Suppression of type 1 pilus assembly in uropathogenic Escherichia coli by chemical inhibition of subunit polymerization

OBJECTIVES: To identify and to characterize small-molecule inhibitors that target the subunit polymerization of the type 1 pilus assembly in uropathogenic Escherichia coli (UPEC). METHODS: Using an SDS-PAGE-based assay, in silico pre-filtered small-molecule compounds were screened for specific inhibitory activity against the critical subunit polymerization step of the chaperone-usher pathway during pilus biogenesis. The biological activity of one of the compounds was validated in assays monitoring UPEC type 1 pilus biogenesis, type 1 pilus-dependent biofilm formation and adherence to human bladder epithelial cells. The time dependence of the in vivo inhibitory activity and the overall effect of the compound on UPEC growth were determined. RESULTS: N-(4-chloro-phenyl)-2-{5-[4-(pyrrolidine-1-sulfonyl)-phenyl]-[1,3,4]oxadiazol-2-yl sulfanyl}-acetamide (AL1) inhibited in vitro pilus subunit polymerization. In bacterial cultures, AL1 disrupted UPEC type 1 pilus biogenesis and pilus-dependent biofilm formation, and resulted in the reduction of bacterial adherence to human bladder epithelial cells, without affecting bacterial cell growth. Bacterial exposure to the inhibitor led to an almost instantaneous loss of type 1 pili. CONCLUSIONS: We have identified and characterized a small molecule that interferes with the assembly of type 1 pili. The molecule targets the polymerization step during the subunit incorporation cycle of the chaperone-usher pathway. Our discovery provides new insight into the design and development of novel anti-virulence therapies targeting key virulence factors of bacterial pathogens

Small Molecule Neuropilin‑1 Antagonists Combine Antiangiogenic and Antitumor Activity with Immune Modulation through Reduction of Transforming Growth Factor Beta (TGFβ) Production in Regulatory T‑Cells

We report the design, synthesis, and biological evaluation of some potent small-molecule neuropilin-1 (NRP1) antagonists. NRP1 is implicated in the immune response to tumors, particularly in Treg cell fragility, required for PD1 checkpoint blockade. The design of these compounds was based on a previously identified compound EG00229. The design of these molecules was informed and supported by X-ray crystal structures. Compound <b>1</b> (EG01377) was identified as having properties suitable for further investigation. Compound <b>1</b> was then tested in several in vitro assays and was shown to have antiangiogenic, antimigratory, and antitumor effects. Remarkably, <b>1</b> was shown to be selective for NRP1 over the closely related protein NRP2. In purified Nrp1⁺, FoxP3⁺, and CD25⁺ populations of Tregs from mice, <b>1</b> was able to block a glioma-conditioned medium-induced increase in TGFβ production. This comprehensive characterization of a small-molecule NRP1 antagonist provides the basis for future in vivo studies

Small Molecule Neuropilin‑1 Antagonists Combine Antiangiogenic and Antitumor Activity with Immune Modulation through Reduction of Transforming Growth Factor Beta (TGFβ) Production in Regulatory T‑Cells

We report the design, synthesis, and biological evaluation of some potent small-molecule neuropilin-1 (NRP1) antagonists. NRP1 is implicated in the immune response to tumors, particularly in Treg cell fragility, required for PD1 checkpoint blockade. The design of these compounds was based on a previously identified compound EG00229. The design of these molecules was informed and supported by X-ray crystal structures. Compound <b>1</b> (EG01377) was identified as having properties suitable for further investigation. Compound <b>1</b> was then tested in several in vitro assays and was shown to have antiangiogenic, antimigratory, and antitumor effects. Remarkably, <b>1</b> was shown to be selective for NRP1 over the closely related protein NRP2. In purified Nrp1⁺, FoxP3⁺, and CD25⁺ populations of Tregs from mice, <b>1</b> was able to block a glioma-conditioned medium-induced increase in TGFβ production. This comprehensive characterization of a small-molecule NRP1 antagonist provides the basis for future in vivo studies