Sulfur(VI) Fluoride Exchange Chemistry in Solid-Phase Synthesis of Compound Arrays: Discovery of Histone Deacetylase Inhibitors.

Multistep synthesis performed on solid support is a powerful means to generate small-molecule libraries for the discovery of chemical probes to dissect biological mechanisms as well as for drug discovery. Therefore, expansion of the collection of robust chemical transformations amenable to solid-phase synthesis is desirable for achieving chemically diverse libraries for biological testing. Here, we show that sulfur(VI) fluoride exchange (SuFEx) chemistry, exemplified by pairing phenols with aryl fluorosulfates, can be used for the solid-phase synthesis of biologically active compounds. As a case study, we designed and synthesized a library of 84 hydroxamic acid-containing small molecules, providing a rich source of inhibitors with diverse selectivity profiles across the human histone deacetylase enzyme family. Among other discoveries, we identified a scaffold that furnished inhibitors of HDAC11 with exquisite selectivity in vitro and a selective inhibitor of HDAC6 that was shown to affect the acetylation of α-tubulin over histone sites H3K18, H3K27, as well as SMC3 in cultured cells. Our results encourage the further use of SuFEx chemistry for the synthesis of diverse small-molecule libraries and provide insight for future design of selective HDAC inhibitors.

Contemporary Applications of Thioamides and Methods for Their Synthesis.

Thioamides are naturally occurring isosteres of amide bonds in which the chalcogen atom of the carbonyl is changed from oxygen to sulfur. This substitution gives rise to altered nucleophilicity and hydrogen bonding properties with importance for both chemical reactivity and non-covalent interactions. As such, thioamides have been introduced into biologically active compounds to achieve improved target affinity and/or stability towards hydrolytic enzymes but have also been applied as probes of protein and peptide folding and dynamics. Recently, a series of new methods have been developed for the synthesis of thioamides as well as their utilization in peptide chemistry. Further, novel strategies for the incorporation of thioamides into proteins have been developed, enabling both structural and functional studies to be performed. In this Review, we highlight the recent developments in the preparation of thioamides and their applications for peptide modification and study of protein function.

Substrates and Cyclic Peptide Inhibitors of the Oligonucleotide-Activated Sirtuin 7.

The sirtuins are NAD+ -dependent lysine deacylases, comprising seven isoforms (SIRT1-7) in humans, which are involved in the regulation of a plethora of biological processes, including gene expression and metabolism. The sirtuins share a common hydrolytic mechanism but display preferences for different ϵ-N-acyllysine substrates. SIRT7 deacetylates targets in nuclei and nucleoli but remains one of the lesser studied of the seven isoforms, in part due to a lack of chemical tools to specifically probe SIRT7 activity. Here we expressed SIRT7 and, using small-angle X-ray scattering, reveal SIRT7 to be a monomeric enzyme with a low degree of globular flexibility in solution. We developed a fluorogenic assay for investigation of the substrate preferences of SIRT7 and to evaluate compounds that modulate its activity. We report several mechanism-based SIRT7 inhibitors as well as de novo cyclic peptide inhibitors selected from mRNA-display library screening that exhibit selectivity for SIRT7 over other sirtuin isoforms, stabilize SIRT7 in cells, and cause an increase in the acetylation of H3 K18.

Aryl Fluorosulfate Based Inhibitors That Covalently Target the SIRT5 Lysine Deacylase.

The sirtuin enzymes are a family of lysine deacylases that regulate gene transcription and metabolism. Sirtuin 5 (SIRT5) hydrolyzes malonyl, succinyl, and glutaryl ϵ-N-carboxyacyllysine posttranslational modifications and has recently emerged as a vulnerability in certain cancers. However, chemical probes to illuminate its potential as a pharmacological target have been lacking. Here we report the harnessing of aryl fluorosulfate-based electrophiles as an avenue to furnish covalent inhibitors that target SIRT5. Alkyne-tagged affinity-labeling agents recognize and capture overexpressed SIRT5 in cultured HEK293T cells and can label SIRT5 in the hearts of mice upon intravenous injection of the compound. This work demonstrates the utility of aryl fluorosulfate electrophiles for targeting of SIRT5 and suggests this as a means for the development of potential covalent drug candidates. It is our hope that these results will serve as inspiration for future studies investigating SIRT5 and general sirtuin biology in the mitochondria.

Investigation of Carboxylic Acid Isosteres and Prodrugs for Inhibition of the Human SIRT5 Lysine Deacylase Enzyme.

Sirtuin 5 (SIRT5) is a protein lysine deacylase enzyme that regulates diverse biology by hydrolyzing ϵ-N-carboxyacyllysine posttranslational modifications in the cell. Inhibition of SIRT5 has been linked to potential treatment of several cancers but potent compounds with activity in cells have been lacking. Here we developed mechanism-based inhibitors that incorporate isosteres of a carboxylic acid residue that is important for high-affinity binding to the enzyme active site. By masking of the tetrazole moiety of the most potent candidate from our initial SAR study, we achieved potent and cytoselective growth inhibition for the treatment of SIRT5-dependent leukemic cancer cell lines in culture. Thus, we provide an efficient, cellularly active small molecule that targets SIRT5, which can help elucidate its function and potential as a future drug target. This work shows that masked isosteres of carboxylic acids are viable chemical motifs for the development of inhibitors that target mitochondrial enzymes, which may have applications beyond the sirtuin field.

Investigating GIPR (ant)agonism: A structural analysis of GIP and its receptor.

The glucose-dependent insulinotropic polypeptide (GIP) is a 42-residue metabolic hormone that is actively being targeted for its regulatory role of glycemia and energy balance. Limited structural data of its receptor has made ligand design tedious. This study investigates the structure and function of the GIP receptor (GIPR), using a homology model based on the GLP-1 receptor. Molecular dynamics combined with in vitro mutational data were used to pinpoint residues involved in ligand binding and/or receptor activation. Significant differences in binding mode were identified for the naturally occurring agonists GIP(1-30)NH2 and GIP(1-42) compared with high potency antagonists GIP(3-30)NH2 and GIP(5-30)NH2. Residues R1832.60, R1902.67, and R3005.40 are shown to be key for activation of the GIPR, and evidence suggests that a disruption of the K293ECL2-E362ECL3 salt bridge by GIPR antagonists strongly reduces GIPR activation. Combinatorial use of these findings can benefit rational design of ligands targeting the GIPR.