Histone deacetylase (HDAC) enzymes are epigenetic regulators that affect diverse protein function by removing acyl groups from lysine side chains throughout the proteome. The most recently discovered human isozyme, HDAC11, differs from other HDACs in substrate preference and tissue expression profile. Elucidation of the biological function of this enzyme has been scarce and only a few chemical probes to help advance this insight have been developed thus far. Here we discovered macrocyclic inhibitors that exhibit selectivity for HDAC11 and penetrate the cytoplasmic membrane in cultured cells as determined by the chloroalkane penetration assay. Our work establishes the combination of de novo macrocycle synthesis with incorporation of N-alkylated hydroxamic acid moieties as a viable strategy for targeting HDAC11. Further, this study demonstrates the potential of applying macrocyclic peptide-based library synthesis to directly furnish high-affinity, cell-permeating ligands. The discovered inhibitors comprise tool compounds for the investigation of the biological function of HDAC11.

Despite recent FDA approvals, Alzheimer's disease (AD) still represents an unmet medical need. Among the different available therapeutic approaches, the development of multitarget molecules represents one of the most widely pursued. In this work, we present a second generation of dual ligands directed toward highly networked targets that are deeply involved in the development of the disease, namely, Histone Deacetylases (HDACs) and Glycogen Synthase Kinase 3β (GSK-3β). The synthesized compounds are highly potent GSK-3β, HDAC2, and HDAC6 inhibitors with IC50 values in the nanomolar range of concentrations. Among them, compound 4 inhibits histone H3 and tubulin acetylation at 0.1 μM concentration, blocks hyperphosphorylation of tau protein, and shows interesting immunomodulatory and neuroprotective properties. These features, together with its ability to cross the blood-brain barrier and its favorable physical-chemical properties, make compound 4 a promising hit for the development of innovative disease-modifying agents.

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 ex-change (SuFEx) chemistry, exemplified by pairing phenols with aryl fluorosulfates, can be used for solid-phase synthesis of biologically active compounds. As a case study, we designed and synthesized a library of 84 hy-droxamic acid containing small molecules, providing a rich source of inhibitors with diverse selectivity pro-files across the human histone deacetylase enzyme family, which is a validated drug target. Among other dis-coveries, we identified a scaffold that furnished inhibitors of HDAC11 with exquisite selectivity in vitro and a selective inhibitor of HDAC6 that was shown to bind this target enzyme selectively over HDAC8 in cells, using cellular thermal shift assays (CETSA). Our results encourage the further use of SuFEx chemistry for the syn-thesis of diverse small molecule libraries, provides insight for future design of selective HDAC inhibitors, and show that CETSA can be applied for evaluation of cellular target engagement of HDAC inhibitors.

Posttranslational modifications alter the structure and function of proteins. Now, genetic code expansion enables encoding of epsilon-N-succinyllysine and epsilon-N-glutaryllysine residues to decipher the effects of these modifications on enzymatic activity, protein-protein interactions and protein-DNA interactions.

The postsynaptic density (PSD) comprises numerous scaffolding proteins, receptors, and signaling molecules that coordinate synaptic transmission in the brain. Postsynaptic density protein 95 (PSD-95) is a master scaffold protein within the PSD and one of its most abundant proteins and therefore constitutes a very attractive biomarker of PSD function and its pathological changes. Here, we exploit a high-affinity inhibitor of PSD-95, AVLX-144, as a template for developing probes for molecular imaging of the PSD. AVLX-144-based probes were labeled with the radioisotopes fluorine-18 and tritium, as well as a fluorescent tag. Tracer binding showed saturable, displaceable, and uneven distribution in rat brain slices, proving effective in quantitative autoradiography and cell imaging studies. Notably, we observed diminished tracer binding in human post-mortem Parkinson’s disease (PD) brain slices, suggesting postsynaptic impairment in PD. We thus offer a suite of translational probes for visualizing and understanding PSD-related pathologies.

Lysine L-lactylation [K(L-la)] is a newly discovered histone mark stimulated under conditions of high glycolysis, such as the Warburg effect. K(L-la) is associated with functions that are different from the widely studied histone acetylation. While K(L-la) can be introduced by the acetyltransferase p300, histone delactylases enzymes remained unknown. Here, we report the systematic evaluation of zinc- and nicotinamide adenine dinucleotide–dependent histone deacetylases (HDACs) for their ability to cleave ε-N-L-lactyllysine marks. Our screens identified HDAC1–3 and SIRT1–3 as delactylases in vitro. HDAC1–3 show robust activity toward not only K(L-la) but also K(D-la) and diverse short-chain acyl modifications. We further confirmed the de-L-lactylase activity of HDACs 1 and 3 in cells. Together, these data suggest that histone lactylation is installed and removed by regulatory enzymes as opposed to spontaneous chemical reactivity. Our results therefore represent an important step toward full characterization of this pathway’s regulatory elements.