Identification of GDC-1971 (RLY-1971), a SHP2 Inhibitor Designed for the Treatment of Solid Tumors.

Protein tyrosine phosphatase SHP2 mediates RAS-driven MAPK signaling and has emerged in recent years as a target of interest in oncology, both for treating with a single agent and in combination with a KRAS inhibitor. We were drawn to the pharmacological potential of SHP2 inhibition, especially following the initial observation that drug-like compounds could bind an allosteric site and enforce a closed, inactive state of the enzyme. Here, we describe the identification and characterization of GDC-1971 (formerly RLY-1971), a SHP2 inhibitor currently in clinical trials in combination with KRAS G12C inhibitor divarasib (GDC-6036) for the treatment of solid tumors driven by a KRAS G12C mutation.

Targeting KRAS Mutant Cancers via Combination Treatment: Discovery of a Pyridopyridazinone pan-RAF Kinase Inhibitor.

Structure-based optimization of a set of aryl urea RAF inhibitors has led to the identification of Type II pan-RAF inhibitor GNE-9815 (7), which features a unique pyrido[2,3-d]pyridazin-8(7H)-one hinge-binding motif. With minimal polar hinge contacts, the pyridopyridazinone hinge binder moiety affords exquisite kinase selectivity in a lipophilic efficient manner. The improved physicochemical properties of GNE-9815 provided a path for oral dosing without enabling formulations. In vivo evaluation of GNE-9815 in combination with the MEK inhibitor cobimetinib demonstrated synergistic MAPK pathway modulation in an HCT116 xenograft mouse model. To the best of our knowledge, GNE-9815 is among the most highly kinase-selective RAF inhibitors reported to date.

Targeting KRAS Mutant Cancers via Combination Treatment: Discovery of a 5-Fluoro-4-(3H)-quinazolinone Aryl Urea pan-RAF Kinase Inhibitor.

Optimization of a series of aryl urea RAF inhibitors led to the identification of type II pan-RAF inhibitor GNE-0749 (7), which features a fluoroquinazolinone hinge-binding motif. By minimizing reliance on common polar hinge contacts, this hinge binder allows for a greater contribution of RAF-specific residue interactions, resulting in exquisite kinase selectivity. Strategic substitution of fluorine at the C5 position efficiently masked the adjacent polar NH functionality and increased solubility by impeding a solid-state conformation associated with stronger crystal packing of the molecule. The resulting improvements in permeability and solubility enabled oral dosing of 7. In vivo evaluation of 7 in combination with the MEK inhibitor cobimetinib demonstrated synergistic pathway inhibition and significant tumor growth inhibition in a KRAS mutant xenograft mouse model.

GNE-781, A Highly Advanced Potent and Selective Bromodomain Inhibitor of Cyclic Adenosine Monophosphate Response Element Binding Protein, Binding Protein (CBP).

Inhibition of the bromodomain of the transcriptional regulator CBP/P300 is an especially interesting new therapeutic approach in oncology. We recently disclosed in vivo chemical tool 1 (GNE-272) for the bromodomain of CBP that was moderately potent and selective over BRD4(1). In pursuit of a more potent and selective CBP inhibitor, we used structure-based design. Constraining the aniline of 1 into a tetrahydroquinoline motif maintained potency and increased selectivity 2-fold. Structure-activity relationship studies coupled with further structure-based design targeting the LPF shelf, BC loop, and KAc regions allowed us to significantly increase potency and selectivity, resulting in the identification of non-CNS penetrant 19 (GNE-781, TR-FRET IC50 = 0.94 nM, BRET IC50 = 6.2 nM; BRD4(1) IC50 = 5100 nΜ) that maintained good in vivo PK properties in multiple species. Compound 19 displays antitumor activity in an AML tumor model and was also shown to decrease Foxp3 transcript levels in a dose dependent manner.

Discovery of a Potent and Selective in Vivo Probe (GNE-272) for the Bromodomains of CBP/EP300.

The single bromodomain of the closely related transcriptional regulators CBP/EP300 is a target of much recent interest in cancer and immune system regulation. A co-crystal structure of a ligand-efficient screening hit and the CBP bromodomain guided initial design targeting the LPF shelf, ZA loop, and acetylated lysine binding regions. Structure-activity relationship studies allowed us to identify a more potent analogue. Optimization of permeability and microsomal stability and subsequent improvement of mouse hepatocyte stability afforded 59 (GNE-272, TR-FRET IC50 = 0.02 µM, BRET IC50 = 0.41 µM, BRD4(1) IC50 = 13 µM) that retained the best balance of cell potency, selectivity, and in vivo PK. Compound 59 showed a marked antiproliferative effect in hematologic cancer cell lines and modulates MYC expression in vivo that corresponds with antitumor activity in an AML tumor model.

A high-content cellular senescence screen identifies candidate tumor suppressors, including EPHA3.

Activation of a cellular senescence program is a common response to prolonged oncogene activation or tumor suppressor loss, providing a physiological mechanism for tumor suppression in premalignant cells. The link between senescence and tumor suppression supports the hypothesis that a loss-of-function screen measuring bona fide senescence marker activation should identify candidate tumor suppressors. Using a high-content siRNA screening assay for cell morphology and proliferation measures, we identify 12 senescence-regulating kinases and determine their senescence marker signatures, including elevation of senescence-associated ß-galactosidase, DNA damage and p53 or p16 (INK4a) expression. Consistent with our hypothesis, SNP array CGH data supports loss of gene copy number of five senescence-suppressing genes across multiple tumor samples. One such candidate is the EPHA3 receptor tyrosine kinase, a gene commonly mutated in human cancer. We demonstrate that selected intracellular EPHA3 tumor-associated point mutations decrease receptor expression level and/or receptor tyrosine kinase (RTK) activity. Our study therefore describes a new strategy to mine for novel candidate tumor suppressors and provides compelling evidence that EPHA3 mutations may promote tumorigenesis only when key senescence-inducing pathways have been inactivated.

Distinct domains within Mash1 and Math1 are required for function in neuronal differentiation versus neuronal cell-type specification.

Many members of the basic helix-loop-helix (bHLH) family of transcription factors play pivotal roles in the development of a variety of tissues and organisms. We identify activities for the neural bHLH proteins Mash1 and Math1 in inducing neuronal differentiation, and in inducing the formation of distinct dorsal interneuron subtypes in the chick neural tube. Although both factors induce neuronal differentiation, each factor has a distinct activity in the type of dorsal interneuron that forms, with overexpression of Math1 increasing dI1 interneurons, and Mash1 increasing dI3 interneurons. Math1 and Mash1 function as transcriptional activators for both of these functions. Furthermore, we define discrete domains within the bHLH motif that are required for these different activities in neural development. Helix 1 of the Mash1 HLH domain is necessary for Mash1 to be able to promote neuronal differentiation, and is sufficient to confer this activity to the non-neural bHLH factor MyoD. In contrast, helix 2 of Math1, and both helix 1 and 2 of Mash1, are the domains required for the neuronal specification activities of these factors. The requirement for distinct domains within the HLH motif of Mash1 and Math1 for driving neuronal differentiation and cell-type specification probably reflects the importance of unique protein-protein interactions involved in these functions.

Zic1 represses Math1 expression via interactions with the Math1 enhancer and modulation of Math1 autoregulation.

Math1 is a basic helix-loop-helix transcription factor expressed in progenitor cells that give rise to dorsal commissural interneurons in the spinal cord, granule cells of the cerebellum, and sensory cells in the inner ear and skin. Transcriptional regulation of this gene is tightly controlled both temporally and spatially during nervous system development. The signals that mediate this regulation are likely integrated at the Math1 enhancer, which is highly conserved among vertebrate species. We have identified the zinc-finger transcription factor Zic1 as a regulator of Math1 expression. Zic1 binds a novel conserved site within the Math1 enhancer, and represses both the expression of endogenous Cath1 (chicken homolog of Math1) and the activity of a Math1 enhancer driven lacZ reporter when expressed in chick neural tubes. Repression by Zic1 blocks the autoregulatory activity of Math1 itself. Although previous reports have shown that Zic1 and Math1 are both induced by BMP signaling, these genes appear to have opposing functions, as Math1 acts to promote neuronal differentiation in the chick neural tube and excess Zic1 appears to block differentiation. Zic1-mediated repression of Cath1 transcription may modulate the temporal switch between the progenitor state and differentiating dorsal cell types during neural tube development.