Development of First-in-Class Dual Sirt2/HDAC6 Inhibitors as Molecular Tools for Dual Inhibition of Tubulin Deacetylation.

Dysregulation of both tubulin deacetylases sirtuin 2 (Sirt2) and the histone deacetylase 6 (HDAC6) has been associated with the pathogenesis of cancer and neurodegeneration, thus making these two enzymes promising targets for pharmaceutical intervention. Herein, we report the design, synthesis, and biological characterization of the first-in-class dual Sirt2/HDAC6 inhibitors as molecular tools for dual inhibition of tubulin deacetylation. Using biochemical in vitro assays and cell-based methods for target engagement, we identified Mz325 (33) as a potent and selective inhibitor of both target enzymes. Inhibition of both targets was further confirmed by X-ray crystal structures of Sirt2 and HDAC6 in complex with building blocks of 33. In ovarian cancer cells, 33 evoked enhanced effects on cell viability compared to single or combination treatment with the unconjugated Sirt2 and HDAC6 inhibitors. Thus, our dual Sirt2/HDAC6 inhibitors are important new tools to study the consequences and the therapeutic potential of dual inhibition of tubulin deacetylation.

Structural Insights into the Substrate Range of a Bacterial Monoamine Oxidase.

Monoamine oxidases (MAOs) play a key role in the breakdown of primary and secondary amines. In eukaryotic organisms, these enzymes are vital to the regulation of monoamine neurotransmitters and the degradation of dietary monoamines. MAOs have also been identified in prokaryotic species, although their role in these organisms is not well understood. Here, we report the biophysical and structural properties of a promiscuous, bacterial MAO from Corynebacterium ammoniagenes (caMAO). caMAO catalyzes the oxidation of a number of monoamine substrates including dopamine and norepinephrine, as well as exhibiting some activity with polyamine substrates such as cadaverine. The X-ray crystal structures of Michaelis complexes with seven substrates show that conserved hydrophobic interactions and hydrogen-bonding pattern (for polar substrates) allow the broad specificity range. The structure of caMAO identifies an unusual cysteine (Cys424) residue in the so-called "aromatic cage", which flanks the flavin isoalloxazine ring in the active site. Site-directed mutagenesis, steady-state kinetics in air-saturated buffer, and UV-vis spectroscopy revealed that Cys424 plays a role in the pH dependence and modulation of electrostatics within the caMAO active site. Notably, bioinformatic analysis shows a propensity for variation at this site within the "aromatic cage" of the flavin amine oxidase (FAO) superfamily. Structural analysis also identified the conservation of a secondary substrate inhibition site, present in a homologous member of the superfamily. Finally, genome neighborhood diagram analysis of caMAO in the context of the FAO superfamily allows us to propose potential roles for these bacterial MAOs in monoamine and polyamine degradation and catabolic pathways related to scavenging of nitrogen.

Structure of the Repurposed Fungal Terpene Cyclase FlvF Implicated in the C-N Bond-Forming Reaction of Flavunoidine Biosynthesis.

The fungal species Aspergillus flavus produces an alkaloid terpenoid, flavunoidine, through a hybrid biosynthetic pathway combining both terpene cyclase and nonribosomal peptide synthetase enzymes. Flavunoidine consists of a tetracyclic, oxygenated sesquiterpene core decorated with dimethyl cadaverine and 5,5-dimethyl-l-pipecolate moieties. Unique to the flavunoidine biosynthetic pathway is FlvF, a putative enzyme implicated in stereospecific C-N bond formation as dimethyl cadaverine is linked to the sesquiterpene core to generate pre-flavunoidine. Here, we report the 2.6 Å resolution crystal structure of FlvF, which adopts the α-helical fold of a class I terpene synthase. However, FlvF is not a terpene synthase, as indicated by its lack of enzymatic activity with farnesyl diphosphate and its lack of signature metal ion binding motifs that would coordinate to catalytic Mg2+ ions. Thus, FlvF is the first example of a protein that adopts a terpene synthase fold but is not a terpene synthase. Two Bis-Tris molecules bind in the active site of FlvF, and the binding of these ligands guided the docking of pre-flavunoidine to generate a model of the enzyme-product complex. Phylogenetic analysis of FlvF and related fungal homologues reveals conservation of residues that interact with the tetracyclic sesquiterpene in this model, but less conservation of residues interacting with the pendant amino moiety. This may hint toward the possibility that alternative amino substrates can be linked to a common sesquiterpene core by FlvF homologues to generate flavunoidine congeners, such as the phospholipase C inhibitor hispidospermidin.

Fast Kinetics Reveals Rate-Limiting Oxidation and the Role of the Aromatic Cage in the Mechanism of the Nicotine-Degrading Enzyme NicA2.

In Pseudomonas putida, the flavoprotein nicotine oxidoreductase (NicA2) catalyzes the oxidation of (S)-nicotine to N-methyl-myosmine, which is nonenzymatically hydrolyzed to pseudooxynicotine. Structural analysis reveals a monoamine oxidase (MAO)-like fold with a conserved FAD-binding domain and variable substrate-binding domain. The flavoenzyme has a unique variation of the classic aromatic cage with flanking residue pair W427/N462. Previous mechanistic studies using O2 as the oxidizing substrate show that NicA2 has a low apparent Km of 114 nM for (S)-nicotine with a very low apparent turnover number (kcat of 0.006 s-1). Herein, the mechanism of NicA2 was analyzed by transient kinetics. Single-site variants of W427 and N462 were used to probe the roles of these residues. Although several variants had moderately higher oxidase activity (7-12-fold), their reductive half-reactions using (S)-nicotine were generally significantly slower than that of wild-type NicA2. Notably, the reductive half-reaction of wild-type NicA2 is 5 orders of magnitude faster than the oxidative half-reaction with an apparent pseudo-first-order rate constant for the reaction of oxygen similar to kcat. X-ray crystal structures of the N462V and N462Y/W427Y variants complexed with (S)-nicotine (at 2.7 and 2.3 Å resolution, respectively) revealed no significant active-site rearrangements. A second substrate-binding site was identified in N462Y/W427Y, consistent with observed substrate inhibition. Together, these findings elucidate the mechanism of a flavoenzyme that preferentially oxidizes tertiary amines with an efficient reductive half-reaction and a very slow oxidative half-reaction when O2 is the oxidizing substrate, suggesting that the true oxidizing agent is unknown.

Catch and Anchor Approach To Combat Both Toxicity and Longevity of Botulinum Toxin A.

Botulinum neurotoxins have remarkable persistence (∼weeks to months in cells), outlasting the small-molecule inhibitors designed to target them. To address this disconnect, inhibitors bearing two pharmacophores-a zinc binding group and a Cys-reactive warhead-were designed to leverage both affinity and reactivity. A series of first-generation bifunctional inhibitors was achieved through structure-based inhibitor design. Through X-ray crystallography, engagement of both the catalytic Zn2+ and Cys165 was confirmed. A second-generation series improved on affinity by incorporating known reversible inhibitor pharmacophores; the mechanism was confirmed by exhaustive dialysis, mass spectrometry, and in vitro evaluation against the C165S mutant. Finally, a third-generation inhibitor was shown to have good cellular activity and low toxicity. In addition to our findings, an alternative method of modeling time-dependent inhibition that simplifies assay setup and allows comparison of inhibition models is discussed.

Bioinformatic Analysis of the Flavin-Dependent Amine Oxidase Superfamily: Adaptations for Substrate Specificity and Catalytic Diversity.

The flavin-dependent amine oxidase (FAO) superfamily consists of over 9000 nonredundant sequences represented in all domains of life. Of the thousands of members identified, only 214 have been functionally annotated to date, and 40 unique structures are represented in the Protein Data Bank. The few functionally characterized members share a catalytic mechanism involving the oxidation of an amine substrate through transfer of a hydride to the FAD cofactor, with differences observed in substrate specificities. Previous studies have focused on comparing a subset of superfamily members. Here, we present a comprehensive analysis of the FAO superfamily based on reaction mechanism and substrate recognition. Using a dataset of 9192 sequences, a sequence similarity network, and subsequently, a genome neighborhood network were constructed, organizing the superfamily into eight subgroups that accord with substrate type. Likewise, through phylogenetic analysis, the evolutionary relationship of subgroups was determined, delineating the divergence between enzymes based on organism, substrate, and mechanism. In addition, using sequences and atomic coordinates of 22 structures from the Protein Data Bank to perform sequence and structural alignments, active-site elements were identified, showing divergence from the canonical aromatic-cage residues to accommodate large substrates. These specificity determinants are held in a structural framework comprising a core domain catalyzing the oxidation of amines with an auxiliary domain for substrate recognition. Overall, analysis of the FAO superfamily reveals a modular fold with cofactor and substrate-binding domains allowing for diversity of recognition via insertion/deletions. This flexibility allows facile evolution of new activities, as shown by reinvention of function between subfamilies.

Crystallography Coupled with Kinetic Analysis Provides Mechanistic Underpinnings of a Nicotine-Degrading Enzyme.

Nicotine oxidoreductase (NicA2) is a bacterial flavoenzyme, which catalyzes the first step of nicotine catabolism by oxidizing S-nicotine into N-methyl-myosmine. It has been proposed as a biotherapeutic for nicotine addiction because of its nanomolar substrate binding affinity. The first crystal structure of NicA2 has been reported, establishing NicA2 as a member of the monoamine oxidase (MAO) family. However, substrate specificity and structural determinants of substrate binding and/or catalysis have not been explored. Herein, analysis of the pH-rate profile, single-turnover kinetics, and binding data establish that pH does not significantly affect the catalytic rate and product release is not rate-limiting. The X-ray crystal structure of NicA2 with S-nicotine refined to 2.65 Å resolution reveals a hydrophobic binding site with a solvent exclusive cavity. Hydrophobic interactions predominantly orient the substrate, promoting the binding of a deprotonated species and supporting a hydride-transfer mechanism. Notably, NicA2 showed no activity against neurotransmitters oxidized by the two isoforms of human MAO. To further probe the substrate range of NicA2, enzyme activity was evaluated using a series of substrate analogues, indicating that S-nicotine is the optimal substrate and substitutions within the pyridyl ring abolish NicA2 activity. Moreover, mutagenesis and kinetic analysis of active-site residues reveal that removal of a hydrogen bond between the pyridyl ring of S-nicotine and the hydroxyl group of T381 has a 10-fold effect on KM, supporting the role of this bond in positioning the catalytically competent form of the substrate. Together, crystallography combined with kinetic analysis provides a deeper understanding of this enzyme's remarkable specificity.

Structural Analysis Provides Mechanistic Insight into Nicotine Oxidoreductase from Pseudomonas putida.

The first structure of nicotine oxidoreductase (NicA2) was determined by X-ray crystallography. Pseudomonas putida has evolved nicotine-degrading activity to provide a source of carbon and nitrogen. The structure establishes NicA2 as a member of the monoamine oxidase family. Residues 1-50 are disordered and may play a role in localization. The nicotine-binding site proximal to the isoalloxazine ring of flavin shows an unusual composition of the classical aromatic cage (W427 and N462). The active site architecture is consistent with the proposed binding of the deprotonated form of the substrate and the flavin-dependent oxidation of the pyrrolidone C-N bond followed by nonenzymatic hydrolysis.