New inhibitor targeting human transcription factor HSF1: effects on the heat shock response and tumor cell survival.

Comparative modeling of the DNA-binding domain of human HSF1 facilitated the prediction of possible binding pockets for small molecules and definition of corresponding pharmacophores. In silico screening of a large library of lead-like compounds identified a set of compounds that satisfied the pharmacophoric criteria, a selection of which compounds was purchased to populate a biased sublibrary. A discriminating cell-based screening assay identified compound 001, which was subjected to systematic analysis of structure-activity relationships, resulting in the development of compound 115 (IHSF115). IHSF115 bound to an isolated HSF1 DNA-binding domain fragment. The compound did not affect heat-induced oligomerization, nuclear localization and specific DNA binding but inhibited the transcriptional activity of human HSF1, interfering with the assembly of ATF1-containing transcription complexes. IHSF115 was employed to probe the human heat shock response at the transcriptome level. In contrast to earlier studies of differential regulation in HSF1-naïve and -depleted cells, our results suggest that a large majority of heat-induced genes is positively regulated by HSF1. That IHSF115 effectively countermanded repression in a significant fraction of heat-repressed genes suggests that repression of these genes is mediated by transcriptionally active HSF1. IHSF115 is cytotoxic for a variety of human cancer cell lines, multiple myeloma lines consistently exhibiting high sensitivity.

Disruption of Proteostasis by Natural Products and Synthetic Compounds That Induce Pervasive Unfolding of Proteins: Therapeutic Implications.

Exposure of many cancer cells, including multiple myeloma cells, to cytotoxic concentrations of natural products celastrol and withaferin A or synthetic compounds of the IHSF series resulted in denaturation of a luciferase reporter protein. Proteomic analysis of detergent-insoluble extract fractions from HeLa-derived cells revealed that withaferin A, IHSF058 and IHSF115 caused denaturation of 915, 722 and 991 of 5132 detected cellular proteins, respectively, of which 440 were targeted by all three compounds. Western blots showed that important fractions of these proteins, in some cases approaching half of total protein amounts, unfolded. Relatively indiscriminate covalent modification of target proteins was observed; 1178 different proteins were modified by IHSF058. Further illustrating the depth of the induced proteostasis crisis, only 13% of these proteins detectably aggregated, and 79% of the proteins that aggregated were not targets of covalent modification. Numerous proteostasis network components were modified and/or found in aggregates. Proteostasis disruption caused by the study compounds may be more profound than that mediated by proteasome inhibitors. The compounds act by a different mechanism that may be less susceptible to resistance development. Multiple myeloma cells were particularly sensitive to the compounds. Development of an additional proteostasis-disrupting therapy of multiple myeloma is suggested.

Small Molecule Neuropilin-1 Antagonists Combine Antiangiogenic and Antitumor Activity with Immune Modulation through Reduction of Transforming Growth Factor Beta (TGFß) 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 1 (EG01377) was identified as having properties suitable for further investigation. Compound 1 was then tested in several in vitro assays and was shown to have antiangiogenic, antimigratory, and antitumor effects. Remarkably, 1 was shown to be selective for NRP1 over the closely related protein NRP2. In purified Nrp1+, FoxP3+, and CD25+ populations of Tregs from mice, 1 was able to block a glioma-conditioned medium-induced increase in TGFß production. This comprehensive characterization of a small-molecule NRP1 antagonist provides the basis for future in vivo studies.

Small molecule inhibitors of the neuropilin-1 vascular endothelial growth factor A (VEGF-A) interaction.

We report the molecular design and synthesis of EG00229, 2, the first small molecule ligand for the VEGF-A receptor neuropilin 1 (NRP1) and the structural characterization of NRP1-ligand complexes by NMR spectroscopy and X-ray crystallography. Mutagenesis studies localized VEGF-A binding in the NRP1 b1 domain and a peptide fragment of VEGF-A was shown to bind at the same site by NMR, providing the basis for small molecule design. Compound 2 demonstrated inhibition of VEGF-A binding to NRP1 and attenuated VEGFR2 phosphorylation in endothelial cells. Inhibition of migration of endothelial cells was also observed. The viability of A549 lung carcinoma cells was reduced by 2, and it increased the potency of the cytotoxic agents paclitaxel and 5-fluorouracil when given in combination. These studies provide the basis for design of specific small molecule inhibitors of ligand binding to NRP1.