A highly selective inhibitor of interleukin-1 receptor-associated kinases 1/4 (IRAK-1/4) delineates the distinct signaling roles of IRAK-1/4 and the TAK1 kinase.

Interleukin-1 receptor-associated kinase-1 (IRAK-1) and IRAK-4, as well as transforming growth factor ß-activated kinase 1 (TAK1), are protein kinases essential for transducing inflammatory signals from interleukin receptors. IRAK family proteins and TAK1 have high sequence identity within the ATP-binding pocket, limiting the development of highly selective IRAK-1/4 or TAK1 inhibitors. Beyond kinase activity, IRAKs and TAK1 act as molecular scaffolds along with other signaling proteins, complicating the interpretation of experiments involving knockin or knockout approaches. In contrast, pharmacological manipulation offers the promise of targeting catalysis-mediated signaling without grossly disrupting the cellular architecture. Recently, we reported the discovery of takinib, a potent and highly selective TAK1 inhibitor that has only marginal activity against IRAK-4. On the basis of the TAK1-takinib complex structure and the structure of IRAK-1/4, here we defined critical contact sites of the takinib scaffold within the nucleotide-binding sites of each respective kinase. Kinase activity testing of takinib analogs against IRAK-4 identified a highly potent IRAK-4 inhibitor (HS-243). In a kinome-wide screen of 468 protein kinases, HS-243 had exquisite selectivity toward both IRAK-1 (IC50 = 24 nm) and IRAK-4 (IC50 = 20 nm), with only minimal TAK1-inhibiting activity (IC50 = 0.5 µm). Using HS-243 and takinib, we evaluated the consequences of cytokine/chemokine responses after selective inhibition of IRAK-1/4 or TAK1 in response to lipopolysaccharide challenge in human rheumatoid arthritis fibroblast-like synoviocytes. Our results indicate that HS-243 specifically inhibits intracellular IRAKs without TAK1 inhibition and that these kinases have distinct, nonredundant signaling roles.

Application of immobilized ATP to the study of NLRP inflammasomes.

The NLRP proteins are a subfamily of the NOD-like receptor (NLR) innate immune sensors that possess an ATP-binding NACHT domain. As the most well studied member, NLRP3 can initiate the assembly process of a multiprotein complex, termed the inflammasome, upon detection of a wide range of microbial products and endogenous danger signals and results in the activation of pro-caspase-1, a cysteine protease that regulates multiple host defense pathways including cytokine maturation. Dysregulated NLRP3 activation contributes to inflammation and the pathogenesis of several chronic diseases, and the ATP-binding properties of NLRPs are thought to be critical for inflammasome activation. In light of this, we examined the utility of immobilized ATP matrices in the study of NLRP inflammasomes. Using NLRP3 as the prototypical member of the family, P-linked ATP Sepharose was determined to be a highly-effective capture agent. In subsequent examinations, P-linked ATP Sepharose was used as an enrichment tool to enable the effective profiling of NLRP3-biomarker signatures with selected reaction monitoring-mass spectrometry (SRM-MS). Finally, ATP Sepharose was used in combination with a fluorescence-linked enzyme chemoproteomic strategy (FLECS) screen to identify potential competitive inhibitors of NLRP3. The identification of a novel benzo[d]imidazol-2-one inhibitor that specifically targets the ATP-binding and hydrolysis properties of the NLRP3 protein implies that ATP Sepharose and FLECS could be applied other NLRPs as well.

Targeting Pim Kinases and DAPK3 to Control Hypertension.

Sustained vascular smooth muscle hypercontractility promotes hypertension and cardiovascular disease. The etiology of hypercontractility is not completely understood. New therapeutic targets remain vitally important for drug discovery. Here we report that Pim kinases, in combination with DAPK3, regulate contractility and control hypertension. Using a co-crystal structure of lead molecule (HS38) in complex with DAPK3, a dual Pim/DAPK3 inhibitor (HS56) and selective DAPK3 inhibitors (HS94 and HS148) were developed to provide mechanistic insight into the polypharmacology of hypertension. In vitro and ex vivo studies indicated that Pim kinases directly phosphorylate smooth muscle targets and that Pim/DAPK3 inhibition, unlike selective DAPK3 inhibition, significantly reduces contractility. In vivo, HS56 decreased blood pressure in spontaneously hypertensive mice in a dose-dependent manner without affecting heart rate. These findings suggest including Pim kinase inhibition within a multi-target engagement strategy for hypertension management. HS56 represents a significant step in the development of molecularly targeted antihypertensive medications.

Synergistic role of HSP90α and HSP90ß to promote myofibroblast persistence in lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of the lung parenchyma, causing significant morbidity through worsening dyspnoea and overall functional decline. IPF is characterised by apoptosis-resistant myofibroblasts, which are a major source for the excessive production of extracellular matrix (ECM) overtaking normal lung tissue. We sought to study the role of heat shock protein (HSP) isoforms HSP90α and HSP90ß, whose distinct roles in lung fibrogenesis remain elusive.We determined the level of circulating HSP90α in IPF patients (n=31) and age-matched healthy controls (n=9) by ELISA. The release of HSP90α and HSP90ß was evaluated in vitro in primary IPF and control lung fibroblasts and ex vivo after mechanical stretch on fibrotic lung slices from rats receiving adenovector-mediated transforming growth factor-ß1.We demonstrate that circulating HSP90α is upregulated in IPF patients in correlation with disease severity. The release of HSP90α is enhanced by the increase in mechanical stress of the fibrotic ECM. This increase in extracellular HSP90α signals through low-density lipoprotein receptor-related protein 1 (LRP1) to promote myofibroblast differentiation and persistence. In parallel, we demonstrate that the intracellular form of HSP90ß stabilises LRP1, thus amplifying HSP90α extracellular action.We believe that the specific inhibition of extracellular HSP90α is a promising therapeutic strategy to reduce pro-fibrotic signalling in IPF.

Near-simultaneous intravital microscopy of glucose uptake and mitochondrial membrane potential, key endpoints that reflect major metabolic axes in cancer.

While the demand for metabolic imaging has increased in recent years, simultaneous in vivo measurement of multiple metabolic endpoints remains challenging. Here we report on a novel technique that provides in vivo high-resolution simultaneous imaging of glucose uptake and mitochondrial metabolism within a dynamic tissue microenvironment. Two indicators were leveraged; 2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) reports on glucose uptake and Tetramethylrhodamine ethyl ester (TMRE) reports on mitochondrial membrane potential. Although we demonstrated that there was neither optical nor chemical crosstalk between 2-NBDG and TMRE, TMRE uptake was significantly inhibited by simultaneous injection with 2-NBDG in vivo. A staggered delivery scheme of the two agents (TMRE injection was followed by 2-NBDG injection after a 10-minute delay) permitted near-simultaneous in vivo microscopy of 2-NBDG and TMRE at the same tissue site by mitigating the interference of 2-NBDG with normal glucose usage. The staggered delivery strategy was evaluated under both normoxic and hypoxic conditions in normal tissues as well as in a murine breast cancer model. The results were consistent with those expected for independent imaging of 2-NBDG and TMRE. This optical imaging technique allows for monitoring of key metabolic endpoints with the unique benefit of repeated, non-destructive imaging within an intact microenvironment.

Takinib, a Selective TAK1 Inhibitor, Broadens the Therapeutic Efficacy of TNF-α Inhibition for Cancer and Autoimmune Disease.

Tumor necrosis factor alpha (TNF-α) has both positive and negative roles in human disease. In certain cancers, TNF-α is infused locally to promote tumor regression, but dose-limiting inflammatory effects limit broader utility. In autoimmune disease, anti-TNF-α antibodies control inflammation in most patients, but these benefits are offset during chronic treatment. TAK1 acts as a key mediator between survival and cell death in TNF-α-mediated signaling. Here, we describe Takinib, a potent and selective TAK1 inhibitor that induces apoptosis following TNF-α stimulation in cell models of rheumatoid arthritis and metastatic breast cancer. We demonstrate that Takinib is an inhibitor of autophosphorylated and non-phosphorylated TAK1 that binds within the ATP-binding pocket and inhibits by slowing down the rate-limiting step of TAK1 activation. Overall, Takinib is an attractive starting point for the development of inhibitors that sensitize cells to TNF-α-induced cell death, with general implications for cancer and autoimmune disease treatment.

Fasnall, a Selective FASN Inhibitor, Shows Potent Anti-tumor Activity in the MMTV-Neu Model of HER2(+) Breast Cancer.

Many tumors are dependent on de novo fatty acid synthesis to maintain cell growth. Fatty acid synthase (FASN) catalyzes the final synthetic step of this pathway, and its upregulation is correlated with tumor aggressiveness. The consequences and adaptive responses of acute or chronic inhibition of essential enzymes such as FASN are not fully understood. Herein we identify Fasnall, a thiophenopyrimidine selectively targeting FASN through its co-factor binding sites. Global lipidomics studies with Fasnall showed profound changes in cellular lipid profiles, sharply increasing ceramides, diacylglycerols, and unsaturated fatty acids as well as increasing exogenous palmitate uptake that is deviated more into neutral lipid formation rather than phospholipids. We also showed that the increase in ceramide levels contributes to some extent in the mediation of apoptosis. Consistent with this mechanism of action, Fasnall showed potent anti-tumor activity in the MMTV-Neu model of HER2(+) breast cancer, particularly when combined with carboplatin.

Identification of an allosteric small-molecule inhibitor selective for the inducible form of heat shock protein 70.

Inducible Hsp70 (Hsp70i) is overexpressed in a wide spectrum of human tumors, and its expression correlates with metastasis, poor outcomes, and resistance to chemotherapy in patients. Identification of small-molecule inhibitors selective for Hsp70i could provide new therapeutic tools for cancer treatment. In this work, we used fluorescence-linked enzyme chemoproteomic strategy (FLECS) to identify HS-72, an allosteric inhibitor selective for Hsp70i. HS-72 displays the hallmarks of Hsp70 inhibition in cells, promoting substrate protein degradation and growth inhibition. Importantly, HS-72 is selective for Hsp70i over the closely related constitutively active Hsc70. Studies with purified protein show HS-72 acts as an allosteric inhibitor, reducing ATP affinity. In vivo HS-72 is well-tolerated, showing bioavailability and efficacy, inhibiting tumor growth and promoting survival in a HER2+ model of breast cancer. The HS-72 scaffold is amenable to resynthesis and iteration, suggesting an ideal starting point for a new generation of anticancer therapeutics targeting Hsp70i.

Fluorescence linked enzyme chemoproteomic strategy for discovery of a potent and selective DAPK1 and ZIPK inhibitor.

DAPK1 and ZIPK (also called DAPK3) are closely related serine/threonine protein kinases that regulate programmed cell death and phosphorylation of non-muscle and smooth muscle myosin. We have developed a fluorescence linked enzyme chemoproteomic strategy (FLECS) for the rapid identification of inhibitors for any element of the purinome and identified a selective pyrazolo[3,4-d]pyrimidinone (HS38) that inhibits DAPK1 and ZIPK in an ATP-competitive manner at nanomolar concentrations. In cellular studies, HS38 decreased RLC20 phosphorylation. In ex vivo studies, HS38 decreased contractile force generated in mouse aorta, rabbit ileum, and calyculin A stimulated arterial muscle by decreasing RLC20 and MYPT1 phosphorylation. The inhibitor also promoted relaxation in Ca(2+)-sensitized vessels. A close structural analogue (HS43) with 5-fold lower affinity for ZIPK produced no effect on cells or tissues. These findings are consistent with a mechanism of action wherein HS38 specifically targets ZIPK in smooth muscle. The discovery of HS38 provides a lead scaffold for the development of therapeutic agents for smooth muscle related disorders and a chemical means to probe the function of DAPK1 and ZIPK across species.

A highly selective Hsp90 affinity chromatography resin with a cleavable linker.

Over 200 proteins have been identified that interact with the protein chaperone Hsp90, a recognized therapeutic target thought to participate in non-oncogene addiction in a variety of human cancers. However, defining Hsp90 clients is challenging because interactions between Hsp90 and its physiologically relevant targets involve low affinity binding and are thought to be transient. Using a chemo-proteomic strategy, we have developed a novel orthogonally cleavable Hsp90 affinity resin that allows purification of the native protein and is quite selective for Hsp90 over its immediate family members, GRP94 and TRAP 1. We show that the resin can be used under low stringency conditions for the rapid, unambiguous capture of native Hsp90 in complex with a native client. We also show that the choice of linker used to tether the ligand to the insoluble support can have a dramatic effect on the selectivity of the affinity media.

Cuticular hydrocarbons of Chagas disease vectors in Mexico

Capillary gas-liquid chromatography was used to analyse the cuticular hydrocarbons of three triatomine species, Triatoma dimidiata, T. barberi and Dipetalogaster maxima, domestic vectors of Chagas disease in Mexico. Mixtures of saturated hydrocarbons of straight and methyl-branched chains were characteristic of the three species, but quantitatively different. Major methylbranched components mostly corresponded to different saturated isomers of monomethyl, dimethyl and trimethyl branched hydrocarbons ranging from 29 to 39 carbon backbones. Sex-dependant, quantitative differences in certain hydrocarbons were apparent in T. dimidiata