David W. Christianson

Histone deacetylase 6 (HDAC6) plays an essential role in the regulation of post-translational lysine acetylation. Myriad biological processes depend on the precise regulation of lysine acetylation-deacetylation cycles, and their dysregulation is linked to human pathologies such as cancer and neurodegeneration. Broad-specificity HDAC inhibitors are efficacious in treating disease, but the inhibitors currently approved for clinical use often cause deleterious side effects due to inhibition of multiple HDAC isozymes as well as other zinc enzymes. Cyclic peptides comprise novel platforms that, in principle, can be tailored for affinity and selectivity. A key advantage of cyclic peptide design is the ability to incorporate both canonical and noncanonical amino acids. As part of our program focusing on cyclic peptide HDAC inhibitors, we now report the 1.9-Å resolution crystal structure of HDAC6 CD2 complexed with macrocyclic nonapeptide 1 . The structure reveals zinc coordination by a noncanonical thiolate-containing amino acid, plus direct and water-mediated enzyme-inhibitor hydrogen bonds that contribute to nanomolar inhibitory potency. Structural comparisons with previously reported HDAC6 CD2 complexes with tetrapeptide and octapeptide macrocycles reveal similar networks of direct and water-mediated interactions. Given the surge of interest in cyclic peptide inhibitors, fueled in part by advances in computational design approaches, the crystal structure of the HDAC6 CD2- 1 complex sets an important foundation for further development of macrocyclic peptide inhibitors. For Table of Contents Use Only:

We herein describe the structure-based design, synthesis and biological evaluation of a new class of histone deacetylase 6 (HDAC6) inhibitors guided by X-ray crystal structures of our previously identified compounds 13-15. X-ray co-crystal structures of second-generation inhibitors 19b (Subseries I) and 20b (Subseries II) within zHDAC6 CD2 confirmed that these compounds behave as highly potent HDAC6 inhibitors with the ability to engage the S531 residue in the L2 loop HDAC6 catalytic domain. Western-blot analysis confirmed the target engagement in cell-based settings, through the increase of acetylated tubulin levels, without significant variation in the histone acetylation status. Compound 20b extensively reduced the viability of A549 cells in both 2- and 3-dimensional cell cultures, through the activation of pro-autophagic pathways. In addition, HDAC6 inhibition impaired the nucleation of stress-granules, confirming the ability of these compounds to modulate liquid−liquid phase separation, an emerging HDAC6-driven hallmark in cancer.

More than one thousand bifunctional terpene synthases combining prenyltransferase and terpene cyclase activities have been identified in bacteria and fungi, but only a handful of enzymes have been identified that combine terpene cyclase activity with a downstream processing activity. Drimenol synthase from the marine bacterium Aquimarina spongiae (AsDMS) consists of a class II terpene cyclase that converts farnesyl diphosphate into drimenyl diphosphate, and a haloacid dehalogenase-like phosphatase that hydrolyzes drimenyl diphosphate to generate the sesquiterpene alcohol drimenol. The first crystal structure of AsDMS to be reported revealed the architecture of domain assembly as well as dimeric quaternary structure, establishing a structural chemical foundation for cyclization and hydrolysis mechanisms [K. R. Osika, M. N. Gaynes, D. W. Christianson (2025) Proc. Natl. Acad. Sci. U.S.A. 122 , e2506584122]. Here, we report crystal structures of the catalytically-inactive double mutant, D33A-D323A AsDMS, complexed with farnesyl diphosphate, geranyl diphosphate, and dimethylallyl diphosphate, which bind in the active sites of both the cyclase and phosphatase domains. Molecular recognition of the diphosphate group dominates binding interactions in both active sites. In the cyclase active site, only farnesyl diphosphate is sufficiently long for its terminal isoprenoid C=C bond to bind adjacent to the catalytic general acid that would initiate the cyclization cascade in the wild-type enzyme. In the phosphatase active site, all isoprenoid diphosphate groups bind similarly, but isoprenoid chain conformations vary. These structures provide a foundation for understanding substrate recognition and catalysis in both active sites. Finally, we present kinetic evidence suggesting that substrate channeling is operative in wild- type AsDMS.

Thioamides are natural post-translational modifications of the peptide backbone and can be introduced synthetically to probe protein folding or functionalize peptides for translational applications. In this work, we demonstrate that thioamide-containing peptides with C-terminal thioesters can be efficiently generated using Knorr pyrazole activation and used in subsequent native chemical ligation reactions to generate thioamide-containing proteins. We compare this method to acyl azide activation and find that both routes provide similar yields. We also investigate ultrasound-mediated desulfurization of the ligation site cysteine for potential advantages over chemical radical initiators. Scaling up our syntheses allows us to study thioamide perturbations to the β-sheet region of the B1 domain of protein G (GB1) as well as β-strand interactions in amyloid fibrils of the Parkinson’s disease protein α-synuclein. In both contexts, we observe dramatic destabilization of the β-sheet networks, manifested in decreased GB1 thermal stability and altered folding and slowed aggregation of α-synuclein. These findings illustrate the impact that a single atom substitution can have on cooperative hydrogen-bonding networks and prompt future study of both systems.

Thioamides are natural post-translational modifications of the peptide backbone and can be introduced synthetically to probe protein folding or functionalize peptides for translational applications. In this work, we demonstrate that thioamide-containing peptides with C-terminal thioesters can be efficiently generated using Knorr pyrazole activation and used in subsequent native chemical ligation reactions to generate thioamide containing proteins. We compare this method to acyl azide activation and find that both routes provide similar yields. We also investigate ultrasound-mediated desulfurization of the ligation site cysteine for potential advantages over chemical radical initiators. Scaling up our syntheses allows us to study thioamide perturbations to the β-sheet region of the B1 domain of protein G (GB1) as well as β-strand interactions in amyloid fibrils of the Parkinson’s disease protein α-synuclein. In both contexts, we observe dramatic destabilization of the β-sheet networks, manifested in decreased GB1 thermal stability and altered folding and slowed aggregation of α-synuclein. These findings illustrate the impact that a single atom substitution can have on cooperative hydrogen bonding networks and prompt future study of both systems.

The eleven known zinc-dependent histone deacetylases (HDACs) catalyze the deacetylation or deacylation of myriad protein and small molecule substrates throughout the cell. The biological functions of HDACs are much more diverse than the name HDAC implies, but this name is nonetheless retained for historical purposes. The chemical mechanism of catalysis is generally conserved among HDAC isozymes: Electrophilic activation of the substrate is achieved by zinc coordination and hydrogen bonding, and nucleophilic activation of a zinc-bound water molecule is enhanced by a general base. Since aberrant activity is observed for specific HDAC isozymes in certain diseases, the development of isozyme-selective inhibitors is a current priority in worldwide medicinal chemistry campaigns. In this review, the biological functions and chemical mechanisms of the HDACs are discussed to establish the molecular context of catalysis and inhibition, particularly as the chemistry of catalysis is harnessed in the development of mechanism-based inhibitors.

Histone deacetylase 6 (HDAC6) is a cytoplasmic enzyme that deacetylates non-histone substrates such as α-tubulin and cortactin. HDAC6 contains two catalytic domains, each containing a catalytic zinc ion, and a zinc-finger ubiquitin-binding domain. We have discovered BAS-2, a selective HDAC6 inhibitor with an isothiouronium core and no obvious zinc-binding group. To define its mechanism, we combine X-ray crystallography, structure-activity-relationships, molecular modeling and mutagenesis. BAS-2 potently inhibits human HDAC6 but it does not inhibit zebrafish HDAC6. Computational modeling highlighted Asp567 in human HDAC6 as critical for BAS-2 recognition and mutational analyses confirmed this. The corresponding zebrafish residue is Asn530 and the crystal structure of the N530D variant zHDAC6 revealed binding of a BAS-2–derived mercaptoacetamide that engages the catalytic zinc via strong thiolate–zinc coordination. Leveraging the orientation of BAS-2 binding, we designed a BAS-2–based proteolysis targeting chimera that induced proteasome-dependent HDAC6 degradation in cells, verified by global proteomics. Collectively, these insights clarify species selectivity and demonstrate that BAS-2 acts as a selective, mechanism-based inhibitor of human HDAC6. These discoveries will aid the development of the next generation of selective HDAC6 inhibitors and degraders.

Difluoromethyl-1,3,4-oxadiazoles are highly selective, mechanism-based inhibitors of histone deacetylase 6. The electrophilic CN bond of the heterocycle undergoes nucleophilic attack by zinc-bound water to yield a zinc-bound acylhydrazide anion.

Boronic acid inhibitors often undergo nucleophilic addition upon binding to an enzyme due to the electrophilicity of the boron atom. A new class of boronic acid inhibitors of human phosphodiesterase 3B (PDE3B) has recently been disclosed, along with the 2.7 Å-resolution crystal structure of PDE3B complexed with inhibitor GSK4394835A [Rowley et al. (2024) Discovery and SAR study of boronic acid-based selective PDE3B inhibitors from a novel DNA-encoded library. J. Med. Chem. 67, 2049–2065]. The crystal structure shows the binding of an intact, unreacted boronic acid, but discrepancies were evident in refinement statistics. Accordingly, we redetermined the structure using structure factor amplitudes deposited in the Protein Data Bank (accession code 8SYC), showing that the boronic acid moiety of GSK4394835A undergoes nucleophilic attack by H737 to form a tetrahedral boronate anion. We refined the structure to convergence with excellent refinement statistics, concluding that GSK4394835A is a reversible covalent inhibitor of PDE3B.