazin-3-one 12 (hCatS IC50 = 430 nM), which also incorporates P3 and P5 binding elements. A crystal structure of 12 bound to Cys25Ser CatS led to the synthesis of the potent diazinone isomers 22 (hCatS IC50 = 60 nM) and 27 (hCatS IC50 = 40 nM).

A series of 2-amino-pyrazolopyridines was designed and synthesized as Polo-like kinase (Plk) inhibitors based on a low micromolar hit. The SAR was developed to provide compounds exhibiting low nanomolar inhibitory activity of Plk1; the phenotype of treated cells is consistent with Plk1 inhibition. A co-crystal structure of one of these compounds with zPlk1 confirms an ATP-competitive binding mode.

Ten oxazoles bearing a C(4) carboxyl group were synthesized and evaluated as transthyretin (TTR) amyloid fibril inhibitors. Substituting aryls at the C(2) position of the oxazole ring reveals that a 3,5-dichlorophenyl substituent significantly reduced amyloidogenesis. The efficacy of these inhibitors was enhanced further by installing an ethyl, a propyl, or a CF(3) group at the C(5) position. The CF(3) substitution at C(5) also improves the TTR binding selectivity over all the other proteins in human blood.