Inhibition of coronavirus HCoV-OC43 by targeting the eIF4F complex.

The translation initiation complex 4F (eIF4F) is a rate-limiting factor in protein synthesis. Alterations in eIF4F activity are linked to several diseases, including cancer and infectious diseases. To this end, coronaviruses require eIF4F complex activity to produce proteins essential for their life cycle. Efforts to target coronaviruses by abrogating translation have been largely limited to repurposing existing eIF4F complex inhibitors. Here, we report the results of a high throughput screen to identify small molecules that disrupt eIF4F complex formation and inhibit coronavirus RNA and protein levels. Of 338,000 small molecules screened for inhibition of the eIF4F-driven, CAP-dependent translation, we identified SBI-1232 and two structurally related analogs, SBI-5844 and SBI-0498, that inhibit human coronavirus OC43 (HCoV-OC43; OC43) with minimal cell toxicity. Notably, gene expression changes after OC43 infection of Vero E6 or A549 cells were effectively reverted upon treatment with SBI-5844 or SBI-0498. Moreover, SBI-5844 or SBI-0498 treatment effectively impeded the eIF4F complex assembly, with concomitant inhibition of newly synthesized OC43 nucleocapsid protein and OC43 RNA and protein levels. Overall, we identify SBI-5844 and SBI-0498 as small molecules targeting the eIF4F complex that may limit coronavirus transcripts and proteins, thereby representing a basis for developing novel therapeutic modalities against coronaviruses.

Allosteric Inhibition of Ubiquitin-like Modifications by a Class of Inhibitor of SUMO-Activating Enzyme.

Ubiquitin-like (Ubl) post-translational modifications are potential targets for therapeutics. However, the only known mechanism for inhibiting a Ubl-activating enzyme is through targeting its ATP-binding site. Here we identify an allosteric inhibitory site in the small ubiquitin-like modifier (SUMO)-activating enzyme (E1). This site was unexpected because both it and analogous sites are deeply buried in all previously solved structures of E1s of ubiquitin-like modifiers (Ubl). The inhibitor not only suppresses SUMO E1 activity, but also enhances its degradation in vivo, presumably due to a conformational change induced by the compound. In addition, the lead compound increased the expression of miR-34b and reduced c-Myc levels in lymphoma and colorectal cancer cell lines and a colorectal cancer xenograft mouse model. Identification of this first-in-class inhibitor of SUMO E1 is a major advance in modulating Ubl modifications for therapeutic aims.

Capzimin is a potent and specific inhibitor of proteasome isopeptidase Rpn11.

The proteasome is a vital cellular machine that maintains protein homeostasis, which is of particular importance in multiple myeloma and possibly other cancers. Targeting of proteasome 20S peptidase activity with bortezomib and carfilzomib has been widely used to treat myeloma. However, not all patients respond to these compounds, and those who do eventually suffer relapse. Therefore, there is an urgent and unmet need to develop new drugs that target proteostasis through different mechanisms. We identified quinoline-8-thiol (8TQ) as a first-in-class inhibitor of the proteasome 19S subunit Rpn11. A derivative of 8TQ, capzimin, shows >5-fold selectivity for Rpn11 over the related JAMM proteases and >2 logs selectivity over several other metalloenzymes. Capzimin stabilized proteasome substrates, induced an unfolded protein response, and blocked proliferation of cancer cells, including those resistant to bortezomib. Proteomic analysis revealed that capzimin stabilized a subset of polyubiquitinated substrates. Identification of capzimin offers an alternative path to develop proteasome inhibitors for cancer therapy.

Inhibition of hematopoietic protein tyrosine phosphatase augments and prolongs ERK1/2 and p38 activation.

The hematopoietic protein tyrosine phosphatase (HePTP) is implicated in the development of blood cancers through its ability to negatively regulate the mitogen-activated protein kinases (MAPKs) ERK1/2 and p38. Small-molecule modulators of HePTP activity may become valuable in treating hematopoietic malignancies such as T cell acute lymphoblastic leukemia (T-ALL) and acute myelogenous leukemia (AML). Moreover, such compounds will further elucidate the regulation of MAPKs in hematopoietic cells. Although transient activation of MAPKs is crucial for growth and proliferation, prolonged activation of these important signaling molecules induces differentiation, cell cycle arrest, cell senescence, and apoptosis. Specific HePTP inhibitors may promote the latter and thereby may halt the growth of cancer cells. Here, we report the development of a small molecule that augments ERK1/2 and p38 activation in human T cells, specifically by inhibiting HePTP. Structure-activity relationship analysis, in silico docking studies, and mutagenesis experiments reveal how the inhibitor achieves selectivity for HePTP over related phosphatases by interacting with unique amino acid residues in the periphery of the highly conserved catalytic pocket. Importantly, we utilize this compound to show that pharmacological inhibition of HePTP not only augments but also prolongs activation of ERK1/2 and, especially, p38. Moreover, we present similar effects in leukocytes from mice intraperitoneally injected with the inhibitor at doses as low as 3 mg/kg. Our results warrant future studies with this probe compound that may establish HePTP as a new drug target for acute leukemic conditions.

Inhibition of the Hematopoietic Protein Tyrosine Phosphatase by Phenoxyacetic Acids.

Protein tyrosine phosphatases (PTPs) have only recently become the focus of attention in the search for novel drug targets despite the fact that they play vital roles in numerous cellular processes and are implicated in many human diseases. The hematopoietic protein tyrosine phosphatase (HePTP) is often found dysregulated in preleukemic myelodysplastic syndrome (MDS), as well as in acute myelogenous leukemia (AML). Physiological substrates of HePTP include the mitogen-activated protein kinases (MAPKs) ERK1/2 and p38. Specific modulators of HePTP catalytic activity will be useful for elucidating mechanisms of MAPK regulation in hematopietic cells, and may also provide treatments for hematopoietic malignancies such as AML. Here we report the discovery of phenoxyacetic acids as inhibitors of HePTP. Structure-activity relationship (SAR) analysis and in silico docking studies reveal the molecular basis of HePTP inhibition by these compounds. We also show that these compounds are able to penetrate cell membranes and inhibit HePTP in human T lymphocytes.