Identification of a novel series of azabenzimidazole-derived inhibitors of spleen tyrosine kinase.

Spleen Tyrosine Kinase (SYK) is a well-studied enzyme with therapeutic applications in oncology and autoimmune diseases. We identified an azabenzimidazole (ABI) series of SYK inhibitors by mining activity data of 86,000 compounds from legacy biochemical assays with SYK and other homologous kinases as target enzymes. A structure-based design and hybridization approach was then used to improve the potency and kinase selectivity of the hits. Lead compound 23 from this novel ABI series has a SYK IC50 = 0.21 nM in a biochemical assay and inhibits growth of SUDHL-4 cells at a GI50 = 210 nM.

Discovery of 2,6-disubstituted pyrazine derivatives as inhibitors of CK2 and PIM kinases.

The design and synthesis of a novel series of 2,6-disubstituted pyrazine derivatives as CK2 kinase inhibitors is described. Structure-guided optimization of a 5-substituted-3-thiophene carboxylic acid screening hit (3a) led to the development of a lead compound (12b), which shows inhibition in both enzymatic and cellular assays. Subsequent design and hybridization efforts also led to the unexpected identification of analogs with potent PIM kinase activity (14f).

Determining direct binders of the Androgen Receptor using a high-throughput Cellular Thermal Shift Assay.

Androgen Receptor (AR) is a key driver in prostate cancer. Direct targeting of AR has valuable therapeutic potential. However, the lack of disease relevant cellular methodologies capable of discriminating between inhibitors that directly bind AR and those that instead act on AR co-regulators has made identification of novel antagonists challenging. The Cellular Thermal Shift Assay (CETSA) is a technology enabling confirmation of direct target engagement with label-free, endogenous protein in living cells. We report the development of the first high-throughput CETSA assay (CETSA HT) to identify direct AR binders in a prostate cancer cell line endogenously expressing AR. Using this approach, we screened a pharmacology library containing both compounds reported to directly engage AR, and compounds expected to target AR co-regulators. Our results show that CETSA HT exclusively identifies direct AR binders, differentiating them from co-regulator inhibitors where other cellular assays measuring functional responses cannot. Using this CETSA HT approach we can derive apparent binding affinities for a range of AR antagonists, which represent an intracellular measure of antagonist-receptor Ki performed for the first time in a label-free, disease-relevant context. These results highlight the potential of CETSA HT to improve the success rates for novel therapeutic interventions directly targeting AR.

Discovery and Optimization of Pyrrolopyrimidine Inhibitors of Interleukin-1 Receptor Associated Kinase 4 (IRAK4) for the Treatment of Mutant MYD88L265P Diffuse Large B-Cell Lymphoma.

Herein we report the optimization of a series of pyrrolopyrimidine inhibitors of interleukin-1 receptor associated kinase 4 (IRAK4) using X-ray crystal structures and structure based design to identify and optimize our scaffold. Compound 28 demonstrated a favorable physicochemical and kinase selectivity profile and was identified as a promising in vivo tool with which to explore the role of IRAK4 inhibition in the treatment of mutant MYD88L265P diffuse large B-cell lymphoma (DLBCL). Compound 28 was shown to be capable of demonstrating inhibition of NF-κB activation and growth of the ABC subtype of DLBCL cell lines in vitro at high concentrations but showed greater effects in combination with a BTK inhibitor at lower concentrations. In vivo, the combination of compound 28 and ibrutinib led to tumor regression in an ABC-DLBCL mouse model.

Target Deconvolution Efforts on Wnt Pathway Screen Reveal Dual Modulation of Oxidative Phosphorylation and SERCA2.

Wnt signaling is critical for development, cell proliferation and differentiation, and mutations in this pathway resulting in constitutive signaling have been implicated in various cancers. A pathway screen using a Wnt-dependent reporter identified a chemical series based on a 1,2,3-thiadiazole-5-carboxamide (TDZ) core with sub-micromolar potency. Herein we report a comprehensive mechanism-of-action deconvolution study toward identifying the efficacy target(s) and biological implication of this chemical series involving bottom-up quantitative chemoproteomics, cell biology, and biochemical methods. Through observing the effects of our probes on metabolism and performing confirmatory cellular and biochemical assays, we found that this chemical series inhibits ATP synthesis by uncoupling the mitochondrial potential. Affinity chemoproteomics experiments identified sarco(endo)plasmic reticulum Ca2+ -dependent ATPase (SERCA2) as a binding partner of the TDZ series, and subsequent validation studies suggest that the TDZ series can act as ionophores through SERCA2 toward Wnt pathway inhibition.

Optimization of Highly Kinase Selective Bis-anilino Pyrimidine PAK1 Inhibitors.

Group I p21-activated kinase (PAK) inhibitors are indicated as important in cancer progression, but achieving high kinase selectivity has been challenging. A bis-anilino pyrimidine PAK1 inhibitor was identified and optimized through structure-based drug design to improve PAK1 potency and achieve high kinase selectivity, giving in vitro probe compound AZ13705339 (18). Reduction of lipophilicity to lower clearance afforded AZ13711265 (14) as an in vivo probe compound with oral exposure in mouse. Such probes will allow further investigation of PAK1 biology.

Potent and Selective CK2 Kinase Inhibitors with Effects on Wnt Pathway Signaling in Vivo.

The Wnt pathway is an evolutionarily conserved and tightly regulated signaling network with important roles in embryonic development and adult tissue regeneration. Impaired Wnt pathway regulation, arising from mutations in Wnt signaling components, such as Axin, APC, and ß-catenin, results in uncontrolled cell growth and triggers oncogenesis. To explore the reported link between CK2 kinase activity and Wnt pathway signaling, we sought to identify a potent, selective inhibitor of CK2 suitable for proof of concept studies in vivo. Starting from a pyrazolo[1,5-a]pyrimidine lead (2), we identified compound 7h, a potent CK2 inhibitor with picomolar affinity that is highly selectivity against other kinase family enzymes and inhibits Wnt pathway signaling (IC50 = 50 nM) in DLD-1 cells. In addition, compound 7h has physicochemical properties that are suitable for formulation as an intravenous solution, has demonstrated good pharmacokinetics in preclinical species, and exhibits a high level of activity as a monotherapy in HCT-116 and SW-620 xenografts.

Discovery of potent KIFC1 inhibitors using a method of integrated high-throughput synthesis and screening.

KIFC1 (HSET), a member of the kinesin-14 family of motor proteins, plays an essential role in centrosomal bundling in cancer cells, but its function is not required for normal diploid cell division. To explore the potential of KIFC1 as a therapeutic target for human cancers, a series of potent KIFC1 inhibitors featuring a phenylalanine scaffold was developed from hits identified through high-throughput screening (HTS). Optimization of the initial hits combined both design-synthesis-test cycles and an integrated high-throughput synthesis and biochemical screening method. An important aspect of this integrated method was the utilization of DMSO stock solutions of compounds registered in the corporate compound collection as synthetic reactants. Using this method, over 1500 compounds selected for structural diversity were quickly assembled in assay-ready 384-well plates and were directly tested after the necessary dilutions. Our efforts led to the discovery of a potent KIFC1 inhibitor, AZ82, which demonstrated the desired centrosome declustering mode of action in cell studies.

Structure and Property Based Design of Pyrazolo[1,5-a]pyrimidine Inhibitors of CK2 Kinase with Activity in Vivo.

In this letter, we describe the design, synthesis, and structure-activity relationship of 5-anilinopyrazolo[1,5-a]pyrimidine inhibitors of CK2 kinase. Property-based optimization of early leads using the 7-oxetan-3-yl amino group led to a series of matched molecular pairs with lower lipophilicity, decreased affinity for human plasma proteins, and reduced binding to the hERG ion channel. Agents in this study were shown to modulate pAKT(S129), a direct substrate of CK2, in vitro and in vivo, and exhibited tumor growth inhibition when administered orally in a murine DLD-1 xenograft.

Discovery of novel benzylidene-1,3-thiazolidine-2,4-diones as potent and selective inhibitors of the PIM-1, PIM-2, and PIM-3 protein kinases.

Novel substituted benzylidene-1,3-thiazolidine-2,4-diones (TZDs) have been identified as potent and highly selective inhibitors of the PIM kinases. The synthesis and SAR of these compounds are described, along with X-ray crystallographic, anti-proliferative, and selectivity data.

Potent and orally bioavailable 8-bicyclo[2.2.2]octylxanthines as adenosine A1 receptor antagonists.

In the search for a selective adenosine A1 receptor antagonist with greater aqueous solubility than the compounds currently in clinical trials as diuretics, a series of 1,4-substituted 8-cyclohexyl and 8-bicyclo[2.2.2]octylxanthines were investigated. The binding affinities of a variety of cyclohexyl and bicyclo[2.2.2]octylxanthines for the rat and human adenosine A1, A2A, A2B, and A3 receptors are presented. Bicyclo[2.2.2]octylxanthine 16 exhibited good pharmaceutical properties and in vivo activity in a rat diuresis model (ED50=0.3 mg/kg po). Optimization of the bridgehead substituent led to propionic acid 29 (BG9928), which retained high potency (hA1, Ki=7 nM) and selectivity for the adenosine A1 receptor (915-fold versus adenosine A2A receptor; 12-fold versus adenosine A2B receptor) with improved oral efficacy in the rat diuresis model (ED50=0.01 mg/kg) as well as high oral bioavailability in rat, dog, and cynomolgus monkey.

Synthesis of [1,2,4]triazolo[1,5-a]pyrazines as adenosine A2A receptor antagonists.

Potent and selective antagonists of the adenosine A2A receptor often contain a nitrogen-rich fused-ring heterocyclic core. Replacement of the core with an isomeric ring system has previously been shown to improve target affinity, selectivity, and in vivo activity. This paper describes the preparation, by a novel route, of A2A receptor antagonists containing the [1,2,4]triazolo[1,5-a]pyrazine nucleus, which is isomeric with the [1,2,4]triazolo[1,5-c]pyrimidine core of a series of known A2A antagonists with in vivo activity in animal models of Parkinson's disease.