Lysine acetylation within the tandem repeat region of cortactin (CTTN) regulates its actin-binding function and has been linked to cancer cell migration and neuronal development. While several lysine deacetylases (KDACs) have been implicated in modulating CTTN acetylation in cells, their site specificity and direct enzymatic roles remain poorly defined. Here, we use genetic code expansion to generate seven site-specifically acetylated CTTN variants and assess their deacetylation by human KDACs in a fully reconstituted in vitro system. Our results identify HDAC6 as the primary CTTN deacetylase, acting via its second catalytic domain (DD2), and demonstrate that SIRT1 and SIRT2 also directly deacetylate CTTN at overlapping sites in an NAD+-dependent manner. In contrast, other zinc-dependent HDACs, including HDAC8, displayed negligible or very weak activity on full-length CTTN. These findings provide new mechanistic insight into KDAC substrate preferences and highlight the value of biochemical reconstitution for dissecting complex acetylation networks.

The sulfonamide function is used extensively as a general building block in various inhibitory scaffolds and, more specifically, as a zinc-binding group (ZBG) of metalloenzyme inhibitors. Here, we provide biochemical, structural, and computational characterization of a metallopeptidase in complex with inhibitors, where the mono- and bisubstituted sulfamide functions are designed to directly engage zinc ions of a bimetallic enzyme site. Structural data showed that while monosubstituted sulfamides coordinate active-site zinc ions via the free negatively charged amino group in a canonical manner, their bisubstituted counterparts adopt an atypical binding pattern divergent from expected positioning of corresponding tetrahedral reaction intermediates. Accompanying quantum mechanics calculations revealed that electroneutrality of the sulfamide function is a major factor contributing to the markedly lower potency of bisubstituted compounds by considerably lowering their interaction energy with the enzyme. Overall, while bisubstituted uncharged sulfamide functions can bolster favorable pharmacological properties of a given inhibitor, their use as ZBGs in metalloenzyme inhibitors might be less advantageous due to their suboptimal metal-ligand properties.

A plethora of compounds can reach our drinking water and possibly affect human health. Still, mostly notorious pollutants like pesticides and disinfection by-products are monitored and regulated. With the increasing availability of high-resolution mass spectrometers (HRMS), non-targeted analyses of environmental samples have become possible. Pilot studies demonstrating the applicability of this approach in the drinking water research field were published. We would like to highlight these studies and appeal to researchers focused on water quality to better exploit the potential of HRMS instruments and broaden the scale of studied pollutants. In addition, the data and experience should be further shared, and the quality standard for the analytical procedures should be set. With advanced knowledge of compounds reaching the drinking water, potential threats would be revealed, and the comprehensive results on water pollution might also act as impulses for associated research branches, including toxicity assessment or development of water treatment technologies, and/or for policy-making.

Human glutamate carboxypeptidase 2 (GCP2) from the M28B metalloprotease group is an important target for therapy in neurological disorders and an established tumor marker. However, its physiological functions remain unclear. To better understand general roles, we used the model organism Caenorhabditis elegans to genetically manipulate its three existing orthologous genes and evaluate the impact on worm physiology. The results of gene knockout studies showed that C. elegans GCP2 orthologs affect the pharyngeal physiology, reproduction, and structural integrity of the organism. Promoter-driven GFP expression revealed distinct localization for each of the three gene paralogs, with gcp-2.1 being most abundant in muscles, intestine, and pharyngeal interneurons, gcp-2.2 restricted to the phasmid neurons, and gcp-2.3 located in the excretory cell. The present study provides new insight into the unique phenotypic effects of GCP2 gene knockouts in C. elegans, and the specific tissue localizations. We believe that elucidation of particular roles in a non-mammalian organism can help to explain important questions linked to physiology of this protease group and in extension to human GCP2 involvement in pathophysiological processes.

Glutamate carboxypeptidase II (GCPII) is a metalloprotease implicated in neurological diseases and prostate oncology. While several classes of potent GCPII-specific inhibitors exist, the development of novel active scaffolds with different pharmacological profiles remains a challenge. Virtual screening followed by in vitro testing is an effective means for the discovery of novel active compounds. Structure- and ligand-based pharmacophore models were created based on a dataset of known GCPII-selective ligands. These models were used in a virtual screening of the SPECS compound library (∼209.000 compounds). Fifty top-scoring virtual hits were further experimentally tested for their ability to inhibit GCPII enzymatic activity in vitro. Six hits were found to have moderate to high inhibitory potency with the best virtual hit, a modified xanthene, inhibiting GCPII with an IC50 value of 353 ± 24 nM. The identification of this novel inhibitory scaffold illustrates the applicability of pharmacophore-based modeling for the discovery of GCPII-specific inhibitors.

Histone deacetylase 6 (HDAC6) is a unique member of the HDAC family of enzymes due to its complex domain organization and cytosolic localization. Experimental data point toward the therapeutic use of HDAC6-selective inhibitors (HDAC6is) for use in both neurological and psychiatric disorders. In this article, we provide side-by-side comparisons of hydroxamate-based HDAC6is frequently used in the field and a novel HDAC6 inhibitor containing the difluoromethyl-1,3,4-oxadiazole function as an alternative zinc-binding group (compound 7). In vitro isotype selectivity screening uncovered HDAC10 as a primary off-target for the hydroxamate-based HDAC6is, while compound 7 features exquisite 10,000-fold selectivity over all other HDAC isoforms. Complementary cell-based assays using tubulin acetylation as a surrogate readout revealed approximately 100-fold lower apparent potency for all compounds. Finally, the limited selectivity of a number of these HDAC6is is shown to be linked to cytotoxicity in RPMI-8226 cells. Our results clearly show that off-target effects of HDAC6is must be considered before attributing observed physiological readouts solely to HDAC6 inhibition. Moreover, given their unparalleled specificity, the oxadiazole-based inhibitors would best be employed either as research tools in further probing HDAC6 biology or as leads in the development of truly HDAC6-specific compounds in the treatment of human disease states.

Human glutamate carboxypeptidase 2 (GCP2) from the M28B metalloprotease group is an important target for therapy in neurological disorders and an established tumor marker. However, its physiological functions remain unclear. To better understand general roles, we used the model organism Caenorhabditis elegans genetically manipulate its three existing orthologous genes and evaluate the impact on worm physiology. The results of gene knockout studies showed that C. elegans GCP2 orthologs affect the pharyngeal physiology, reproduction, and structural integrity of the organism. Promoter-driven GFP expression revealed distinct localization for each of the three gene paralogs, with gcp-2 . 1 being most abundant in muscles, intestine, and pharyngeal interneurons, gcp-2 . 2 restricted to the phasmid neurons, and gcp-2 . 3 located in the excretory cell. This study provides new insight into the unique phenotypic effects of GCP2 gene knockouts in C. elegans , and the specific tissue localizations. We believe that elucidation of particular roles in a non-mammalian organism can help to explain important questions linked to human GCP2 physiology and in extension to GCP2 involvement in pathophysiological processes.

Histone deacetylase (HDAC) inhibitors used in the clinic typically contain a hydroxamate zinc-binding group (ZBG). However, more recent work has shown that the use of alternative ZBGs, and, in particular, the heterocyclic oxadiazoles, can confer higher isoenzyme selectivity and more favorable ADMET profiles. Herein, we report on the synthesis and biochemical, crystallographic, and computational characterization of a series of oxadiazole-based inhibitors selectively targeting the HDAC6 isoform. Surprisingly, but in line with a very recent finding reported in the literature, a crystal structure of the HDAC6/inhibitor complex revealed that hydrolysis of the oxadiazole ring transforms the parent oxadiazole into an acylhydrazide through a sequence of two hydrolytic steps. An identical cleavage pattern was also observed both in vitro using the purified HDAC6 enzyme as well as in cellular systems. By employing advanced quantum and molecular mechanics (QM/MM) and QM calculations, we elucidated the mechanistic details of the two hydrolytic steps to obtain a comprehensive mechanistic view of the double hydrolysis of the oxadiazole ring. This was achieved by fully characterizing the reaction coordinate, including identification of the structures of all intermediates and transition states, together with calculations of their respective activation (free) energies. In addition, we ruled out several (intuitively) competing pathways. The computed data (ΔG‡ ≈ 21 kcal·mol–1 for the rate-determining step of the overall dual hydrolysis) are in very good agreement with the experimentally determined rate constants, which a posteriori supports the proposed reaction mechanism. We also clearly (and quantitatively) explain the role of the −CF3 or −CHF2 substituent on the oxadiazole ring, which is a prerequisite for hydrolysis to occur. Overall, our data provide compelling evidence that the oxadiazole warheads can be efficiently transformed within the active sites of target metallohydrolases to afford reaction products possessing distinct selectivity and inhibition profiles.

Quantum and molecular mechanics (QM/MM) and QM-only (cluster model) modeling techniques represent the two workhorses in mechanistic understanding of enzyme catalysis. One of the stringent tests for QM/MM and/or QM approaches is to provide quantitative answers to real-world biochemical questions, such as the effect of single-point mutations on enzyme kinetics. This translates into predicting the relative activation energies to 1-2 kcal·mol-1 accuracy; such predictions can be used for the rational design of novel enzyme variants with desired/improved characteristics. Herein, we employ glutamate carboxypeptidase II (GCPII), a dizinc metallopeptidase, also known as the prostate specific membrane antigen, as a model system. The structure and activity of this major cancer antigen have been thoroughly studied, both experimentally and computationally, which makes it an ideal model system for method development. Its reaction mechanism is quite well understood: the reaction coordinate comprises a "tetrahedral intermediate" and two transition states and experimental activation Gibbs free energy of ∼17.5 kcal·mol-1 can be inferred for the known kcat ≈ 1 s-1. We correlate experimental kinetic data (including the E424H variant, newly characterized in this work) for various GCPII mutants (kcat = 8.6 × 10-5 s-1 to 2.7 s-1) with the energy profiles calculated by QM/MM and QM-only (cluster model) approaches. We show that the near-quantitative agreement between the experimental values and the calculated activation energies (ΔH⧧) can be obtained and recommend the combination of the two protocols: QM/MM optimized structures and cluster model (QM) energetics. The trend in relative activation energies is mostly independent of the QM method (DFT functional) used. Last but not least, a satisfactory correlation between experimental and theoretical data allows us to provide qualitative and fairly simple explanations of the observed kinetic effects which are thus based on a rigorous footing.

Glutamate carboxypeptidase 2 (GCP2) belongs to the M28B metalloprotease subfamily encompassing a variety of zinc-dependent exopeptidases that can be found in many eukaryotes, including unicellular organisms. Limited information exists on the physiological functions of GCP2 orthologs in mammalian tissues outside of the brain and intestine, and such data are completely absent for non-mammalian species. Here, we investigate GCP2 orthologs found in trematodes, not only as putative instrumental molecules for defining their basal function(s) but also as drug targets. Identified genes encoding M28B proteases Schistosoma mansoni and Fasciola hepatica genomes were analyzed and annotated. Homology modeling was used to create three-dimensional models of SmM28B and FhM28B proteins using published X-ray structures as the template. For S. mansoni, RT-qPCR was used to evaluate gene expression profiles, and, by RNAi, we exploited the possible impact of knockdown on the viability of worms. Enzymes from both parasite species were cloned for recombinant expression. Polyclonal antibodies raised against purified recombinant enzymes and RNA probes were used for localization studies in both parasite species. Single genes encoding M28B metalloproteases were identified in the genomes of S. mansoni and F. hepatica. Homology models revealed the conserved three-dimensional fold as well as the organization of the di-zinc active site. Putative peptidase activities of purified recombinant proteins were assayed using peptidic libraries, yet no specific substrate was identified, pointing towards the likely stringent substrate specificity of the enzymes. The orthologs were found to be localized in reproductive, digestive, nervous, and sensory organs as well as parenchymal cells. Knockdown of gene expression by RNAi silencing revealed that the genes studied were non-essential for trematode survival under laboratory conditions, reflecting similar findings for GCP2 KO mice. Our study offers the first insight to our knowledge into M28B protease orthologs found in trematodes. Conservation of their three-dimensional structure, as well as tissue expression pattern, suggests that trematode GCP2 orthologs may have functions similar to their mammalian counterparts and can thus serve as valuable models for future studies aimed at clarifying the physiological role(s) of GCP2 and related subfamily proteases.

Class IIa histone deacetylases (HDACs) play critical roles in vertebrate development and physiology, yet direct evidence of their intrinsic deacetylase activity and on substrate specificity regarding the peptide sequence is still missing. In this study, we designed and synthesized a combinatorial peptide library allowing us to profile class IIa HDACs sequence specificity at positions +3 through -3 from the central lysine modified by the well-accepted trifluoroacetyl function. Our data revealed a strong preference for bulky aromatic acids directly flanking the central trifluoroacetyllysine, while all class IIa HDACs disfavor positively charged residues and proline at the +1/-1 positions. The chemical nature of amino acid residues N-terminally to the central trifluoroacetyllysine has a more profound effect on substrate recognition as compared to residues located C-terminally. These findings were validated by designing selected favored and disfavored peptide sequences, with the favored ones are accepted with catalytic efficacy of 75 000 and 525 000 M-1 s(-1) for HDAC7 and HDAC5, respectively. Results reported here could help in developing class IIa HDACs inhibitors and also in the search for new natural class IIa HDACs substrates.

In humans, disruptions in the heme biosynthetic pathway are associated with various types of porphyrias, including variegate porphyria that results from the decreased activity of protoporphyrinogen oxidase IX (PPO; E.C.1.3.3.4), the enzyme catalyzing the penultimate step of the heme biosynthesis. Here we report the generation and characterization of human cell lines, in which PPO was inactivated using the CRISPR/Cas9 system. The PPO knock-out (PPO-KO) cell lines are viable with the normal proliferation rate and show massive accumulation of protoporphyrinogen IX, the PPO substrate. Observed low heme levels trigger a decrease in the amount of functional heme containing respiratory complexes III and IV and overall reduced oxygen consumption rates. Untargeted proteomics further revealed dysregulation of 22 cellular proteins, including strong upregulation of 5-aminolevulinic acid synthase, the major regulatory protein of the heme biosynthesis, as well as additional ten targets with unknown association to heme metabolism. Importantly, knock-in of PPO into PPO-KO cells rescued their wild-type phenotype, confirming the specificity of our model. Overall, our model system exploiting a non-erythroid human U-2 OS cell line reveals physiological consequences of the PPO ablation at the cellular level and can serve as a tool to study various aspects of dysregulated heme metabolism associated with variegate porphyria.

Histone deacetylase 6 (HDAC6) is a promising therapeutic target for the treatment of neurodegenerative disorders. SW-100 (1a), a phenylhydroxamate-based HDAC6 inhibitor (HDAC6i) bearing a tetrahydroquinoline (THQ) capping group, is a highly potent and selective HDAC6i that was shown to be effective in mouse models of Fragile X syndrome and Charcot-Marie-Tooth disease type 2A (CMT2A). In this study, we report the discovery of a new THQ-capped HDAC6i, termed SW-101 (1s), that possesses excellent HDAC6 potency and selectivity, together with markedly improved metabolic stability and druglike properties compared to SW-100 (1a). X-ray crystallography data reveal the molecular basis of HDAC6 inhibition by SW-101 (1s). Importantly, we demonstrate that SW-101 (1s) treatment elevates the impaired level of acetylated α-tubulin in the distal sciatic nerve, counteracts progressive motor dysfunction, and ameliorates neuropathic symptoms in a CMT2A mouse model bearing mutant MFN2. Taken together, these results bode well for the further development of SW-101 (1s) as a disease-modifying HDAC6i.

Variegate porphyria is caused by mutations in the protoporphyrinogen oxidase IX (PPOX, EC 1.3.3.4) gene, resulting in reduced overall enzymatic activity of PPOX in human tissues. Recently, we have identified the His333Arg mutation in the PPOX protein (PPOX(H333R)) as a putative founder mutation in the Moroccan Jewish population. Herein we report the molecular characterization of PPOX(H333R) in vitro and in cells. Purified recombinant PPOX(H333R) did not show any appreciable enzymatic activity in vitro, corroborating the clinical findings. Biophysical experiments and molecular modeling revealed that PPOX(H333R) is not folded properly and fails to adopt its native functional three-dimensional conformation due to steric clashes in the vicinity of the active site of the enzyme. On the other hand, PPOX(H333R) subcellular distribution, as evaluated by live-cell confocal microscopy, is unimpaired suggesting that the functional three-dimensional fold is not required for efficient transport of the polypeptide chain into mitochondria. Overall, the data presented here provide molecular underpinnings of the pathogenicity of PPOX(H333R) and might serve as a blueprint for deciphering whether a given PPOX variant represents a disease-causing mutation.

In silico methods like molecular docking and pharmacophore modeling are established strategies in lead identification. Their successful application for finding new active molecules for a target is reported by a plethora of studies. However, once a potential lead is identified, lead optimization, with the focus on improving potency, selectivity, or pharmacokinetic parameters of a parent compound, is a much more complex task. Even though in silico molecular modeling methods could contribute a lot of time and cost-saving by rationally filtering synthetic optimization options, they are employed less widely in this stage of research. In this review, we highlight studies that have successfully used computer-aided SAR analysis in lead optimization and want to showcase sound methodology and easily accessible in silico tools for this purpose.

Surgery is an efficient way to treat localized prostate cancer (PCa), however, it is challenging to demarcate rapidly and accurately the tumor boundary intraoperatively, as existing tumor detection methods are seldom performed in real-time. To overcome those limitations, we develop a fluorescent molecular rotor that specifically targets the prostate-specific membrane antigen (PSMA), an established marker for PCa. The probes have picomolar affinity (IC50 = 63-118 pM) for PSMA and generate virtually instantaneous onset of robust fluorescent signal proportional to the concentration of the PSMA-probe complex. In vitro and ex vivo experiments using PCa cell lines and clinical samples, respectively, indicate the utility of the probe for biomedical applications, including real-time monitoring of endocytosis and tumor staging. Experiments performed in a PCa xenograft model reveal suitability of the probe for imaging applications in vivo. Detection of the tumour boundary in prostate cancer is required for surgery. Here the authors present a fluorescent molecular rotor probe to target a prostate cancer marker, prostate-specific membrane antigen (PSMA), which they use in a xenograft mouse model to show it can be used for in vivo imaging.

Prostate-specific membrane antigen (PSMA) is an excellent biomarker for the early diagnosis of prostate cancer progression and metastasis. The most promising PSMA-targeted agents in the clinical phase are based on the Lys-urea-Glu motif, in which Lys and Glu are α-(l)-amino acids. In this study, we aimed to determine the effect of β- and γ-amino acids in the S1 pocket on the binding affinity for PSMA. We synthesized and evaluated the β- and γ-amino acid analogues with (S)- or (R)-configuration with keeping α-(l)-Glu as the S1'-binding pharmacophore. The structure-activity relationship studies identified that compound 13c, a β-amino acid analogue with (R)-configuration, exhibited the most potent PSMA inhibitory activity with an IC50 value of 3.97 nM. The X-ray crystal structure of PSMA in complex with 13c provided a mechanistic basis for the stereochemical preference of PSMA, which can guide the development of future PSMA inhibitors.

Histone deacetylase 6 (HDAC6) is a multidomain cytosolic enzyme having tubulin deacetylase activity that has been unequivocally assigned to the second of the tandem catalytic domains. However, virtually no information exists on the contribution of other HDAC6 domains on tubulin recognition. Here, using recombinant protein expression, site-directed mutagenesis, fluorimetric and biochemical assays, microscale thermophoresis, and total internal reflection fluorescence microscopy, we identified the N-terminal, disordered region of HDAC6 as a microtubule-binding domain and functionally characterized it to the single-molecule level. We show that the microtubule-binding motif spans two positively charged patches comprising residues Lys-32 to Lys-58. We found that HDAC6-microtubule interactions are entirely independent of the catalytic domains and are mediated by ionic interactions with the negatively charged microtubule surface. Importantly, a crosstalk between the microtubule-binding domain and the deacetylase domain was critical for recognition and efficient deacetylation of free tubulin dimers both in vitro and in vivo. Overall, our results reveal that recognition of substrates by HDAC6 is more complex than previously appreciated and that domains outside the tandem catalytic core are essential for proficient substrate deacetylation.