Development of Selective Phosphatidylinositol 5-Phosphate 4-Kinase γ Inhibitors with a Non-ATP-competitive, Allosteric Binding Mode.

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are emerging as attractive therapeutic targets in diseases, such as cancer, immunological disorders, and neurodegeneration, owing to their central role in regulating cell signaling pathways that are either dysfunctional or can be modulated to promote cell survival. Different modes of binding may enhance inhibitor selectivity and reduce off-target effects in cells. Here, we describe efforts to improve the physicochemical properties of the selective PI5P4Kγ inhibitor, NIH-12848 (1). These improvements enabled the demonstration that this chemotype engages PI5P4Kγ in intact cells and that compounds from this series do not inhibit PI5P4Kα or PI5P4Kß. Furthermore, the first X-ray structure of PI5P4Kγ bound to an inhibitor has been determined with this chemotype, confirming an allosteric binding mode. An exemplar from this chemical series adopted two distinct modes of inhibition, including through binding to a putative lipid interaction site which is 18 Å from the ATP pocket.

Alkynyl Benzoxazines and Dihydroquinazolines as Cysteine Targeting Covalent Warheads and Their Application in Identification of Selective Irreversible Kinase Inhibitors.

With a resurgence in interest in covalent drugs, there is a need to identify new moieties capable of cysteine bond formation that are differentiated from commonly employed systems such as acrylamide. Herein, we report on the discovery of new alkynyl benzoxazine and dihydroquinazoline moieties capable of covalent reaction with cysteine. Their utility as alternative electrophilic warheads for chemical biological probes and drug molecules is demonstrated through site-selective protein modification and incorporation into kinase drug scaffolds. A potent covalent inhibitor of JAK3 kinase was identified with superior selectivity across the kinome and improvements in in vitro pharmacokinetic profile relative to the related acrylamide-based inhibitor. In addition, the use of a novel heterocycle as a cysteine reactive warhead is employed to target Cys788 in c-KIT, where acrylamide has previously failed to form covalent interactions. These new reactive and selective heterocyclic warheads supplement the current repertoire for cysteine covalent modification while avoiding some of the limitations generally associated with established moieties.

Structure-Based Design and Pharmacokinetic Optimization of Covalent Allosteric Inhibitors of the Mutant GTPase KRASG12C.

Attempts to directly drug the important oncogene KRAS have met with limited success despite numerous efforts across industry and academia. The KRASG12C mutant represents an "Achilles heel" and has recently yielded to covalent targeting with small molecules that bind the mutant cysteine and create an allosteric pocket on GDP-bound RAS, locking it in an inactive state. A weak inhibitor at this site was optimized through conformational locking of a piperazine-quinazoline motif and linker modification. Subsequent introduction of a key methyl group to the piperazine resulted in enhancements in potency, permeability, clearance, and reactivity, leading to identification of a potent KRASG12C inhibitor with high selectivity and excellent cross-species pharmacokinetic parameters and in vivo efficacy.

Heparan sulfates are critical regulators of the inhibitory megakaryocyte-platelet receptor G6b-B.

The immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B is critical for platelet production and activation. Loss of G6b-B results in severe macrothrombocytopenia, myelofibrosis and aberrant platelet function in mice and humans. Using a combination of immunohistochemistry, affinity chromatography and proteomics, we identified the extracellular matrix heparan sulfate (HS) proteoglycan perlecan as a G6b-B binding partner. Subsequent in vitro biochemical studies and a cell-based genetic screen demonstrated that the interaction is specifically mediated by the HS chains of perlecan. Biophysical analysis revealed that heparin forms a high-affinity complex with G6b-B and mediates dimerization. Using platelets from humans and genetically modified mice, we demonstrate that binding of G6b-B to HS and multivalent heparin inhibits platelet and megakaryocyte function by inducing downstream signaling via the tyrosine phosphatases Shp1 and Shp2. Our findings provide novel insights into how G6b-B is regulated and contribute to our understanding of the interaction of megakaryocytes and platelets with glycans.

Discovery of N-(4-{[5-Fluoro-7-(2-methoxyethoxy)quinazolin-4-yl]amino}phenyl)-2-[4-(propan-2-yl)-1 H-1,2,3-triazol-1-yl]acetamide (AZD3229), a Potent Pan-KIT Mutant Inhibitor for the Treatment of Gastrointestinal Stromal Tumors.

While the treatment of gastrointestinal stromal tumors (GISTs) has been revolutionized by the application of targeted tyrosine kinase inhibitors capable of inhibiting KIT-driven proliferation, diverse mutations to this kinase drive resistance to established therapies. Here we describe the identification of potent pan-KIT mutant kinase inhibitors that can be dosed without being limited by the tolerability issues seen with multitargeted agents. This effort focused on identification and optimization of an existing kinase scaffold through the use of structure-based design. Starting from a series of previously reported phenoxyquinazoline and quinoline based inhibitors of the tyrosine kinase PDGFRα, potency against a diverse panel of mutant KIT driven Ba/F3 cell lines was optimized, with a particular focus on reducing activity against a KDR driven cell model in order to limit the potential for hypertension commonly seen in second and third line GIST therapies. AZD3229 demonstrates potent single digit nM growth inhibition across a broad cell panel, with good margin to KDR-driven effects. Selectivity over KDR can be rationalized predominantly by the interaction of water molecules with the protein and ligand in the active site, and its kinome selectivity is similar to the best of the approved GIST agents. This compound demonstrates excellent cross-species pharmacokinetics, shows strong pharmacodynamic inhibition of target, and is active in several in vivo models of GIST.

Potent and selective bivalent inhibitors of BET bromodomains.

Proteins of the bromodomain and extraterminal (BET) family, in particular bromodomain-containing protein 4 (BRD4), are of great interest as biological targets. BET proteins contain two separate bromodomains, and existing inhibitors bind to them monovalently. Here we describe the discovery and characterization of probe compound biBET, capable of engaging both bromodomains simultaneously in a bivalent, in cis binding mode. The evidence provided here was obtained in a variety of biophysical and cellular experiments. The bivalent binding results in very high cellular potency for BRD4 binding and pharmacological responses such as disruption of BRD4-mediator complex subunit 1 foci with an EC50 of 100 pM. These compounds will be of considerable utility as BET/BRD4 chemical probes. This work illustrates a novel concept in ligand design-simultaneous targeting of two separate domains with a drug-like small molecule-providing precedent for a potentially more effective paradigm for developing ligands for other multi-domain proteins.

Discovery of a novel allosteric inhibitor-binding site in ERK5: comparison with the canonical kinase hinge ATP-binding site.

MAP kinases act as an integration point for multiple biochemical signals and are involved in a wide variety of cellular processes such as proliferation, differentiation, regulation of transcription and development. As a member of the MAP kinase family, ERK5 (MAPK7) is involved in the downstream signalling pathways of various cell-surface receptors, including receptor tyrosine kinases and G protein-coupled receptors. In the current study, five structures of the ERK5 kinase domain co-crystallized with ERK5 inhibitors are reported. Interestingly, three of the compounds bind at a novel allosteric binding site in ERK5, while the other two bind at the typical ATP-binding site. Binding of inhibitors at the allosteric site is accompanied by displacement of the P-loop into the ATP-binding site and is shown to be ATP-competitive in an enzymatic assay of ERK5 kinase activity. Kinase selectivity data show that the most potent allosteric inhibitor exhibits superior kinase selectivity compared with the two inhibitors that bind at the canonical ATP-binding site. An analysis of these structures and comparison with both a previously published ERK5-inhibitor complex structure (PDB entry 4b99) and the structures of three other kinases (CDK2, ITK and MEK) in complex with allosteric inhibitors are presented.

Landscape of activating cancer mutations in FGFR kinases and their differential responses to inhibitors in clinical use.

Frequent genetic alterations discovered in FGFRs and evidence implicating some as drivers in diverse tumors has been accompanied by rapid progress in targeting FGFRs for anticancer treatments. Wider assessment of the impact of genetic changes on the activation state and drug responses is needed to better link the genomic data and treatment options. We here apply a direct comparative and comprehensive analysis of FGFR3 kinase domain variants representing the diversity of point-mutations reported in this domain. We reinforce the importance of N540K and K650E and establish that not all highly activating mutations (for example R669G) occur at high-frequency and conversely, that some "hotspots" may not be linked to activation. Further structural characterization consolidates a mechanistic view of FGFR kinase activation and extends insights into drug binding. Importantly, using several inhibitors of particular clinical interest (AZD4547, BGJ-398, TKI258, JNJ42756493 and AP24534), we find that some activating mutations (including different replacements of the same residue) result in distinct changes in their efficacy. Considering that there is no approved inhibitor for anticancer treatments based on FGFR-targeting, this information will be immediately translatable to ongoing clinical trials.

Pyrimidinone nicotinamide mimetics as selective tankyrase and wnt pathway inhibitors suitable for in vivo pharmacology.

The canonical Wnt pathway plays an important role in embryonic development, adult tissue homeostasis, and cancer. Germline mutations of several Wnt pathway components, such as Axin, APC, and ß-catenin, can lead to oncogenesis. Inhibition of the poly(ADP-ribose) polymerase (PARP) catalytic domain of the tankyrases (TNKS1 and TNKS2) is known to inhibit the Wnt pathway via increased stabilization of Axin. In order to explore the consequences of tankyrase and Wnt pathway inhibition in preclinical models of cancer and its impact on normal tissue, we sought a small molecule inhibitor of TNKS1/2 with suitable physicochemical properties and pharmacokinetics for hypothesis testing in vivo. Starting from a 2-phenyl quinazolinone hit (compound 1), we discovered the pyrrolopyrimidinone compound 25 (AZ6102), which is a potent TNKS1/2 inhibitor that has 100-fold selectivity against other PARP family enzymes and shows 5 nM Wnt pathway inhibition in DLD-1 cells. Moreover, compound 25 can be formulated well in a clinically relevant intravenous solution at 20 mg/mL, has demonstrated good pharmacokinetics in preclinical species, and shows low Caco2 efflux to avoid possible tumor resistance mechanisms.

Investigation of potential bioisosteric replacements for the carboxyl groups of peptidomimetic inhibitors of protein tyrosine phosphatase 1B: identification of a tetrazole-containing inhibitor with cellular activity.

Protein tyrosine phosphatases (PTPs) constitute a diverse family of enzymes that, together with protein tyrosine kinases, control the level of intracellular tyrosine phosphorylation, thus regulating many cellular functions. PTP1B negatively regulates insulin signaling, in part, by dephosphorylating key tyrosine residues within the regulatory domain of the beta-subunit of the insulin receptor, thereby attenuating receptor kinase activity. Inhibitors of PTP1B would therefore have the potential of prolonging the phosphorylated (activated) state of the insulin receptor and are anticipated to be a novel treatment of the insulin resistance characteristic of type 2 diabetes. We previously reported a series of small molecular weight peptidomimetics as competitive inhibitors of PTP1B, with the most active analogues having K(i) values in the low nanomolar range. Furthermore, we confirmed that the O-carboxymethyl salicylic acid moiety is a remarkably effective novel phosphotyrosine mimetic. Because of the low cell permeability of this compound class, it was important to investigate the possibility of replacing one or both of the remaining carboxyl groups while maintaining PTP1B inhibitory activity. The analogues described herein further support the importance of an acidic functionality at both positions of the tyrosine head moiety. An important discovery was the ortho tetrazole analogue 29 (K(i) = 2.0 microM), which was equipotent to the dicarboxylic acid analogue 2 (K(i) = 2.0 microM). Solution of the X-ray cocrystal structure of the ortho tetrazole analogue 29 bound to PTP1B revealed that the tetrazole moiety is well-accommodated in the active site and binds in a fashion similar to the ortho carboxylate analogue 2 reported previously. This novel monocarboxylic acid analogue revealed significantly higher Caco-2 cell permeability as compared to all previous compounds. Furthermore, compound 29 exhibited modest enhancement of insulin-stimulated 2-deoxyglucose uptake by L6 myocytes.

Synthesis and biological activity of a novel class of small molecular weight peptidomimetic competitive inhibitors of protein tyrosine phosphatase 1B.

Protein tyrosine phosphatase 1B (PTP1B) negatively regulates insulin signaling in part by dephosphorylating key tyrosine residues within the regulatory domain of the beta-subunit of the insulin receptor (IR), thereby attenuating receptor tyrosine kinase activity. Inhibition of PTP1B is therefore anticipated to improve insulin resistance and has recently become the focus of discovery efforts aimed at identifying new drugs to treat type II diabetes. We previously reported that the tripeptide Ac-Asp-Tyr(SO(3)H)-Nle-NH(2) is a surprisingly effective inhibitor of PTP1B (K(i) = 5 microM). With the goal of improving the stability and potency of this lead, as well as attenuating its peptidic character, an analogue program was undertaken. Specific elements of the initial phase of this program included replacement of the N- and C-termini with non-amino acid components, modification of the tyrosine subunit, and replacement of the tyrosine sulfate with other potential phosphate mimics. The most potent analogue arising from this effort was triacid 71, which inhibits PTP1B competitively with a K(i) = 0.22 microM without inhibiting SHP-2 or LAR at concentrations up to 100 microM. Overall, the inhibitors generated in this work showed little or no enhancement of insulin signaling in cellular assays. However, potential prodrug triester 70 did induce enhancements in 2-deoxyglucose uptake into two different cell lines with concomitant augmentation of the tyrosine phosphorylation levels of insulin-signaling molecules. Key elements of the overall SAR reported herein include confirmation of the effectiveness and remarkable PTP1B-specificity of the novel tyrosine phosphate bioisostere, O-carboxymethyl salicylic acid; demonstration that the tyrosine skeleton is optimal relative to closely related structures; replacement of the p-1 aspartic acid with phenylalanine with little effect on activity; and demonstration that inhibitory activity can be maintained in the absence of an N-terminal carboxylic acid. An X-ray cocrystal structure of an analogue bearing a neutral N-terminus (69) bound to PTP1B is reported that confirms a mode of binding similar to that of peptidic substrates.