Catalytic Zinc Ion Research Articles

Zinc-Binding Oligonucleotide Backbone Modifications for Targeting a DNA-Processing Metalloenzyme.

A series of chemically-modified oligonucleotides for targeting the DNA repair nuclease SNM1A have been designed and synthesised. Each oligonucleotide contains a modified internucleotide linkage designed to both mimic the native phosphodiester backbone and chelate to the catalytic zinc ion(s) in the SNM1A active site. Dinucleoside phosphoramidites containing urea, squaramide, sulfanylacetamide, and sulfinylacetamide linkages were prepared and employed successfully in solid-phase oligonucleotide synthesis. All the modified oligonucleotides were found to interact with SNM1A in a gel electrophoresis-based assay, demonstrating the first examples of inhibition of DNA damage repair enzymes for many of these groups in oligonucleotides. One strand containing a sulfinylacetamide-linkage was found to have the strongest interaction with SNM1A and was further tested in a real-time fluorescence assay. This allowed an IC50 value of 231 nM to be determined, significantly lower than previously reported substrate-mimics targeting this enzyme. It is expected that these modified oligonucleotides will serve as a scaffold for the future development of fluorescent or biotin-labelled probes for the in vivo study of DNA repair processes.

Comparative Structure-Based Virtual Screening Utilizing Optimized AlphaFold Model Identifies Selective HDAC11 Inhibitor.

HDAC11 is a class IV histone deacylase with no crystal structure reported so far. The catalytic domain of HDAC11 shares low sequence identity with other HDAC isoforms, which makes conventional homology modeling less reliable. AlphaFold is a machine learning approach that can predict the 3D structure of proteins with high accuracy even in absence of similar structures. However, the fact that AlphaFold models are predicted in the absence of small molecules and ions/cofactors complicates their utilization for drug design. Previously, we optimized an HDAC11 AlphaFold model by adding the catalytic zinc ion and minimization in the presence of reported HDAC11 inhibitors. In the current study, we implement a comparative structure-based virtual screening approach utilizing the previously optimized HDAC11 AlphaFold model to identify novel and selective HDAC11 inhibitors. The stepwise virtual screening approach was successful in identifying a hit that was subsequently tested using an in vitro enzymatic assay. The hit compound showed an IC50 value of 3.5 µM for HDAC11 and could selectively inhibit HDAC11 over other HDAC subtypes at 10 µM concentration. In addition, we carried out molecular dynamics simulations to further confirm the binding hypothesis obtained by the docking study. These results reinforce the previously presented AlphaFold optimization approach and confirm the applicability of AlphaFold models in the search for novel inhibitors for drug discovery.

Au Nanorods Activated the Zn/Ce Composites with Cancer Cell Specific Cytotoxicity for Enhanced Chemodynamic Therapy.

Chemodynamic therapy based on the Fenton reaction has been developed as an extremely promising modality for cancer therapeutics. In this study, a core-shell structure nanoplatform was constructed by a Au nanorod externally encapsulating Ce/Zn-based composites (ACZO). The nanoparticles can catalyze the generation of reactive oxygen species (ROS) under acidic conditions and effectively consume existing glutathione (GSH) to destroy the redox balance within the tumor. Moreover, the decomposition of the nanocomplexes under acidic conditions releases large amounts of zinc ions, leading to zinc overload in cancer cells. The photothermal effect generated by the Au nanorods not only provides photothermal therapy (PTT) but also augments the catalytic reaction and ions action mentioned above. This facile strategy to improve the efficacy of chemodynamic therapy by the photothermal enhancement of catalytic activity and zinc ion release provides a promising perspective for potential tumor treatment.

Utilization of AlphaFold models for drug discovery: Feasibility and challenges. Histone deacetylase 11 as a case study

Histone deacetylase 11 (HDAC11), an enzyme that cleaves acyl groups from acylated lysine residues, is the sole member of class IV of HDAC family with no reported crystal structure so far. The catalytic domain of HDAC11 shares low sequence identity with other HDAC isoforms which complicates the conventional template-based homology modeling. AlphaFold is a neural network machine learning approach for predicting the 3D structures of proteins with atomic accuracy even in absence of similar structures. However, the structures predicted by AlphaFold are missing small molecules as ligands and cofactors. In our study, we first optimized the HDAC11 AlphaFold model by adding the catalytic zinc ion followed by assessment of the usability of the model by docking of the selective inhibitor FT895. Minimization of the optimized model in presence of transplanted inhibitors, which have been described as HDAC11 inhibitors, was performed. Four complexes were generated and proved to be stable using three replicas of 50 ns MD simulations and were successfully utilized for docking of the selective inhibitors FT895, MIR002 and SIS17. For SIS17, The most reasonable pose was selected based on structural comparison between HDAC6, HDAC8 and the HDAC11 optimized AlphaFold model. The manually optimized HDAC11 model is thus able to explain the binding behavior of known HDAC11 inhibitors and can be used for further structure-based optimization.

Discovery of Potent Angiotensin-Converting Enzyme Inhibitors in Pomegranate as a Treatment for Hypertension.

Pomegranate (Punica granatum L.) is associated with numerous health benefits due to its high levels of antioxidant polyphenolic substances. Since pomegranate extract has been shown to inhibit angiotensin-converting enzyme (ACE), the potential inhibitory effect of most of its main constituents against ACE is unknown. Therefore, we tested the activities of 24 major compounds, the majority of which significantly inhibited ACE. Notably, pedunculagin, punicalin, and gallagic acid were the most effective ACE inhibitors with IC50 values of 0.91, 1.12, and 1.77 μM, respectively. As demonstrated in molecular docking studies, compounds block ACE by forming multiple hydrogen bonds and hydrophobic interactions with catalytic residues and zinc ions in ACE's C- and N-domains, consequently inhibiting ACE's catalytic activity. Also, the most active pedunculagin stimulated nitric oxide (NO) production, activated the endothelial nitric oxide synthase enzyme (eNOS), and significantly increased eNOS protein expression levels up to 5.3-fold in EA.hy926 cells. Furthermore, pedunculagin increased in cellular calcium (Ca2+) concentration promoted eNOS enzyme activation and reduced the production of reactive oxygen species (ROS). In addition, the active compounds improved glucose uptake in insulin-resistant C2C12 skeletal muscle cells in a dose-dependent manner. The results of these computational, in vitro, and cellular experiments provide further evidence to the traditional medicine that involves using pomegranates to treat cardiovascular diseases like hypertension.

Oligomer nanoparticle release from polylactic acid plastics catalysed by gut enzymes triggers acute inflammation.

The health risks of exposure to 'eco-friendly' biodegradable plastics of anthropogenic origin and their effects on the gastrointestinal tract are largely unknown. Here we demonstrate that the enzymatic hydrolysis of polylactic acid microplastics generated nanoplastic particles by competing for triglyceride-degrading lipase during gastrointestinal processes. Nanoparticle oligomers were formed by hydrophobically driven self-aggregation. In a mouse model, polylactic acid oligomers and their nanoparticles bioaccumulated in the liver, intestine and brain. Hydrolysed oligomers caused intestinal damage and acute inflammation. A large-scale pharmacophore model revealed that oligomers interacted with matrix metallopeptidase 12. Mechanistically, high binding affinity (Kd = 13.3 μmol l-1) of oligomers to the catalytic zinc-ion finger domain led to matrix metallopeptidase 12 inactivation, which might mediate the adverse bowel inflammatory effects after exposure to polylactic acid oligomers. Biodegradable plastics are considered to be a solution to address environmental plastic pollution. Thus, understanding the gastrointestinal fates and toxicities of bioplastics will provide insights into potential health risks.

Structural insights into latency of the metallopeptidase ulilysin (lysargiNase) and its unexpected inhibition by a sulfonyl-fluoride inhibitor of serine peptidases.

Peptidases are regulated by latency and inhibitors, as well as compatibilization and cofactors. Ulilysin from Methanosarcina acetivorans, also called lysargiNase, is an archaeal metallopeptidase (MP) that is biosynthesized as a zymogen with a 60-residue N-terminal prosegment (PS). In the presence of calcium, it self-activates to yield the mature enzyme, which specifically cleaves before basic residues and thus complements trypsin in proteomics workflows. Here, we obtained a low-resolution crystal structure of proulilysin, in which 28 protomers arranged as 14 dimers form a continuous double helix of 544 Å pitch that parallels cell axis b of the crystal. The PS includes two α-helices and obstructs the active-site cleft of the catalytic domain (CD) by traversing it in the opposite orientation of a substrate, and a cysteine blocks the catalytic zinc according to a "cysteine-switch mechanism". Moreover, the PS interacts through its first helix with an "S-loop" of the CD, which acts as an "activation segment" that lacks one of two essential calcium cations. Upon PS removal during maturation, the S-loop adopts its competent conformation and binds the second calcium ion. Next, we found that in addition to general MP inhibitors, ulilysin was competitively and reversibly inhibited by 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF; Ki = 4 μM). This is a compound that normally forms an irreversible covalent complex with serine peptidases but does not inhibit MPs. A high-resolution crystal structure of the complex revealed that the inhibitor penetrates the specificity pocket of ulilysin. A primary amine of the inhibitor salt-bridges an aspartate at the pocket bottom, thus mimicking the basic side chain of substrates. In contrast, the sulfonyl fluoride warhead is not involved and the catalytic zinc ion is freely accessible. Thus, the usage of inhibitor cocktails of peptidases, which typically contain AEBSF at ∼25-fold higher concentrations than the determined Ki, should be avoided when working with ulilysin. Finally, the structure of the complex, which occurred as a crystallographic dimer recurring in previous mature ulilysin structures, unveiled an N-terminal product fragment that delineated the non-primed side of the cleft. These results complement prior structures of ulilysin with primed-side product fragments and inhibitors.

Design and search for prospective diuretics (CA II Inhibitors) among aroylhydrazones of esters quinone oxime using in silico and in vivo methodology

The design and search for new selective inhibitors of CA II with a better pharmacological profile, which would cause minimal electrolyte disturbances in the body, remains an urgent problem of medical chemistry and pharmacology today. It is important that the discovered new classes of inhibitors do not always contain the main “pharmacophoric” function (sulfamide), which is characteristic of “classic” drugs (Acetazolamide, Methazolamide, Ethoxzolamide, Dorzolamide and others), but are derivatives of phenols, polyamines, coumarins/thiocoumarins, ureas, thioureas, hydroxamates, etc. These molecules also bind in the active site of the enzyme, but do not interact directly with the catalytic zinc ion or interact through zinc-coordinated water molecules/hydroxide ion. However, this leads to an increase in their selectivity and, as a result, pharmacological action. Continuing the search for compounds that affect urination, we were interested in aroylhydrazones of esters of quinone oxime. Firstly, they are characterized by certain structural features (dynamic and geometric isomerism); secondly, they exhibit redox properties; thirdly, the presence of aromatic fragments makes it possible to create a voluminous combinatorial library for analysis. These compounds are ligands in complexation reactions, and an additional increase in the number of hydrogen acceptors in the molecule due to structural modification will improve ligand-enzymatic interactions with carbonic anhydrase (CAII) and, as a result, reveal new promising diuretics. The aim – design and search for potential diuretics (CA II inhibitors) among aroylhydrazones of esters of quinone oxime using in silico, traditional synthesis and in vivo methodologies. Methods of organic synthesis, physico-chemical methods of analysis of organic compounds (NMR 1H-spectroscopy, elemental analysis). Prediction of affinity to the biological target, prediction of toxicity and lipophilicity of the combinatorial library of benzohydrazides O-aroyl esters of quinone oxime using computer services. The study of compounds affecting the excretory function of rat kidneys was carried out according to the generally accepted method of E.B.Berkhin with water load. The investigation of the probable mechanism was carried out using flexible molecular docking, as an approach to search for molecules that have affinity for human carbonic anhydrase type II (CA II). Macromolecular data of the crystal structure of CA II (PDB ID – 3HS4) were downloaded from the Protein Data Bank (PDB). The design was developed and the search for diuretic agents among benzohydrazides of O-aroyl esters of quinone oximes was developed using in silico methods (prediction of affinity, lipophilicity, toxicity and enzyme-ligand interactions), traditional organic synthesis, and in vivo methods (effect on excretory function of rat kidneys). The synthesis of benzohydrazides of O-aroyl esters of quinone oxime was carried out by the interaction of aroylhydrazines with 4-[(aroylimino)]cyclohexa-2,5-dien-1-ones. The structure of the synthesized compounds was confirmed by elemental analysis and 1H NMR spectra. Studies of the effect of synthesized compounds on the excretory function of rat kidneys allowed us to identify a number of promising compounds among aroylhydrazones of quinonexime esters, which increase daily diuresis by 54.2-352.8% compared to the control group. At the same time, it was established that the most active was N'-(4-[(2-chlorobenzoyloxy)imino]cyclohexa-2,5-dien-1-ylidene)-3-nitrobenzohydrazide, which increased daily diuresis by 352.8% in comparison with the control group, while exceeding the effect of “Hydrochlorothiazide” (170.8%). The developed and implemented strategy for the search for diuretics among benzohydrazides of O-aroylesters of quinone oxime allowed the identification of an effective compound, which in terms of diuretic effect exceeds the comparison drug “Hydrochlorothiazide”. Visualization of the molecular docking of the active compounds showed that their geometry makes it difficult to place them in the pocket of the active site of CA II, but the pronounced diuretic effect can also be associated with their ability to form coordination bonds with the zinc cation. The obtained results justify the further targeted search for potential diuretics among this class of compounds for a more detailed understanding and study of the mechanism of action.

Salvianolic Acid B Strikes Back: New Evidence in the Modulation of Expression and Activity of Matrix Metalloproteinase 9 in MDA-MB-231 Human Breast Cancer Cells

Salvianolic acid B (SalB) is a bioactive compound from Salviae miltiorrhizae, one of the most important traditional herbal medicines widely used in several countries for the treatment of cardiovascular diseases. The aim of this study was to evaluate the in vitro effect of SalB on the expression and the activity of matrix metalloproteinase 9 (MMP-9), a zinc-dependent proteolytic enzyme, in human MDA-MB-231 breast cancer cells. This cellular model is characterized by a marked invasive phenotype, supported by a high constitutive expression of MMPs, especially gelatinases. SalB was first of all evaluated by in silico approaches primarily aimed at predicting the main pharmacokinetic parameters. The most favorable interaction between the natural compound and MMP-9 was instead tested by molecular docking analysis that was subsequently verified by an enzymatic inhibition assay. MDA-MB-231 cells were treated with SalB 5 µM and 50 µM for 24 h and 48 h. The conditioned media obtained from treated cells were then analyzed by gelatin zymography and reverse zymography to, respectively, evaluate the MMP-9 activity and the presence of TIMP-1. The expression of the enzyme was then evaluated by Western blot on conditioned media and by analysis of transcripts through reverse transcriptase-polymerase chain reaction (RT-PCR). The in silico approach showed the ability of SalB to interact with the catalytic zinc ion of the enzyme, with a plausible competitive mode of action. The analysis of conditioned culture media showed a reduction in MMP-9 activity and the concomitant decrease in the enzyme concentration, partially confirmed by analysis of transcripts. SalB showed the ability to modulate the function of MMP-9 in MDA-MB-231 cells. To our knowledge, this is the first time in which the role of SalB on MMP-9 in a highly invasive cellular model is investigated. The obtained results impose further and more specific evaluations in order to obtain a better understanding of the biochemical mechanisms that regulate the interaction between this natural compound and the MMP-9.

Optimization of 1,2,4-Triazole-3-thiones toward Broad-Spectrum Metallo-β-lactamase Inhibitors Showing Potent Synergistic Activity on VIM- and NDM-1-Producing Clinical Isolates.

Metallo-β-lactamases (MBLs) contribute to the resistance of Gram-negative bacteria to carbapenems, last-resort antibiotics at hospital, and MBL inhibitors are urgently needed to preserve these important antibacterial drugs. Here, we describe a series of 1,2,4-triazole-3-thione-based inhibitors displaying an α-amino acid substituent, which amine was mono- or disubstituted by (hetero)aryl groups. Compounds disubstituted by certain nitrogen-containing heterocycles showed submicromolar activities against VIM-type enzymes and strong NDM-1 inhibition (Ki = 10-30 nM). Equilibrium dialysis, native mass spectrometry, isothermal calorimetry (ITC), and X-ray crystallography showed that the compounds inhibited both VIM-2 and NDM-1 at least partially by stripping the catalytic zinc ions. These inhibitors also displayed a very potent synergistic activity with meropenem (16- to 1000-fold minimum inhibitory concentration (MIC) reduction) against VIM-type- and NDM-1-producing ultraresistant clinical isolates, including Enterobacterales and Pseudomonas aeruginosa. Furthermore, selected compounds exhibited no or moderate toxicity toward HeLa cells, favorable absorption, distribution, metabolism, excretion (ADME) properties, and no or modest inhibition of several mammalian metalloenzymes.

Phage-assisted, active site-directed ligand evolution of a potent and selective histone deacetylase 8 inhibitor.

Phage-assisted, active site-directed ligand evolution (PADLE) is a recently developed technique that uses an amber codon-encoded noncanonical amino acid (ncAA) as an anchor to direct phage-displayed peptides to a target for an enhanced ligand identification process. 2-Amino-8-oxodecanoic acid (Aoda) is a ketone-containing ncAA residue in the macrocyclic peptide natural product apicidin that is a pan-inhibitor of Zn2+ -dependent histone deacetylases (HDACs). Its ketone serves as an anchoring point to coordinate the catalytic zinc ion in HDACs. Using a previously evolved N𝜀 -acetyl-lysyl-tRNA synthetase in combination with tRNAPyl , we showed that Aoda was efficiently incorporated into proteins in Escherichia coli by amber suppression. By propagating an amber codon-obligate phagemid library in E. coli encoding Aoda, we generated an Aoda-containing phage-displayed peptide library. Using this library to conduct PADLE against HDAC8 revealed a 7-mer peptide GH8P01F1 with Aoda-flanking amino acid residues that matched existing peptide sequences in identified HDAC8 substrates. Switching Aoda in GH8P01F1 to a more Zn2+ -chelating ncAA S-2-amino-8-hydroxyamino-8-oxooctanoic acid (Asuha) led to an extremely potent compound GH8HA01, which has an HDAC8-inhibition Ki value of 0.67 nM. GH8HA01 and its 5-mer truncation analogue Ac-GH8HA01Δ1Δ7 that has an HDAC8-inhibition Ki value of 0.31 nM are two of the most potent HDAC8 inhibitors that have been developed. Furthermore, both are highly selective against HDAC8 compared with other HDACs tested, demonstrating the great potential of using PADLE to identify highly potent and selective ligands for targets with conserved active sites among homologues.

Expansion of the Catalytic Repertoire of Alcohol Dehydrogenases in Plant Metabolism**

Medium-chain alcohol dehydrogenases (ADHs) comprise a highly conserved enzyme family that catalyse the reversible reduction of aldehydes. However, recent discoveries in plant natural product biosynthesis suggest that the catalytic repertoire of ADHs has been expanded. Here we report the crystal structure of dihydroprecondylocarpine acetate synthase (DPAS), an ADH that catalyses the non-canonical 1,4-reduction of an α,β-unsaturated iminium moiety. Comparison with structures of plant-derived ADHs suggest the 1,4-iminium reduction does not require a proton relay or the presence of a catalytic zinc ion in contrast to canonical 1,2-aldehyde reducing ADHs that require the catalytic zinc and a proton relay. Furthermore, ADHs that catalysed 1,2-iminium reduction required the presence of the catalytic zinc and the loss of the proton relay. This suggests how the ADH active site can be modified to perform atypical carbonyl reductions, providing insight into how chemical reactions are diversified in plant metabolism.

Expansion of the Catalytic Repertoire of Alcohol Dehydrogenases in Plant Metabolism.

Medium-chain alcohol dehydrogenases (ADHs) comprise a highly conserved enzyme family that catalyse the reversible reduction of aldehydes. However, recent discoveries in plant natural product biosynthesis suggest that the catalytic repertoire of ADHs has been expanded. Here we report the crystal structure of dihydroprecondylocarpine acetate synthase (DPAS), an ADH that catalyses the non-canonical 1,4-reduction of an α,β-unsaturated iminium moiety. Comparison with structures of plant-derived ADHs suggest the 1,4-iminium reduction does not require a proton relay or the presence of a catalytic zinc ion in contrast to canonical 1,2-aldehyde reducing ADHs that require the catalytic zinc and a proton relay. Furthermore, ADHs that catalysed 1,2-iminium reduction required the presence of the catalytic zinc and the loss of the proton relay. This suggests how the ADH active site can be modified to perform atypical carbonyl reductions, providing insight into how chemical reactions are diversified in plant metabolism.

Targeting Aggrecanases for Osteoarthritis Therapy: From Zinc Chelation to Exosite Inhibition.

Osteoarthritis (OA) is the most common degenerative joint disease. In 1999, two members of the A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) family of metalloproteinases, ADAMTS4 and ADAMTS5, or aggrecanases, were identified as the enzymes responsible for aggrecan degradation in cartilage. The first aggrecanase inhibitors targeted the active site by chelation of the catalytic zinc ion. Due to the generally disappointing performance of zinc-chelating inhibitors in preclinical and clinical studies, inhibition strategies tried to move away from the active-site zinc in order to improve selectivity. Exosite inhibitors bind to proteoglycan-binding residues present on the aggrecanase ancillary domains (called exosites). While exosite inhibitors are generally more selective than zinc-chelating inhibitors, they are still far from fulfilling their potential, partly due to a lack of structural and functional data on aggrecanase exosites. Filling this gap will inform the design of novel potent, selective aggrecanase inhibitors.

Phytochemical Analysis and Screening of Acetylcholinesterase and Carbonic Anhydrase I and II Isoenzymes Inhibitory Effect of Heptaptera triquetra (Vent.) Tutin Root

Alzheimer's disease (AD) is characterized by progressive memory loss, deterioration of other cognitive functions, and inability to perform activities of daily living. Inhibiting the AChE enzyme causes Ach accumulation in cholinergic synapses and is expected to increase cognitive functions. Carbonic anhydrase enzymes (CAs) are ubiquitous in all living organisms. They have crucial physiological and pathological roles. CA inhibitors bind to catalytic zinc ion in the active site of CA isoenzymes and block their activity. The clinical use of CAIs had been established as antiglaucoma, anticonvulsant agents, diuretics, and anti-obesity drugs, in the management of mountain sickness, gastric and duodenal ulcers, neurological disorders, osteoporosis, and tumors. To evaluate the bioactive profile of dichloromethane extract prepared from Heptaptera triquetra root, isolation studies, AChE, and hCA I and II inhibitory activities were performed. According to isolation studies, one fatty acid, coniferyl palmitate (1); four sesquiterpene coumarins, umbelliprenin (2), badrakemin acetate (4), colladonin (5), karatavicinol (6); and two sterols, stigmasterol (3a), β-sitosterol (3b) were isolated. The dichloromethane extract and all isolated compounds showed high potency against all enzymes (except badrakemin acetate for AChE) when compared to standards. Umbelliprenin (2) with IC50 value of 31.500 nM against hCA I, colladonin (5) with IC50 value of 36.473 nM against hCA II and stigmasterol (3a), and β-sitosterol (3b) mixture with IC50 value 9.000 nM against AChE demonstrated the best activity.

Identification of Zinc-Binding Inhibitors of Matrix Metalloproteinase-9 to Prevent Cancer Through Deep Learning and Molecular Dynamics Simulation Approach.

The overexpression of matrix metalloproteinase-9 (MMP-9) is associated with tumor development and angiogenesis, and hence, it has been considered an attractive drug target for anticancer therapy. To assist in drug design endeavors for MMP-9 targets, an in silico study was presented to investigate whether our compounds inhibit MMP-9 by binding to the catalytic domain, similar to their inhibitor or not. For that, in the initial stage, a deep-learning algorithm was used for the predictive modeling of the CHEMBL321 dataset of MMP-9 inhibitors. Several regression models were built and evaluated based on R2, MAE MSE, RMSE, and Loss. The best model was utilized to screen the drug bank database containing 9,102 compounds to seek novel compounds as MMP-9 inhibitors. Then top high score compounds were selected for molecular docking based on the comparison between the score of the reference molecule. Furthermore, molecules having the highest docking scores were selected, and interaction mechanisms with respect to S1 pocket and catalytic zinc ion of these compounds were also discussed. Those compounds, involving binding to the catalytic zinc ion and the S1 pocket of MMP-9, were considered preferentially for molecular dynamics studies (100 ns) and an MM-PBSA (last 30 ns) analysis. Based on the results, we proposed several novel compounds as potential candidates for MMP-9 inhibition and investigated their binding properties with MMP-9. The findings suggested that these compounds may be useful in the design and development of MMP-9 inhibitors in the future.

Structural Insights for Core Scaffold and Substrate Specificity of B1, B2, and B3 Metallo-β-Lactamases.

Metallo-β-lactamases (MBLs) hydrolyze almost all β-lactam antibiotics, including penicillins, cephalosporins, and carbapenems; however, no effective inhibitors are currently clinically available. MBLs are classified into three subclasses: B1, B2, and B3. Although the amino acid sequences of MBLs are varied, their overall scaffold is well conserved. In this study, we systematically studied the primary sequences and crystal structures of all subclasses of MBLs, especially the core scaffold, the zinc-coordinating residues in the active site, and the substrate-binding pocket. We presented the conserved structural features of MBLs in the same subclass and the characteristics of MBLs of each subclass. The catalytic zinc ions are bound with four loops from the two central β-sheets in the conserved αβ/βα sandwich fold of MBLs. The three external loops cover the zinc site(s) from the outside and simultaneously form a substrate-binding pocket. In the overall structure, B1 and B2 MBLs are more closely related to each other than they are to B3 MBLs. However, B1 and B3 MBLs have two zinc ions in the active site, while B2 MBLs have one. The substrate-binding pocket is different among all three subclasses, which is especially important for substrate specificity and drug resistance. Thus far, various classes of β-lactam antibiotics have been developed to have modified ring structures and substituted R groups. Currently available structures of β-lactam-bound MBLs show that the binding of β-lactams is well conserved according to the overall chemical structure in the substrate-binding pocket. Besides β-lactam substrates, B1 and cross-class MBL inhibitors also have distinguished differences in the chemical structure, which fit well to the substrate-binding pocket of MBLs within their inhibitory spectrum. The systematic structural comparison among B1, B2, and B3 MBLs provides in-depth insight into their substrate specificity, which will be useful for developing a clinical inhibitor targeting MBLs.

5'-Phosphorylation Increases the Efficacy of Nucleoside Inhibitors of the DNA Repair Enzyme SNM1A.

Certain cancers exhibit upregulation of DNA interstrand crosslink repair pathways, which contributes to resistance to crosslinking chemotherapy drugs and poor prognoses. Inhibition of enzymes implicated in interstrand crosslink repair is therefore a promising strategy for improving the efficacy of cancer treatment. One such target enzyme is SNM1A, a zinc co-ordinating 5'-3' exonuclease. Previous studies have demonstrated the feasibility of inhibiting SNM1A using modified nucleosides appended with zinc-binding groups. In this work, we sought to develop more effective SNM1A inhibitors by exploiting interactions with the phosphate-binding pocket adjacent to the enzyme's active site, in addition to the catalytic zinc ions. A series of nucleoside derivatives bearing phosphate moieties at the 5'-position, as well as zinc-binding groups at the 3'-position, were prepared and tested in gel-electrophoresis and real-time fluorescence assays. As well as investigating novel zinc-binding groups, we found that incorporation of a 5'-phosphate dramatically increased the potency of the inhibitors.

Het(aryl)isatin to het(aryl)aminoindoline scaffold hopping: A route to selective inhibitors of matrix metalloproteinases

Matrix metalloproteinases (MMPs) are a large family of zinc-dependent endoproteases known to exert multiple regulatory roles in tumor progression. A variety of chemical classes have been explored for targeting individual MMP isoforms. In the present study, we further developed our isatin based scaffold BB0223107 capable of binding to and inactivating MMP-2 in a zinc-independent manner (Agamennone et al., 2016). Forty four new compounds were synthesized based on the modified BB0223107 . All compounds were tested in enzyme inhibition assays against MMP-2, −8 and −13. SAR studies demonstrated that 5-het(aryl)-3-aminoindolin-2-ones ( 37 – 39 ) were active toward MMP-2 and MMP-13. The most potent compounds 33 and 37 displayed an IC 50 of 3 µM against MMP-13 and showed a negligible activity toward MMP-8; almost all new compounds were inactive toward MMP-8. Replacement of the isatin ring with a biaryl system (compound 33 ) did not decrease the potency against MMP-13 but reduced the selectivity. Structure-based computational studies were carried out to rationalize the inhibitory activity data. The analysis of binding geometries confirmed that all fragments occupied the S1′ site in the three enzymes while no ligand was able to bind the catalytic zinc ion. To the best of our knowledge, this is the first example of 3-aminoindolin-2-one-based MMP inhibitors that, based on the computer modeling study, do not coordinate the zinc ion. Thus, the het(aryl)-3-aminoindolin-2-one derivatives emerge as a drug-like and promising chemotype that, along with the hetaryl variations, represents an alternative and thrifty tool for chemical space exploration aimed at MMP inhibitor design.

A multiscale approach to predict the binding mode of metallo beta-lactamase inhibitors.

Antibiotic resistance is a major threat to global public health. β-lactamases, which catalyze breakdown of β-lactam antibiotics, are a principal cause. Metallo β-lactamases (MBLs) represent a particular challenge because they hydrolyze almost all β-lactams and to date no MBL inhibitor has been approved for clinical use. Molecular simulations can aid drug discovery, for example, predicting inhibitor complexes, but empirical molecular mechanics (MM) methods often perform poorly for metalloproteins. Here we present a multiscale approach to model thiol inhibitor binding to IMP-1, a clinically important MBL containing two catalytic zinc ions, and predict the binding mode of a 2-mercaptomethyl thiazolidine (MMTZ) inhibitor. Inhibitors were first docked into the IMP-1 active site, testing different docking programs and scoring functions on multiple crystal structures. Complexes were then subjected to molecular dynamics (MD) simulations and subsequently refined through QM/MM optimization with a density functional theory (DFT) method, B3LYP/6-31G(d), increasing the accuracy of the method with successive steps. This workflow was tested on two IMP-1:MMTZ complexes, for which it reproduced crystallographically observed binding, and applied to predict the binding mode of a third MMTZ inhibitor for which a complex structure was crystallographically intractable. We also tested a 12-6-4 nonbonded interaction model in MD simulations and optimization with a SCC-DFTB QM/MM approach. The results show the limitations of empirical models for treating these systems and indicate the need for higher level calculations, for example, DFT/MM, for reliable structural predictions. This study demonstrates a reliable computational pipeline that can be applied to inhibitor design for MBLs and other zinc-metalloenzyme systems.