TAK1 inhibition leads to RIPK1-dependent apoptosis in immune-activated cancers.

Poor survival and lack of treatment response in glioblastoma (GBM) is attributed to the persistence of glioma stem cells (GSCs). To identify novel therapeutic approaches, we performed CRISPR/Cas9 knockout screens and discovered TGFβ activated kinase (TAK1) as a selective survival factor in a significant fraction of GSCs. Loss of TAK1 kinase activity results in RIPK1-dependent apoptosis via Caspase-8/FADD complex activation, dependent on autocrine TNFα ligand production and constitutive TNFR signaling.

Selective targeting of Plasmodium falciparum Hsp90 disrupts the 26S proteasome.

The molecular chaperone heat shock protein 90 (Hsp90) has an essential but largely undefined role in maintaining proteostasis in Plasmodium falciparum, the most lethal malaria parasite. Herein, we identify BX-2819 and XL888 as potent P. falciparum (Pf)Hsp90 inhibitors.

Targeting Borrelia burgdorferi HtpG with a berserker molecule, a strategy for anti-microbial development.

Conventional antimicrobial discovery relies on targeting essential enzymes in pathogenic organisms, contributing to a paucity of new antibiotics to address resistant strains. Here, by targeting a non-essential enzyme, Borrelia burgdorferi HtpG, to deliver lethal payloads, we expand what can be considered druggable within any pathogen. We synthesized HS-291, an HtpG inhibitor tethered to the photoactive toxin verteporfin.

Expression of membrane Hsp90 is a molecular signature of T cell activation.

Heat shock protein 90 (Hsp90) maintains cellular proteostasis during stress and has been under investigation as a therapeutic target in cancer for over two decades. We and others have identified a membrane expressed form of Hsp90 (mHsp90) that previously appeared to be restricted to rapidly proliferating cells exhibiting a metastatic phenotype. Here, we used HS-131, a fluor-tethered mHsp90 inhibitor, to quantify the effect of T cell activation on the expression of mHsp90 in human and mouse T cells.

Combination of a novel heat shock protein 90-targeted photodynamic therapy with PD-1/PD-L1 blockade induces potent systemic antitumor efficacy and abscopal effect against breast cancers.

Background: We previously demonstrated potent antitumor activity against human breast cancer xenografts using photodynamic therapy (PDT) targeting a novel tumor-specific photosensitizer (HS201), which binds heat shock protein 90 (HS201-PDT). However, induction of systemic antitumor immunity by HS201-PDT alone or by the combination strategy with immune checkpoint blockade has yet to be determined.

Methods: Using unilateral and bilateral implantation models of syngeneic breast tumors (E0771, MM3MG-HER2, and JC-HER3) in mice, we assessed whether HS201-PDT could induce local and systemic antitumor immunity.

HSP90-Specific nIR Probe Identifies Aggressive Prostate Cancers: Translation from Preclinical Models to a Human Phase I Study.

A noninvasive test to discriminate indolent prostate cancers from lethal ones would focus treatment where necessary while reducing overtreatment. We exploited the known activity of heat shock protein 90 (Hsp90) as a chaperone critical for the function of numerous oncogenic drivers, including the androgen receptor and its variants, to detect aggressive prostate cancer. We linked a near-infrared fluorescing molecule to an HSP90 binding drug and demonstrated that this probe (designated HS196) was highly sensitive and specific for detecting implanted prostate cancer cell lines with greater uptake by more aggressive subtypes.

Targeting therapy-resistant prostate cancer via a direct inhibitor of the human heat shock transcription factor 1.

Heat shock factor 1 (HSF1) is a cellular stress-protective transcription factor exploited by a wide range of cancers to drive proliferation, survival, invasion, and metastasis. Nuclear HSF1 abundance is a prognostic indicator for cancer severity, therapy resistance, and shortened patient survival. The gene was amplified, and nuclear HSF1 abundance was markedly increased in prostate cancers and particularly in neuroendocrine prostate cancer (NEPC), for which there are no available treatment options.

Heat shock protein 90-targeted photodynamic therapy enables treatment of subcutaneous and visceral tumors.

Photodynamic therapy (PDT) ablates malignancies by applying focused near-infrared (nIR) light onto a lesion of interest after systemic administration of a photosensitizer (PS); however, the accumulation of existing PS is not tumor-exclusive. We developed a tumor-localizing strategy for PDT, exploiting the high expression of heat shock protein 90 (Hsp90) in cancer cells to retain high concentrations of PS by tethering a small molecule Hsp90 inhibitor to a PS (verteporfin, VP) to create an Hsp90-targeted PS (HS201). HS201 accumulates to a greater extent than VP in breast cancer cells both in vitro and in vivo, resulting in increased treatment efficacy of HS201-PDT in various human breast cancer xenografts regardless of molecular and clinical subtypes.

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 β-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.

Plasmodium PK9 Inhibitors Promote Growth of Liver-Stage Parasites.

There is a scarcity of pharmacological tools to interrogate protein kinase function in Plasmodium parasites, the causative agent of malaria. Among Plasmodium's protein kinases, those characterized as atypical represent attractive drug targets as they lack sequence similarity to human proteins. Here, we describe takinib as a small molecule to bind the atypical P.

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.

Identification of Hsp90 Inhibitors with Anti-Plasmodium Activity.

Malaria remains a global health burden partly due to parasite resistance to first-line therapeutics. The molecular chaperone heat shock protein 90 (Hsp90) has emerged as an essential protein for blood-stage parasites, but details about its function during malaria's elusive liver stage are unclear. We used target-based screens to identify compounds that bind to and human Hsp90, which revealed insights into chemotypes with species-selective binding.

Detection of HSP90 Identifies Breast Cancers with Aggressive Behavior.

Hsp90, a chaperone to numerous molecular pathways in malignant cells, is elevated in aggressive breast cancers. We hypothesized that identifying breast cells with elevated Hsp90 activity in situ could result in early detection of aggressive breast cancers. We exploited the uptake of an Hsp90 inhibitor by malignant cells to create an imaging probe (HS131) of Hsp90 activity by linking it to a near-infrared (nIR) dye.

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.

The FNIP co-chaperones decelerate the Hsp90 chaperone cycle and enhance drug binding.

Heat shock protein-90 (Hsp90) is an essential molecular chaperone in eukaryotes involved in maintaining the stability and activity of numerous signalling proteins, also known as clients. Hsp90 ATPase activity is essential for its chaperone function and it is regulated by co-chaperones. Here we show that the tumour suppressor FLCN is an Hsp90 client protein and its binding partners FNIP1/FNIP2 function as co-chaperones.

An inducible heat shock protein 70 small molecule inhibitor demonstrates anti-dengue virus activity, validating Hsp70 as a host antiviral target.

An estimated three billion people are at risk of Dengue virus (DENV) infection worldwide and there are currently no approved therapeutic interventions for DENV infection. Due to the relatively small size of the DENV genome, DENV is reliant on host factors throughout the viral life cycle. The inducible form of Heat Shock Protein 70 (Hsp70i) has been implicated as a host factor in DENV pathogenesis, however the complete role remains to be elucidated.

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.

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.

Optical and radioiodinated tethered Hsp90 inhibitors reveal selective internalization of ectopic Hsp90 in malignant breast tumor cells.

Inhibitors of heat-shock protein 90 (Hsp90) have demonstrated an unusual selectivity for tumor cells despite its ubiquitous expression. This phenomenon has remained unexplained, but could be influenced by ectopically expressed Hsp90 in tumors. In this work, we synthesized Hsp90 inhibitors that can carry optical or radioiodinated probes via a polyethyleneglycol tether.

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.

Discovery of novel aminoquinazolin-7-yl 6,7-dihydro-indol-4-ones as potent, selective inhibitors of heat shock protein 90.

A novel class of Hsp90 inhibitors, structurally distinct from previously reported scaffolds, was developed from rational design and optimization of a compound library screen hit. These aminoquinazoline derivatives, represented by compound 15 (SNX-6833) or 1-(2-amino-4-methylquinazolin-7-yl)-3,6,6-trimethyl-6,7-dihydro-1H-indol-4(5H)-one, selectively bind to Hsp90 and inhibit its cellular activities at concentrations as low as single digit nanomolar.

Application of chemoproteomics to drug discovery: identification of a clinical candidate targeting hsp90.

A chemoproteomics-based drug discovery strategy is presented that utilizes a highly parallel screening platform, encompassing more than 1000 targets, with a focused chemical library prior to target selection. This chemoproteomics-based process enables a data-driven selection of both the biological target and chemical hit after the screen is complete. The methodology has been exemplified for the purine binding proteome (proteins utilizing ATP, NAD, FAD).

Discovery of novel 2-aminobenzamide inhibitors of heat shock protein 90 as potent, selective and orally active antitumor agents.

A novel class of heat shock protein 90 (Hsp90) inhibitors was developed from an unbiased screen to identify protein targets for a diverse compound library. These indol-4-one and indazol-4-one derived 2-aminobenzamides showed strong binding affinity to Hsp90, and optimized analogues exhibited nanomolar antiproliferative activity across multiple cancer cell lines. Heat shock protein 70 (Hsp70) induction and specific client protein degradation in cells on treatment with the inhibitors supported Hsp90 inhibition as the mechanism of action.