Discovery of lirafugratinib (RLY-4008), a highly selective irreversible small-molecule inhibitor of FGFR2.

Fibroblast growth factor receptor (FGFR) kinase inhibitors have been shown to be effective in the treatment of intrahepatic cholangiocarcinoma and other advanced solid tumors harboring FGFR2 alterations, but the toxicity of these drugs frequently leads to dose reduction or interruption of treatment such that maximum efficacy cannot be achieved. The most common adverse effects are hyperphosphatemia caused by FGFR1 inhibition and diarrhea due to FGFR4 inhibition, as current therapies are not selective among the FGFRs. Designing selective inhibitors has proved difficult with conventional approaches because the orthosteric sites of FGFR family members are observed to be highly similar in X-ray structures. In this study, aided by analysis of protein dynamics, we designed a selective, covalent FGFR2 inhibitor. In a key initial step, analysis of long-timescale molecular dynamics simulations of the FGFR1 and FGFR2 kinase domains allowed us to identify differential motion in their P-loops, which are located adjacent to the orthosteric site. Using this insight, we were able to design orthosteric binders that selectively and covalently engage the P-loop of FGFR2. Our drug discovery efforts culminated in the development of lirafugratinib (RLY-4008), a covalent inhibitor of FGFR2 that shows substantial selectivity over FGFR1 (~250-fold) and FGFR4 (~5,000-fold) in vitro, causes tumor regression in multiple FGFR2-altered human xenograft models, and was recently demonstrated to be efficacious in the clinic at doses that do not induce clinically significant hyperphosphatemia or diarrhea.

Discovery and Clinical Proof-of-Concept of RLY-2608, a First-in-Class Mutant-Selective Allosteric PI3Kα Inhibitor That Decouples Antitumor Activity from Hyperinsulinemia.

PIK3CA (PI3Kα) is a lipid kinase commonly mutated in cancer, including ∼40% of hormone receptor-positive breast cancer. The most frequently observed mutants occur in the kinase and helical domains. Orthosteric PI3Kα inhibitors suffer from poor selectivity leading to undesirable side effects, most prominently hyperglycemia due to inhibition of wild-type (WT) PI3Kα. Here, we used molecular dynamics simulations and cryo-electron microscopy to identify an allosteric network that provides an explanation for how mutations favor PI3Kα activation. A DNA-encoded library screen leveraging electron microscopy-optimized constructs, differential enrichment, and an orthosteric-blocking compound led to the identification of RLY-2608, a first-in-class allosteric mutant-selective inhibitor of PI3Kα. RLY-2608 inhibited tumor growth in PIK3CA-mutant xenograft models with minimal impact on insulin, a marker of dysregulated glucose homeostasis. RLY-2608 elicited objective tumor responses in two patients diagnosed with advanced hormone receptor-positive breast cancer with kinase or helical domain PIK3CA mutations, with no observed WT PI3Kα-related toxicities. SIGNIFICANCE: Treatments for PIK3CA-mutant cancers are limited by toxicities associated with the inhibition of WT PI3Kα. Molecular dynamics, cryo-electron microscopy, and DNA-encoded libraries were used to develop RLY-2608, a first-in-class inhibitor that demonstrates mutant selectivity in patients. This marks the advance of clinical mutant-selective inhibition that overcomes limitations of orthosteric PI3Kα inhibitors. See related commentary by Gong and Vanhaesebroeck, p. 204 . See related article by Varkaris et al., p. 227 . This article is featured in Selected Articles from This Issue, p. 201.

Which Small Molecule? Selecting Chemical Probes for Use in Cancer Research and Target Validation.

Small-molecule chemical "probes" complement the use of molecular biology techniques to explore, validate, and generate hypotheses on the function of proteins in diseases such as cancer. Unfortunately, the poor selection and use of small-molecule reagents can lead to incorrect conclusions. Here, we illustrate examples of poor chemical tools and suggest best practices for the selection, validation, and use of high-quality chemical probes in cancer research. We also note the complexity associated with tools for novel drug modalities, exemplified by protein degraders, and provide advice and resources to facilitate the independent identification of appropriate small-molecule probes by researchers. SIGNIFICANCE: Validation of biological targets and pathways will be aided by a shared understanding of the criteria of potency, selectivity, and target engagement associated with small-molecule reagents ("chemical probes") that enable that work. Interdisciplinary collaboration between cancer biologists, medicinal chemists, and chemical biologists and the awareness of available resources will reduce misleading data generation and interpretation, strengthen data robustness, and improve productivity in academic and industrial research.

Expanding Chemical Probe Space: Quality Criteria for Covalent and Degrader Probes.

Within druggable target space, new small-molecule modalities, particularly covalent inhibitors and targeted degraders, have expanded the repertoire of medicinal chemists. Molecules with such modes of action have a large potential not only as drugs but also as chemical probes. Criteria have previously been established to describe the potency, selectivity, and properties of small-molecule probes that are qualified to enable the interrogation and validation of drug targets. These definitions have been tailored to reversibly acting modulators but fall short in their applicability to other modalities. While initial guidelines have been proposed, we delineate here a full set of criteria for the characterization of covalent, irreversible inhibitors as well as heterobifunctional degraders ("proteolysis-targeting chimeras", or PROTACs) and molecular glue degraders. We propose modified potency and selectivity criteria compared to those for reversible inhibitors. We discuss their relevance and highlight examples of suitable probe and pathfinder compounds.

Principles of Kinase Allosteric Inhibition and Pocket Validation.

With several marketed drugs, allosteric inhibition of kinases has translated to pharmacological effects and clinical benefits comparable to those from orthosteric inhibition. However, despite much effort over more than 20 years, the number of kinase targets associated with FDA-approved allosteric drugs is limited, suggesting the challenges in identifying and validating allosteric inhibitors. Here we review the principles of allosteric inhibition, summarize the discovery of allosteric MEK1/2 and BCR-ABL1 inhibitors, and discuss the approaches to screening and demonstrating the functional activity of allosteric pocket ligands.

A First-in-Class, Highly Selective and Cell-Active Allosteric Inhibitor of Protein Arginine Methyltransferase 6.

Protein arginine methyltransferase 6 (PRMT6) catalyzes monomethylation and asymmetric dimethylation of arginine residues in various proteins, plays important roles in biological processes, and is associated with multiple cancers. To date, a highly selective PRMT6 inhibitor has not been reported. Here we report the discovery and characterization of a first-in-class, highly selective allosteric inhibitor of PRMT6, (R)-2 (SGC6870). (R)-2 is a potent PRMT6 inhibitor (IC50 = 77 ± 6 nM) with outstanding selectivity for PRMT6 over a broad panel of other methyltransferases and nonepigenetic targets. Notably, the crystal structure of the PRMT6-(R)-2 complex and kinetic studies revealed (R)-2 binds a unique, induced allosteric pocket. Additionally, (R)-2 engages PRMT6 and potently inhibits its methyltransferase activity in cells. Moreover, (R)-2's enantiomer, (S)-2 (SGC6870N), is inactive against PRMT6 and can be utilized as a negative control. Collectively, (R)-2 is a well-characterized PRMT6 chemical probe and a valuable tool for further investigating PRMT6 functions in health and disease.

LLY-283, a Potent and Selective Inhibitor of Arginine Methyltransferase 5, PRMT5, with Antitumor Activity.

Protein arginine methyltransferase 5 (PRMT5) is a type II arginine methyltransferase that catalyzes the formation of symmetric dimethylarginine in a number of nuclear and cytoplasmic proteins. Although the cellular functions of PRMT5 have not been fully unraveled, it has been implicated in a number of cellular processes like RNA processing, signal transduction, and transcriptional regulation. PRMT5 is ubiquitously expressed in most tissues and its expression has been shown to be elevated in several cancers including breast cancer, gastric cancer, glioblastoma, and lymphoma. Here, we describe the identification and characterization of a novel and selective PRMT5 inhibitor with potent in vitro and in vivo activity. Compound 1 (also called LLY-283) inhibited PRMT5 enzymatic activity in vitro and in cells with IC50 of 22 ± 3 and 25 ± 1 nM, respectively, while its diastereomer, compound 2 (also called LLY-284), was much less active. Compound 1 also showed antitumor activity in mouse xenografts when dosed orally and can serve as an excellent probe molecule for understanding the biological function of PRMT5 in normal and cancer cells.

Characterization of LY3023414, a Novel PI3K/mTOR Dual Inhibitor Eliciting Transient Target Modulation to Impede Tumor Growth.

The PI3K/AKT/mTOR pathway is among the most frequently altered pathways in cancer cell growth and survival. LY3023414 is a complex fused imidazoquinolinone with high solubility across a wide pH range designed to inhibit class I PI3K isoforms and mTOR kinase. Here, we describe the in vitro and in vivo activity of LY3023414. LY3023414 was highly soluble at pH 2-7. In biochemical testing against approximately 266 kinases, LY3023414 potently and selectively inhibited class I PI3K isoforms, mTORC1/2, and DNA-PK at low nanomolar concentrations. In vitro, inhibition of PI3K/AKT/mTOR signaling by LY3023414 caused G1 cell-cycle arrest and resulted in broad antiproliferative activity in cancer cell panel screens. In vivo, LY3023414 demonstrated high bioavailability and dose-dependent dephosphorylation of PI3K/AKT/mTOR pathway downstream substrates such as AKT, S6K, S6RP, and 4E-BP1 for 4 to 6 hours, reflecting the drug's half-life of 2 hours. Of note, equivalent total daily doses of LY3023414 given either once daily or twice daily inhibited tumor growth to similar extents in multiple xenograft models, indicating that intermittent target inhibition is sufficient for antitumor activity. In combination with standard-of-care drugs, LY3023414 demonstrated additive antitumor activity. The novel, orally bioavailable PI3K/mTOR inhibitor LY3023414 is highly soluble and exhibits potent in vivo efficacy via intermittent target inhibition. It is currently being evaluated in phase I and II trials for the treatment of human malignancies. Mol Cancer Ther; 15(10); 2344-56. ©2016 AACR.

Beyond PAINs: Chemotype Sensitivity of Protein Methyltransferases in Screens.

Screening of the relatively new target class, the lysine and arginine methyltransferases (MTases), presents unique challenges in the identification and confirmation of active chemical matter. Examination of high throughput screening data generated using Scintillation Proximity Assay (SPA) format for a number of protein MTase targets reveals sensitivity to both the known pan assay interference compounds (PAINS) and also other scaffolds not currently precedented as assay interferers. We find that, in general, true actives show significant selectivity within the MTase family. With the exception of slight modifications of SAM-like compounds, scaffolds that are observed frequently in multiple MTase assays should be viewed with caution and should be carefully validated before following up.

Structure-guided expansion of kinase fragment libraries driven by support vector machine models.

This work outlines a new de novo design process for the creation of novel kinase inhibitor libraries. It relies on a profiling paradigm that generates a substantial amount of kinase inhibitor data from which highly predictive QSAR models can be constructed. In addition, a broad diversity of X-ray structure information is needed for binding mode prediction. This is important for scaffold and substituent site selection. Borrowing from FBDD, the process involves fragmentation of known actives, proposition of binding mode hypotheses for the fragments, and model-driven recombination using a pharmacophore derived from known kinase inhibitor structures. The support vector machine method, using Merck atom pair derived fingerprint descriptors, was used to build models from activity from 6 kinase assays. These models were qualified prospectively by selecting and testing compounds from the internal compound collection. Overall hit and enrichment rates of 82% and 2.5%, respectively, qualified the models for use in library design. Using the process, 7 novel libraries were designed, synthesized and tested against these same 6 kinases. The results showed excellent results, yielding a 92% hit rate for the 179 compounds that made up the 7 libraries. The results of one library designed to include known literature compounds, as well as an analysis of overall substituent frequency, are discussed.

Novel antiproliferative antitumor agents.

Small molecules with potent and selective antitumor activity continue to be identified by screening in cellular assays and to be entered into clinical development, and in some cases small molecules are progressed despite the fact that the mechanism of action is unknown. Current examples of drugs with unknown mechanism of action include LY-573636 (Eli Lilly & Co), CHS-828 and SPC-595 (Sunesis Pharmaceutical Co Ltd). Early discovery and the elucidation of structure-activity relationships may be in part facilitated by the fact that the compounds must be relatively soluble and permeable to cells to demonstrate activity, although the challenges of optimizing absorption, distribution, metabolism and excretion/pharmacokinetic properties, toxicity and clinical activity remain similar to compounds developed under the 'targeted therapy' paradigm.

Complete inhibition of anisomycin and UV radiation but not cytokine induced JNK and p38 activation by an aryl-substituted dihydropyrrolopyrazole quinoline and mixed lineage kinase 7 small interfering RNA.

Mixed lineage kinase 7 (MLK7) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the pro-apoptotic signaling pathways p38 and JNK. A library of potential kinase inhibitors was screened, and a series of dihydropyrrolopyrazole quinolines was identified as highly potent inhibitors of MLK7 in vitro catalytic activity. Of this series, an aryl-substituted dihydropyrrolopyrazole quinoline (DHP-2) demonstrated an IC50 of 70 nM for inhibition of pJNK formation in COS-7 cell MLK7/JNK co-transfection assays. In stimulated cells, DHP-2 at 200 nM or MLK7 small interfering RNA completely blocked anisomycin and UV induced but had no effect on interleukin-1beta or tumor necrosis factor-alpha-induced p38 and JNK activation. Additionally, the compound blocked anisomycin and UV-induced apoptosis in COS-7 cells. Heart tissue homogenates from MLK7 transgenic mice treated with DHP-2 at 30 mg/kg had reduced JNK and p38 activation with no apparent effect on ERK activation, demonstrating that this compound can be used to block MLK7-driven MAPK pathway activation in vivo. Taken together, these data demonstrate that MLK7 is the MAPKKK required for modulation of the stress-activated MAPKs downstream of anisomycin and UV stimulation and that DHP-2 can be used to block MLK7 pathway activation in cells as well as in vivo.

Acyl sulfonamide anti-proliferatives. Part 2: activity of heterocyclic sulfonamide derivatives.

The anti-proliferative activity of acylated heterocyclic sulfonamides is described in Vascular Endothelial Growth Factor-dependent Human Umbilical Vascular Endothelial Cells (VEGF-HUVEC) and in HCT116 tumor cells in a soft agar diffusion assay.

Acyl sulfonamide anti-proliferatives: benzene substituent structure-activity relationships for a novel class of antitumor agents.

Two closely related diaryl acylsulfonamides were recently reported as potent antitumor agents against a broad spectrum of human tumor xenografts (colon, lung, breast, ovary, and prostate) in nude mice. Especially intriguing was their activity against colorectal cancer xenografts. In this paper, rapid parallel synthesis along with traditional medicinal chemistry techniques were used to quickly delineate the structure-activity relationships of the substitution patterns in both phenyl rings of the acylsufonamide anti-proliferative scaffold. Although the molecular target of the compounds remains unclear, we determined that the vascular endothelial growth factor-dependent human umbilical vein endothelial cells assay in combination with a soft agar disk diffusion assay allowed for optimization of potency in the series. The pharmacokinetic properties and in vivo activity in an HCT116 xenograft model are reported for representative compounds.

Acyclic stereoselective boron alkylation reactions for the asymmetric synthesis of beta-substituted alpha-amino acid derivatives.

Optically active syn- or anti-beta-substituted-alpha-amino acid derivatives are prepared in 94 to >/=99% ee and 66-98% ds by reaction of the Schiff base acetate of glycine tert-butyl ester with chiral, nonracemic B-alkyl-9-BBN derivatives in the presence of the Cinchona alkaloid, cinchonidine (CdOH) or cinchonine (CnOH), base, and lithium chloride.

Computational investigation of the role of fluoride in Tamao oxidations.

The Tamao oxidation of alkoxysilanes was investigated computationally to determine the role of fluoride, a key additive, in this reaction. A sequence of fluoride equilibria as well as possible transition states, mediated by basic and neutral peroxide, respectively, were examined, and a potential energy surface was calculated which was consistent with the typical synthetic methods required for the conversion of alkoxysilanes to alcohols.

Solid-phase synthesis and investigation of benzofurans as selective estrogen receptor modulators.

A library of benzofurans was prepared by solid-phase synthesis methods, and several analogues were identified as potent ligands for the estrogen receptors ER-alpha and ER-beta, with some compounds having selectivity for ER-alpha. Analogues designed to more closely mimic Raloxifene were less effective. Certain benzofurans were effective in a bone pit assay, but were characterized as agonists in a MCF-7 breast tumor cell proliferation assay.