The calcium release activated calcium (CRAC) channel is highly expressed in T lymphocytes and plays a critical role in regulating T cell proliferation and functions including activation of the transcription factor nuclear factor of activated T cells (NFAT), cytokine production and cytotoxicity. The CRAC channel consists of the Orai pore subunit and STIM (stromal interacting molecule) endoplasmic reticulum calcium sensor. Loss of CRAC channel mediated calcium signaling has been identified as an underlying cause of severe combined immunodeficiency (SCID), leading to drastically weakened immunity against infections. Gain-of-function mutations in Orai and STIM have been associated with tubular aggregated myopathy (TAM), a skeletal muscle disease. While a number of small molecules have shown activity in inhibiting the CRAC signaling pathway, the usefulness of those tool compounds is limited by their off-target activity against TRPM4 and TRPM7 ion channels, high lipophilicity, and a lack of understanding of their mechanism of action. We report structure-activity relationship (SAR) studies that resulted in the characterization of compound 4k [1-(cyclopropylmethyl)-N-(3-fluoropyridin-4-yl)-1H-indazole-3-carboxamie] as a fast onset, reversible, and selective CRAC channel blocker. 4k fully blocked the CRAC current (IC50: 4.9 μM) and the nuclear translocation of NFAT at 30 and 10 μM, respectively, without affecting the electrophysiological function of TRPM4 and TRPM7 channels. Computational modeling appears to support its direction binding to Orai proteins that form the transmembrane CRACchannel.

Receptor-interacting serine/threonine kinase 2 (RIPK2) is a vital immunomodulator that plays critical roles in nucleotide-binding oligomerization domain 1 (NOD1), NOD2, and Toll-like receptors (TLRs) signaling. Stimulated NOD1 and NOD2 interact with RIPK2 and lead to the activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPK), followed by the production of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-12/23. Defects in NOD/RIPK2 signaling are associated with numerous inflammatory diseases, including asthma, sarcoidosis, inflammatory bowel disease (Crohn’s disease and ulcerative colitis), multiple sclerosis, and Blau syndrome. As RIPK2 is a crucial element of innate immunity, small molecules regulating RIPK2 functions are attractive to establish novel immunotherapies. The increased interest in developing RIPK2 inhibitors has led to the clinical investigations of novel drug candidates. In this review, we attempt to summarize recent advances in the development of RIPK2 inhibitors and degraders.

Tranexamic Acid (TA) is an anti-fibrinolytic agent that inhibits Plasminogen activation and is used to control bleeding. Past studies have suggested that the ability of TA to block Plasminogen activation may be useful for ablating the growth or invasion of cancer. Based on the similarity of TA to the amino acid Lysine we hypothesize that TA might exhibit novel mechanisms of action independent of blocking Plasminogen activation. Analysis of a series of signaling pathways indicated that TA inhibits phosphorylation of S6K1 and STAT3 on sites required for their activation and reduces expression of the MYC oncogene in a concentration-dependent manner. Interestingly, MYC transformed MCF10A human mammary epithelial cells exhibited enhanced sensitivity to TA-mediated reduction of cell viability, suggesting that TA may exhibit selectivity for MYC overexpressing breast cancers. Treatment of breast tumor bearing mice with TA strongly blocked tumor growth and was associated with extensive cancer cell death compared with control tumors. Analysis of extracts from TA or vehicle treated tumors showed that TA blocked S6K1 phosphorylation in vivo in a statistically significant manner. Since S6K1 phosphorylation is regulated by amino acid levels, we hypothesized that due to the structural similarity between TA and Lysine and Arginine, TA may block Lysine or Arginine uptake by blocking cationic amino acid transporters (CATs). Molecular docking simulations utilizing the structure of the bacterial CAT predicted that TA binds to CAT with affinity similar to that of Lysine. Amino acid uptake experiments performed with radiolabeled Arginine and Lysine showed that TA blocked both Arginine and Lysine absorption by cancer cells in a concentration-dependent manner. Together, these results indicate a novel mechanism by which TA inhibits uptake of Arginine and Lysine, triggering loss of S6K1 activity in parallel with tumor growth inhibition. Initial studies suggest the feasibility of designing TA analogs with significantly improved potency for amino acid transporters, and with corresponding increases in selective toxicity to breast cancer cells. Based on these novel mechanisms of TA action, we expect TA to exhibit anti-cancer activity against a broad range of human malignancies. Citation Format: Brian K. Law, Mary E. Law, Amanda F. Ghilardi, Elham Yaaghubi, Brad J. Davis, Zaafir M. Dulloo, Mengxiong Wang, Olga A. Guryanova, Coy D. Heldermon, Stephan C. Jahn, Ronald K. Castellano. Inhibition of amino acid transporters as a novel mechanism of action of the repurposed anti-cancer agent tranexamic acid. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4879.

Previous studies indicated that compounds termed Disulfide bond Disrupting Agents (DDAs) exhibit anti-cancer activity that is associated with downregulation of EGFR/HER1, HER2, and HER3, and activation of Death Receptors 4 and 5 (DR4/5). DDA-induced HER1-3 downregulation is preceded by disulfide-mediated oligomerization. In contrast, DDA-mediated DR4/5 oligomerization results in DR5 upregulation, and activation of DR4/5 pro-apoptotic signaling through Caspases 8 and 3. However, the precise mechanisms by which altered disulfide bonding stabilizes and activates DR5 are unknown. A recent report indicated that the extracellular domain of DR5 acts in an auto-inhibitory manner to prevent DR5 oligomerization and pro-apoptotic signaling in the absence of its ligand, TRAIL. A subsequent paper showed that the DR5 auto-inhibitory domain is a positive patch consisting of three basic residues. Importantly, the structure of the auto-inhibitory loop is formed by two disulfide bonds. We hypothesize that DDAs disrupt the disulfide bonds that make up the auto-inhibitory loop, resulting in DR5 oligomerization, and activation of Caspase 8/3-driven apoptosis in a TRAIL-independent manner. Due to their novel mechanisms of action, DDAs may overcome the pharmacological liabilities that have limited the efficacy of TRAIL analogs and DR5 agonist antibodies. Another unanswered question is how precisely DDAs alter DR5 and EGFR disulfide bonding. The direct targets of DDA action were revealed through affinity purification studies with biotinylated-DDA analogs. These studies identified the protein disulfide isomerases AGR2, ERp44, and PDIA1 as DDA target proteins, explaining how DDAs alter DR5 and EGFR disulfide bonding patterns. Consistent with this interpretation, knockdown of AGR2 or ERp44, or expression of catalytically null AGR2 or ERp44 mutants, mimicked DDAs in inducing disulfide-mediated DR5 oligomerization and Caspase 8 activation. Together, these results demonstrate a fundamentally novel, ligand-independent mechanism for activation of DR5 through DDA-mediated inhibition of the PDIs AGR2, ERp44, and PDIA1. Significantly, DDAs are the first identified active site inhibitors of AGR2 and ERp44. Citation Format: Brian K. Law, Mary E. Law, Elham Yaaghubi, Amanda Ghilardi, Brad J. Davis, Renan Ferreira, Samantha Eggleston, Jade Nguyen, Grace Alexandrow, Jin Koh, Sixue Chen, Chi-Wu Chiang, Coy Heldermon, Peter Norgaard, Ronald K. Castallano, Zaafir M. Dulloo. Disulfide isomerases AGR2, ERp44, and PDIA1 maintain death receptor 5 in an auto-inhibited, monomeric form. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6148.

Introduction Malignant pleural mesothelioma (MPM) is a neoplasm with dismal prognosis and notorious resistance to the standard therapeutics cisplatin and pemetrexed. Chalcone derivatives are efficacious anti-cancer agents with minimal toxicity and have, therefore, gained pharmaceutical interest. Here, we investigated the efficacy of CIT-026 and CIT-223, two indolyl-chalcones (CITs), to inhibit growth and viability of MPM cells and defined the mechanism by which the compounds induce cell death. Methods The effects of CIT-026 and CIT-223 were analyzed in five MPM cell lines, using viability, immunofluorescence, real-time cell death monitoring, and tubulin polymerization assays, along with siRNA knockdown. Phospho-kinase arrays and immunoblotting were used to identify signaling molecules that contribute to cell death. Results CIT-026 and CIT-223 were toxic in all cell lines at sub-micromolar concentrations, in particular in MPM cells resistant to cisplatin and pemetrexed, while normal fibroblasts were only modestly affected. Both CITs targeted tubulin polymerization via (1) direct interaction with tubulin and (2) phosphorylation of microtubule regulators STMN1, CRMP2 and WNK1. Formation of aberrant tubulin fibers caused abnormal spindle morphology, mitotic arrest and apoptosis. CIT activity was not reduced in CRMP2-negative and STMN1-silenced MPM cells, indicating that direct tubulin targeting is sufficient for toxic effects of CITs. Discussion CIT-026 and CIT-223 are highly effective inducers of tumor cell apoptosis by disrupting microtubule assembly, with only modest effects on non-malignant cells. CITs are potent anti-tumor agents against MPM cells, in particular cells resistant to standard therapeutics, and thus warrant further evaluation as potential small-molecule therapeutics in MPM.

Breast cancer mortality remains unacceptably high, indicating a need for safer and more effective therapeutic agents. Disulfide bond Disrupting Agents (DDAs) were previously identified as a novel class of anticancer compounds that selectively kill cancers that overexpress the Epidermal Growth Factor Receptor (EGFR) or its family member HER2. DDAs kill EGFR+ and HER2+ cancer cells via the parallel downregulation of EGFR, HER2, and HER3 and activation/oligomerization of Death Receptors 4 and 5 (DR4/5). However, the mechanisms by which DDAs mediate these effects are unknown. Affinity purification analyses employing biotinylated-DDAs reveal that the Protein Disulfide Isomerase (PDI) family members AGR2, PDIA1, and ERp44 are DDA target proteins. Further analyses demonstrate that shRNA-mediated knockdown of AGR2 and ERp44, or expression of ERp44 mutants, enhance basal DR5 oligomerization. DDA treatment of breast cancer cells disrupts PDIA1 and ERp44 mixed disulfide bonds with their client proteins. Together, the results herein reveal DDAs as the first small molecule, active site inhibitors of AGR2 and ERp44, and demonstrate roles for AGR2 and ERp44 in regulating the activity, stability, and localization of DR4 and DR5, and activation of Caspase 8.

Reported are structure-property-function relationships associated with a class of cyclic thiosulfonate molecules-disulfide-bond disrupting agents (DDAs)-with the ability to downregulate the Epidermal Growth Factor Receptor (HER) family in parallel and selectively induce apoptosis of EGFR+ or HER2+ breast cancer cells. Recent findings have revealed that the DDA mechanism of action involves covalent binding to the thiol(ate) from the active site cysteine residue of members of the protein disulfide isomerase (PDI) family. Reported is how structural modifications to the pharmacophore can alter the anticancer activity of cyclic thiosulfonates by tuning the dynamics of thiol-thiosulfonate exchange reactions, and the studies reveal a correlation between the biological potency and thiol-reactivity. Specificity of the cyclic thiosulfonate ring-opening reaction by a nucleophilic attack can be modulated by substituent addition to a parent scaffold. Lead compound optimization efforts are also reported, and have resulted in a considerable decrease of the IC50 /IC90 values toward HER-family overexpressing breast cancer cells.

Tranexamic Acid (TA) is a clinically used antifibrinolytic agent that acts as a Lys mimetic to block binding of Plasminogen with Plasminogen activators, preventing conversion of Plasminogen to its proteolytically activated form, Plasmin. Previous studies suggested that TA may exhibit anticancer activity by blockade of extracellular Plasmin formation. Plasmin-mediated cleavage of the CDCP1 protein may increase its oncogenic functions through several downstream pathways. Results presented herein demonstrate that TA blocks Plasmin-mediated excision of the extracellular domain of the oncoprotein CDCP1. In vitro studies indicate that TA reduces the viability of a broad array of human and murine cancer cell lines, and breast tumor growth studies demonstrate that TA reduces cancer growth in vivo. Based on the ability of TA to mimic Lys and Arg, we hypothesized that TA may perturb multiple processes that involve Lys/Arg-rich protein sequences, and that TA may alter intracellular signaling pathways in addition to blocking extracellular Plasmin production. Indeed, TA-mediated suppression of tumor cell viability is associated with multiple biochemical actions, including inhibition of protein synthesis, reduced activating phosphorylation of STAT3 and S6K1, decreased expression of the MYC oncoprotein, and suppression of Lys acetylation. Further, TA inhibited uptake of Lys and Arg by cancer cells. These findings suggest that TA or TA analogs may serve as lead compounds and inspire the production of new classes of anticancer agents that function by mimicking Lys and Arg.

Affiliations Department of Pharmacology & Therapeutics, University of Florida, Gainesville, FL 32610 USA. Department of Chemistry, University of Florida, Gainesville, FL 32611 USA. Proteomics and Mass Spectrometry Facility, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL 32610 USA. Department of Biology, Genetics Institute, University of Florida, Gainesville, FL 32610 USA Institute of Molecular Medicine, College of Medicine and Center for Infectious Disease and Signaling Research, National Cheng Kung University, Tainan, Taiwan. Department of Medicine, University of Florida, Gainesville, FL 32610 USA. UF-Health Cancer Center, University of Florida, Gainesville, FL 32610 USA. Department of Pathology, Copenhagen University Hospital Herlev, DK-2730 Herlev, Denmark.

Breast cancer mortality remains unacceptably high, indicating a need for safer and more effective therapeutic agents. Disulfide bond Disrupting Agents (DDAs) were previously identified as a novel class of anticancer compounds that selectively kill cancers that overexpress the Epidermal Growth Factor Receptor (EGFR) or its family member HER2. DDAs kill EGFR+ and HER2+ cancer cells via the parallel downregulation of EGFR, HER2, and HER3 and activation/oligomerization of Death Receptors 4 and 5 (DR4/5). However, the mechanisms by which DDAs mediate these effects are unknown. Affinity purification analyses employing biotinylated-DDAs reveal that the Protein Disulfide Isomerase (PDI) family members AGR2, AGR3, PDIA1, and ERp44 are DDA target proteins. Further analyses demonstrate that shRNA-mediated knockdown of AGR2 and ERp44, or expression of ERp44 mutants, enhance basal and DDA-induced DR5 oligomerization. DDA treatment of breast cancer cells disrupts PDIA1 and ERp44 mixed disulfide bonds with their client proteins. Together, these results reveal DDAs as the first small molecule, active site inhibitors of AGR2 and ERp44, demonstrate a role for AGR2 and ERp44 in regulating the activity, stability, and localization of DR4 and DR5, and nominate ERp44 as a new molecular target for anticancer therapeutics.

Disulfide bond Disrupting Agents (DDAs) are a new chemical class of agents recently shown to have activity against breast tumors in animal models. However, it is unknown how DDAs trigger cancer cell death without affecting nontransformed cells. As demonstrated here, DDAs are the first compounds identified that upregulate the TRAIL receptor DR5 through both transcriptional and posttranscriptional mechanisms. At the protein level, DDAs alter DR5 disulfide bonding to increase steady-state DR5 levels and oligomerization, leading to downstream Caspase 8 and 3 activation. DDAs and TRAIL synergize to kill cancer cells and are cytotoxic to HER2+ cancer cells with acquired resistance to the EGFR/HER2 tyrosine kinase inhibitor. Investigation of the mechanisms responsible for DDA selectivity for cancer cells reveals that DDA-induced upregulation of DR5 is enhanced in the context of EGFR overexpression, and DDA-induced cytotoxicity is strongly amplified by MYC overexpression. Together, the results demonstrate selective DDA lethality against oncogene-transformed cells, DDA-mediated DR5 upregulation and protein stabilization, and DDAs against drug-resistant and metastatic cancer cells. DDAs thus represent a new therapeutic approach to cancer therapy.

While HER2 and EGFR are overexpressed in breast cancers and multiple other types of tumors, the use of EGFR and/or HER2 inhibitors have failed to cure many cancer patients, largely because cancers acquire resistance to HER2/EGFR-specific drugs. Cancers that overexpress the HER-family proteins EGFR, HER2, and HER3 are uniquely sensitive to agents that disrupt HER2 and EGFR protein folding. We previously showed that disruption of disulfide bond formation by Disulfide Disrupting Agents (DDAs) kills HER2/EGFR overexpressing cells through multiple mechanisms. Herein, we show that interference with proline isomerization in HER2/EGFR overexpressing cells also induces cancer cell death. The peptidyl-prolyl isomerase inhibitor Cyclosporine A (CsA) selectively kills EGFR+ or HER2+ breast cancer cells in vitro by activating caspase-dependent apoptotic pathways. Further, CsA synergizes with the DDA tcyDTDO to kill HER2/EGFR overexpressing cells in vitro and the two agents cooperate to kill HER2+ tumors in vivo. There is a critical need for novel strategies to target HER2+ and EGFR+ cancers that are resistant to currently available mechanism-based agents. Drugs that target HER2/EGFR protein folding, including DDAs and CsA, have the potential to kill cancers that overexpress EGFR or HER2 through the induction of proteostatic synthetic lethality.

Disulfide bond-disrupting agents (DDAs) are a new chemical class of agents recently shown to have activity against breast tumors in animal models. Blockade of tumor growth is associated with downregulation of EGFR, HER2, and HER3 and reduced Akt phosphorylation, as well as the induction of endoplasmic reticulum stress. However, it is not known how DDAs trigger cancer cell death without affecting nontransformed cells. As demonstrated here, DDAs are the first compounds identified that upregulate the TRAIL receptor DR5 through transcriptional and post-transcriptional mechanisms to activate the extrinsic cell death pathway. At the protein level, DDAs alter DR5 disulfide bonding to increase steady-state DR5 levels and oligomerization, leading to downstream caspase 8 and 3 activation. DDAs and TRAIL synergize to kill cancer cells and are cytotoxic to HER2+ cancer cells with acquired resistance to the EGFR/HER2 tyrosine kinase inhibitor Lapatinib. Investigation of the mechanisms responsible for DDA selectivity for cancer cells reveals that DDA-induced upregulation of DR5 is enhanced in the context of EGFR overexpression. DDA-induced cytotoxicity is strongly amplified by MYC overexpression. This is consistent with the known potentiation of TRAIL-mediated cell death by MYC. Together, the results demonstrate selective DDA lethality against oncogene-transformed cells, DDA-mediated DR5 upregulation, and protein stabilization, and that DDAs have activity against drug-resistant cancer cells. Our results indicate that DDAs are unique in causing DR5 accumulation and oligomerization and inducing downstream caspase activation and cancer cell death through mechanisms involving altered DR5 disulfide bonding. DDAs thus represent a new therapeutic approach to cancer therapy.