BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by obliterative vascular remodeling of the small pulmonary arteries (PAs) and progressive increase in pulmonary vascular resistance leading to right ventricular failure. Although several drugs are approved for the treatment of PAH, mortality rates remain high. Accumulating evidence supports a pathological function of integrins in vessel remodeling, which are gaining renewed interest as drug targets. However, their role in PAH remains largely unexplored. METHODS: The expression of the RGD (arginylglycylaspartic acid)–binding integrin α5β1 was assessed in PAs, PA smooth muscle cells, and PA endothelial cells from patients with PAH and controls using NanoString, immunoblotting, and Mesoscale Discovery assays. RNA sequencing was conducted to identify gene networks regulated by α5β1 inhibition in PAH PA smooth muscle cells. The therapeutic efficacy of α5β1 inhibition was evaluated using a novel small molecule inhibitor and selective neutralizing antibodies in Sugen/hypoxia and monocrotaline rat models, with validation by an external contract research organization. Comparisons were made against standard-of-care therapies (ie, macitentan, tadalafil) and sotatercept and efficacy was assessed using echocardiographic, hemodynamic, and histological assessments. Ex vivo studies using human precision-cut lung slices were performed to further assess the effects of α5β1 inhibition on pulmonary vascular remodeling. RESULTS: We found that the arginine-glycine-aspartate RGD-binding integrin α5β1 is upregulated in PA endothelial cells and PA smooth muscle cells from patients with PAH and remodeled PAs from animal models. Blockade of the integrin α5β1 or depletion of the α5 subunit downregulated FOXM1 (forkhead box protein M1)–regulated gene networks, resulting in mitotic defects and inhibition of the pro-proliferative and apoptosis-resistant phenotype of PAH cells. We demonstrated that α5β1 integrin blockade safely attenuates pulmonary vascular remodeling and improves hemodynamics and right ventricular function and matched or exceeded the efficacy of standard of care and sotatercept in multiple preclinical models. Ex vivo studies further validated its potential in reversing advanced remodeling in human precision-cut lung slices. CONCLUSIONS: These findings establish α5β1 integrin as a pivotal driver of PAH pathology and we propose its inhibition as a novel, safe, and effective therapeutic strategy for PAH.

Inhibition of integrin αvβ6 is a promising approach to the treatment of fibrotic disease such as idiopathic pulmonary fibrosis. Screening a small library combining head groups that stabilize the bent-closed conformation of integrin αIIbβ3 with αv integrin binding motifs resulted in the identification of hit compounds that bind the bent-closed conformation of αvβ6. Crystal structures of these compounds bound to αvβ6 and related integrins revealed opportunities to increase potency and selectivity, and these efforts were accelerated using accurate free energy perturbation (FEP+) calculations. Optimization of PK parameters including permeability, bioavailability, clearance, and half-life resulted in the discovery of development candidate MORF-627, a highly selective inhibitor of αvβ6 that stabilizes the bent-closed conformation and has good oral PK. Unfortunately, the compound showed toxicity in a 28-day NHP safety study, precluding further development. Nevertheless, MORF-627 is a useful tool compound for studying the biology of integrin αvβ6.

Pulmonary arterial hypertension (PAH) is characterized by the narrowing of pulmonary arteries (PAs), causing right ventricular (RV) failure and premature death. Pulmonary artery smooth muscle cells (PASMCs) from PAH patients exhibit increased proliferation and resistance to apoptosis in response to extracellular matrix remodeling. Integrins are known to be involved in all these processes. Our hypothesis is that integrin signaling promotes PASMCs proliferation and apoptosis resistance, leading to PA remodeling, RV maladaptive hypertrophy and fibrosis, resulting in RV failure in PAH. Using NanoString, we identified members of the fibronectin-binding integrins (FnBIs) family as the most abundantly expressed in PAH-PASMCs. We confirmed the changes in FnBIs expression levels by Western blot (WB) in distal PAs, PASMCs and decompensated RVs from PAH patients compared to controls. Pharmacological inhibition of FnBIs decreased PAH-PASMCs proliferation (WB PLK1; Ki67), resistance to apoptosis (WB Survivin; Annexin V) and was associated with a decreased activation of FnBIs downstream signaling pathways FAK and ILK. In adult rat cardiomyocytes and human RV fibroblasts (RVFbs), FnBIs inhibition decreased phenylephrine-induced hypertrophy (f-actin labelling) and TGFβ1-induced RVFbs activation (WB αSMA, COL1). In both monocrotaline and sugen/hypoxia rats, pharmacological inhibition of FnBIs alone or in combination with macitentan and tadalafil improved RV function (mPAP, CO, TAPSE) and vascular remodeling (EVG). In the PA banding rat model, inhibition of FnBIs attenuated RV failure (CO, TAPSE, RVEDP). In conclusion, FnBIs signaling promotes maladaptive remodeling of the pulmonary vasculature and RV in PAH.

Spleen tyrosine kinase (SYK) is a non-receptor cytoplasmic kinase. Due to its pivotal role in B cell receptor and Fc-receptor signalling, inhibition of SYK has been a target of interest in a variety of diseases. Herein, we report the use of structure-based drug design to discover a series of potent macrocyclic inhibitors of SYK, with excellent kinome selectivity and in vitro metabolic stability. We were able to remove hERG inhibition through the optimization of physical properties, and utilized a pro-drug strategy to address permeability challenges.

Abstract Introduction We explored whether integrin αvβ8 inhibition potentiates immune checkpoint blockade (ICB) in syngeneic orthotopic models of breast cancer. Integrin αvβ8 mediates cell type specific and tissue localized activation of TGFβ1/3 to regulate the immune system. For example, αvβ8 expressed on dendritic cells (DC) in the intestine has been shown to be a key mediator of tolerance, maintaining gut immunologic homeostasis. Methods Efficacy was evaluated in combination with anti-PD-1 in EMT6 and PyMT breast cancer syngeneic mouse models. A potent αvβ8 inhibitor was orally administered at 60mg/kg BID for 21 days. Anti-αvβ8 or non-isoform specific anti-TGFβ mAbs were dosed TIWx3 at 7 and 10mg/kg, respectively. Tumor volumes are presented as mean±SEM. Statistics were performed by t test, one-way ANOVA, or log-rank test. Flow cytometry and transcriptome analysis on bulk and single-cell levels were used to assess the mechanism of action in EMT6. Results A similar αvβ8 expression pattern on DC, macrophages and regulatory T cells (Treg) was observed in mouse models and human tumors. Combination of oral αvβ8 inhibitor with anti-PD-1 was efficacious in the primary ICB resistant EMT6 model and resulted in superior tumor regression during treatment (p=0.0003) and improved survival with 5/12 complete responders relative to 0/12 in anti-PD-1 alone. Across studies the αvβ8 inhibitor phenocopied the results obtained with αvβ8 and TGFβ mAbs (n=3 independent studies). Complete responders re-challenged 89 days after treatment with EMT6 or 4T1 tumors showed no EMT6 tumor growth, suggesting the combination induced long-term immunologic memory. Analysis of tumors by flow cytometry showed combination resulted in increased CD8 T cell infiltrates (p=0.0006), T cell activation (CD8+CD69+, p=0.0194) and IFN-γ expression (CD8+IFN-γ+, p=0.0021). Single cell transcriptomic analysis of lymph nodes showed that αvβ8 inhibition potentiated DC co-stimulation (Cd40, Cd83/6) and migration (Ccr7, Cxcl16, Ccl22). Moreover, combination treatment led to tumor infiltrated Treg dysfunction including downregulation of Ctla4, Il10 and Tigit, and upregulation of Ifn-γ. The observation of these anti-tolerance and pro-inflammatory signatures in DC and Treg has not been described previously. Anti-tumor efficacy was driven by immune-mediated mechanisms as confirmed by a CD8 depletion study. Efficacy was confirmed in PyMT breast cancer model. Conclusions An αvβ8 inhibitor in combination with anti-PD-1 showed efficacy in syngeneic mouse models, supported by increased T cell infiltrates and evidence of reduced tumor tolerance. These results show that an orally administered αvβ8 targeted inhibitor is a potent modulator of anti-tumor immune response acting across the immunologic synapse, and is a promising therapeutic approach to ICB refractory tumors. Citation Format: Natalia J. Reszka-Blanco, Vinod Yadav, Megan Krumpoch, Laura Cappellucci, Dan Cui, James E. Dowling, Elizabeth Gwara, Bryce Harrison, Dooyoung Lee, Fu-Yang Lin, Lia Luus, Meghan Monroy, Terence I. Moy, Eugene Nebelitsky, Qi Qiao, Andrew Sullivan, Dawn Troast, Blaise Lippa, Bruce Rogers, Adrian S. Ray. Inhibition of integrin αvβ8 enhances immune checkpoint induced anti-tumor immunity by acting across immunologic synapse in syngeneic models of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1559.

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 GI(50) = 210 nM.

Spleen tyrosine kinase (SYK) is a non-receptor cytosolic kinase. Due to its pivotal role in B cell receptor and Fc-receptor signaling, inhibition of SYK has been targeted in a variety of disease areas. Herein, we report the optimization of a series of potent and selective SYK inhibitors, focusing on improving metabolic stability, pharmacokinetics and hERG inhibition. As a result, we identified 30, which exhibited no hERG activity but unfortunately was poorly absorbed in rats and mice. We also identified a SYK chemical probe, 17, which exhibits excellent potency at SYK, and an adequate rodent PK profile to support in vivo efficacy/PD studies.

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).

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.

The front cover picture shows the process of photocrosslinking a small molecule functionalized with a diaziridino group to its putative target upon UV irradiation, a critical step in photoaffinity chemical proteomics and target deconvolution. Though not intended to reflect the mode of binding, this depiction shows the photocrosslinking between a 1,2,3-thiadiazole-5-carboxamide (TDZ) series with SERCA2, one of the binding partners identified after enrichment and mass spectrometry analysis. Cellular effects observed with the TDZ series are consistent with mitochondrial dysfunction and SERCA engagement and modulation being mechanisms of actions. Front cover art created by Matias Casás-Selves and Julie Grouleff. More information can be found in the Full Paper by Andrew X. Zhang, et al. on page 917 in Issue 12, 2017 (DOI: 10.1002/cmdc.201700028).

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.

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.

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 β-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.

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.