Tight-binding Inhibitor Research Articles

In silico study of selective inhibition mechanism of S-adenosyl-L-methionine analogs for human DNA methyltransferase 3A

Epigenetic mechanisms leading to transcriptional regulation, including DNA methylation, are frequently dysregulated in diverse cancers. Interfering with aberrant DNA methylation performed by DNA cytosine methyltransferases (DNMTs) is a clinically validated approach. In particular, the selective inhibition of the de novo DNMT3A and DNMT3B enzymes, whose expression is limited to early embryogenesis, adult stem cells, and in cancers, is particularly attractive; such selectivity is likely to attenuate the dose limiting toxicity shown by current, non-selective DNMT inhibitors. We use molecular dynamics (MD) based computational analysis to study known small molecule binders of DNMT3A, then propose reversible, tight binding, and selective inhibitors that exploit the Asn1192/Arg688 difference between the maintenance DNMT1 and DNMT3A near the active site. A similar strategy exploiting the presence of a unique active site cysteine Cys666 is used to propose DNMT3A-selective irreversible inhibitors. We report our results of relative binding energies of the known and proposed compounds estimated using MM/GBSA and umbrella sampling (US) techniques, and our evaluation of other end-point binding free energy calculation methods for these receptors. These calculations offer insight into the potential for small molecules to selectively target the active site of DNMT3A.

Role of Half-of-Sites Reactivity and Inter-Subunit Communications in DAHP Synthase Catalysis and Regulation.

α-Carboxyketose synthases, including 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase (DAHPS), are long-standing targets for inhibition. They are challenging targets to create tight-binding inhibitors against, and inhibitors often display half-of-sites binding and partial inhibition. Half-of-sites inhibition demonstrates the existence of inter-subunit communication in DAHPS. We used X-ray crystallography and spatially resolved hydrogen-deuterium exchange (HDX) to reveal the structural and dynamic bases for inter-subunit communication in Escherichia coli DAHPS(Phe), the isozyme that is feedback-inhibited by phenylalanine. Crystal structures of this homotetrameric (dimer-of-dimers) enzyme are invariant over 91% of its sequence. Three variable loops make up 8% of the sequence and are all involved in inter-subunit contacts across the tight-dimer interface. The structures have pseudo-twofold symmetry indicative of inter-subunit communication across the loose-dimer interface, with the diagonal subunits B and C always having the same conformation as each other, while subunits A and D are variable. Spatially resolved HDX reveals contrasting responses to ligand binding, which, in turn, affect binding of the second substrate, erythrose-4-phosphate (E4P). The N-terminal peptide, M1-E12, and the active site loop that binds E4P, F95-K105, are key parts of the communication network. Inter-subunit communication appears to have a catalytic role in all α-carboxyketose synthase families and a regulatory role in some members.

The Discovery of the Mode of Action of Nitisinone.

This review briefly discusses the discovery of the mode of action of the triketone herbicide, 2-(2-nitro-4-trifluormethylbenzoyl)-1,3-cyclohexanedione and its use as a drug Nitisinone for the treatment of inborn errors of tyrosine metabolism. Nitisinone is a potent reversible tight-binding inhibitor of the enzyme 4-hydroxyphenylpyruvate dioxygenase, involved in the catabolism of the amino acid tyrosine. Nitisinone is used to treat the rare disease hereditary tyrosinaemia type 1 where the last enzyme in the breakdown of tyrosine, fumarylacetoacetase is deficient. Nitisinone is also used to treat patients with alkaptonuria where the enzyme homogentisic acid oxidase is deficient. Articles in this issue discuss metabolites of tyrosine catabolism in healthy patients and those with alkaptonuria.

Design, synthesis, and characterization of novel fluorogenic substrates of the proprotein convertases furin, PC1/3, PC2, PC5/6, and PC7

Proprotein convertases (PCs) are involved in the pathogenesis of various diseases, making them promising drug targets. Most assays for PCs have been performed with few standard substrates, regardless of differences in cleavage efficiencies. Derived from studies on substrate-analogue inhibitors, 11 novel substrates were synthesized and characterized with five PCs. H-Arg-Arg-Tle-Lys-Arg-AMC is the most efficiently cleaved furin substrate based on its kcat/KM value. Due to its higher kcat value, acetyl-Arg-Arg-Tle-Arg-Arg-AMC was selected for further measurements to demonstrate the benefit of this improved substrate. Compared to our standard conditions, its use allowed a 10-fold reduction of the furin concentration, which enabled Ki value determinations of previously described tight-binding inhibitors under classical conditions. Under these circumstances, a slow-binding behavior was observed for the first time with inhibitor MI-1148. In addition to furin, four additional PCs were used to characterize these substrates. The most efficiently cleaved PC1/3 substrate was acetyl-Arg-Arg-Arg-Tle-Lys-Arg-AMC. The highest kcat/KM values for PC2 and PC7 were found for the N-terminally unprotected analogue of this substrate, although other substrates possess higher kcat values. The highest efficiency for PC5/6A was observed for the substrate acetyl-Arg-Arg-Tle-Lys-Arg-AMC. In summary, we have identified new substrates for furin, PC1/3, PC2, and PC7 suitable for improved enzyme-kinetic measurements.

Novel Insights on Human Carbonic Anhydrase Inhibitors Based on Coumalic Acid: Design, Synthesis, Molecular Modeling Investigation, and Biological Studies.

Human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms IX and XII are overexpressed in solid hypoxic tumors, and they are considered as prognostic tools and therapeutic targets for cancer. Based on a molecular simplification of the well-known coumarin scaffold, we developed a new series of derivatives of the pyran-2-one core. The new compounds are endowed with potent and selective inhibitory activity against the tumor-related hCA isoforms IX and XII, in the low nanomolar range, whereas they are inactive against the two cytosolic off-targets hCA I and II. The compounds exhibiting the best hCA inhibition were further investigated against the breast adenocarcinoma cell line (MCF7) in hypoxic conditions, evaluating their ability to eventually synergize with doxorubicin. The compounds’ biocompatibility on healthy cells was also tested and confirmed on Human Gingival Fibroblasts (HGFs). Furthermore, the possible binding mode of all compounds to the active site of the tumor-associated human CA IX was investigated by computational techniques which predicted the binding conformations and the persistency of binding poses within the active site of the enzyme, furnishing relevant data for the design of tight binding inhibitors.

Discovery of the first tight‐binding reversible antagonists of Hedgehog protein autoprocessing

Hedgehog (Hh) signaling ligands (HhN) are expressed as precursors with an adjacent C‐terminal autoprocessing domain, HhC. Autoprocessing activity of HhC releases and cholesterylates the Hh ligand thereby activating the HhN for full biological activity. Inhibitors of HhC activity are of interest as probes of the Hh signaling pathway, and HhC inhibitors may also offer new therapeutic approaches to suppress oncogenic signaling activity of HhN. The intramolecular nature of the HhN/HhC system poses a challenge for reversible inhibitors, and to date most antagonists of HhC bind the active site covalently. Here we report we report the discovery and optimization of the first tight‐binding reversible inhibitor of HhC. Using a light‐to‐dark FRET activity assay for Drosophila melanogaster HhC, we screened a focused library of 1187 sterol‐like analogs in search of HhC inhibitors. Four structurally related hits were identified and further validated by dose‐response assays. The most effective inhibitor from the screen had an IC50 value of 2x10‐6M. A panel of analogs were prepared and tested for SAR analysis resulting in compound tBT‐HBT, a derivative with a sub‐micromolar IC50of 3x10‐7 M. Inhibition appears to be mediated by a previously unidentified allosteric binding site on HhC. Kinetic analysis, photoaffinity labeling, computational modeling, and point mutagenesis studies suggest that the binding site is nearby, but not overlapping with the cholesterol binding site of HhC.

Dual Reversible Coumarin Inhibitors Mutually Bound to Monoamine Oxidase B and Acetylcholinesterase Crystal Structures.

Multitarget directed ligands (MTDLs) represent a promising frontier in tackling the complexity of multifactorial pathologies. The synergistic inhibition of monoamine oxidase B (MAO B) and acetylcholinesterase (AChE) is believed to provide a potentiated effect in the treatment of Alzheimer’s disease. Among previously reported micromolar or sub-micromolar coumarin-bearing dual inhibitors, compound 1 returned a tight-binding inhibition of MAO B (Ki = 4.5 μM) and a +5.5 °C increase in the enzyme Tm value. Indeed, the X-ray crystal structure revealed that binding of 1 produces unforeseen conformational changes at the MAO B entrance cavity. Interestingly, 1 showed great shape complementarity with the AChE enzymatic gorge, being deeply buried from the catalytic anionic subsite (CAS) to the peripheral anionic subsite (PAS) and causing significant structural changes in the active site. These findings provide structural templates for further development of dual MAO B and AChE inhibitors.

Cryo-electron microscopy reveals how acetogenins inhibit mitochondrial respiratory complex I

Mitochondrial complex I (NADH:ubiquinone oxidoreductase), a crucial enzyme in energy metabolism, captures the redox potential energy from NADH oxidation/ubiquinone reduction to create the proton motive force used to drive ATP synthesis in oxidative phosphorylation. High-resolution single-particle electron cryo-EM analyses have provided detailed structural knowledge of the catalytic machinery of complex I, but not of the molecular principles of its energy transduction mechanism. Although ubiquinone is considered to bind in a long channel at the interface of the membrane-embedded and hydrophilic domains, with channel residues likely involved in coupling substrate reduction to proton translocation, no structures with the channel fully occupied have yet been described. Here, we report the structure (determined by cryo-EM) of mouse complex I with a tight-binding natural product acetogenin inhibitor, which resembles the native substrate, bound along the full length of the expected ubiquinone-binding channel. Our structure reveals the mode of acetogenin binding and the molecular basis for structure–activity relationships within the acetogenin family. It also shows that acetogenins are such potent inhibitors because they are highly hydrophobic molecules that contain two specific hydrophilic moieties spaced to lock into two hydrophilic regions of the otherwise hydrophobic channel. The central hydrophilic section of the channel does not favor binding of the isoprenoid chain when the native substrate is fully bound but stabilizes the ubiquinone/ubiquinol headgroup as it transits to/from the active site. Therefore, the amphipathic nature of the channel supports both tight binding of the amphipathic inhibitor and rapid exchange of the ubiquinone/ubiquinol substrate and product.

Allosteric Inhibition of Acinetobacter baumannii ATP Phosphoribosyltransferase by Protein:Dipeptide and Protein:Protein Interactions.

ATP phosphoribosyltransferase (ATPPRT) catalyzes the first step of histidine biosynthesis in bacteria, namely, the condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate (PRPP) to generate N1-(5-phospho-β-d-ribosyl)-ATP (PRATP) and pyrophosphate. Catalytic (HisGS) and regulatory (HisZ) subunits assemble in a hetero-octamer where HisZ activates HisGS and mediates allosteric inhibition by histidine. In Acinetobacter baumannnii, HisGS is necessary for the bacterium to persist in the lung during pneumonia. Inhibition of ATPPRT is thus a promising strategy for specific antibiotic development. Here, A. baumannii ATPPRT is shown to follow a rapid equilibrium random kinetic mechanism, unlike any other ATPPRT. Histidine noncompetitively inhibits ATPPRT. Binding kinetics indicates histidine binds to free ATPPRT and to ATPPRT:PRPP and ATPPRT:ATP binary complexes with similar affinity following a two-step binding mechanism, but with distinct kinetic partition of the initial enzyme:inhibitor complex. The dipeptide histidine-proline inhibits ATPPRT competitively and likely uncompetitively, respectively, against PRPP and ATP. Rapid kinetics analysis shows His-Pro binds to the ATPPRT:ATP complex via a two-step binding mechanism. A related HisZ that shares 43% sequence identity with A. baumannii HisZ is a tight-binding allosteric inhibitor of A. baumannii HisGS. These findings lay the foundation for inhibitor design against A. baumannii ATPPRT.

Discovery of tight-binding competitive inhibitors of dipeptidyl peptidase IV

Dipeptidyl peptidase IV (DPP-IV, EC 3.4.14.5) is an abundant serine aminopeptidase that preferentially cleaves N-terminal Xaa-Pro or Xaa-Ala dipeptides from oligopeptides. Inhibitors of DPP-IV activity are used for treating type 2 diabetes mellitus and other diseases. DPP-IV is also involved in tumor progression. We identified four new non-peptide tight-binding competitive inhibitors of porcine DPP-IV by virtual screening and enzymatic assays. Molecular docking simulations supported the competitive behavior, and the selectivity of one of the compounds in the DPP-IV family. Since three of these inhibitors are also aminopeptidase N (APN) inhibitors, we tested their impact on APN+/DPP-IV+ and DPP-IV+ human tumor cells' viability. Using kinetic assays, we determined that HL-60 tumor cells express both APN and DPP-IV activities and that MDA-MB-231 tumor cells express DPP-IV activity. The inhibitors had a slight inhibitory effect on human HEK-293 cell viability but reduced the viability of APN+/DPP-IV+ and DPP-IV+ human tumor cells more potently. Remarkably, the intraperitoneal injection of these compounds inhibited DPP-IV activity in rat brain, liver, and pancreas. In silico studies suggested inhibitors binding to serum albumin contribute to blood-brain barrier crossing. The spectrum of action of some of these compounds may be useful for niche applications.

Joseph R. Bertino: In Memoriam (1930–2021)

Joseph R. Bertino: In Memoriam (1930–2021)

Identification of a substrate-like cleavage-resistant thrombin inhibitor from the saliva of the flea Xenopsylla cheopis

The salivary glands of the flea Xenopsylla cheopis, a vector of the plague bacterium, Yersinia pestis, express proteins and peptides thought to target the hemostatic and inflammatory systems of its mammalian hosts. Past transcriptomic analyses of salivary gland tissue revealed the presence of two similar peptides (XC-42 and XC-43) having no extensive similarities to any other deposited sequences. Here we show that these peptides specifically inhibit coagulation of plasma and the amidolytic activity of α-thrombin. XC-43, the smaller of the two peptides, is a fast, tight-binding inhibitor of thrombin with a dissociation constant of less than 10 pM. XC-42 exhibits similar selectivity as well as kinetic and binding properties. The crystal structure of XC-43 in complex with thrombin shows that despite its substrate-like binding mode, XC-43 is not detectably cleaved by thrombin and that it interacts with the thrombin surface from the enzyme catalytic site through the fibrinogen-binding exosite I. The low rate of hydrolysis was verified in solution experiments with XC-43, which show the substrate to be largely intact after 2 h of incubation with thrombin at 37 °C. The low rate of XC-43 cleavage by thrombin may be attributable to specific changes in the catalytic triad observable in the crystal structure of the complex or to extensive interactions in the prime sites that may stabilize the binding of cleavage products. Based on the increased arterial occlusion time, tail bleeding time, and blood coagulation parameters in rat models of thrombosis XC-43 could be valuable as an anticoagulant.

Mapping Chromone-3-Phenylcarboxamide Pharmacophore: Quid Est Veritas?

Chromone-3-phenylcarboxamides (Crom-1 and Crom-2) were identified as potent, selective, and reversible inhibitors of human monoamine oxidase B (hMAO-B). Since they exhibit some absorption, distribution, metabolism, and excretion (ADME)-toxicity liabilities, new derivatives were synthesized to map the chemical structural features that compose the pharmacophore, a process vital for lead optimization. Structure-activity relationship data, supported by molecular docking studies, provided a rationale for the contribution of the heterocycle's rigidity, the carbonyl group, and the benzopyran heteroatom for hMAO-B inhibitory activity. From the study, N-(3-chlorophenyl)-4H-thiochromone-3-carboxamide (31) (hMAO-B IC50 = 1.52 ± 0.15 nM) emerged as a reversible tight binding inhibitor with an improved pharmacological profile. In in vitro ADME-toxicity studies, compound 31 showed a safe cytotoxicity profile in Caco-2, SH-SY5Y, HUVEC, HEK-293, and MCF-7 cells, did not present cardiotoxic effects, and did not affect P-gp transport activity. Compound 31 also protected SH-SY5Y cells from iron(III)-induced damage. Collectively, these studies highlighted compound 31 as the first-in-class and a suitable candidate for in vivo preclinical investigation.

Synthesis and biological evaluation of 3-styrylchromone derivatives as selective monoamine oxidase B inhibitors

A series of 3-styrylchromone derivatives was synthesized and evaluated for monoamine oxidase (MAO) A and B inhibitory activities. Most of all derivatives inhibited MAO-B selectively, except compound 21. Compound 19, which had a methoxy group at R2 on the chromone ring and chlorine at R4 on phenyl ring, potently inhibited MAO-B, with an IC50 value of 2.2 nM. Compound 1 showed the highest MAO-B selectivity, with a selectivity index of >3700. Further analysis of these compounds indicated that compounds 1 and 19 were reversible and mixed-type MAO-B inhibitors, suggesting that their mode of action may be through tight-binding inhibition to MAO-B.Quantitative structure–activity relationship (QSAR) analyses of the 3-styrylchromone derivatives were conducted using their pIC50 values, through Molecular Operating Environment (MOE) and Dragon. There were 1796 descriptors of MAO-B inhibitory activity, which showed significant correlations (P < 0.05). Further investigation of the 3-styrylchromone structures as useful scaffolds was performed through three-dimensional-QSAR studies using AutoGPA, which is based on the molecular field analysis algorithm using MOE. The MAO-B inhibitory activity model constructed using pIC50 value index exhibited a determination coefficients (R2) of 0.972 and a Leave-One-Out cross-validated determination coefficients (Q2) of 0.914.These data suggest that the 3-styrylchromone derivatives assessed herein may be suitable for the design and development of novel MAO inhibitors.

Accurate quantitative determination of affinity and binding kinetics for tight binding inhibition of xanthine oxidase

The accurate quantitative determination of affinity and binding kinetics (BK) for tight binding inhibition is extraordinary important from both the continuous optimization of compounds, particularly in developing structure–activity relationships (SAR), and the prediction of in vivo target occupancy (TO). Due to the unique properties for tight binding inhibition that the inhibitors are characterized by the ultrahigh-affinity, relatively fast association to the target enzyme combined with extremely slow dissociation of the inhibitor-enzyme binary complex, the classical steady state equilibrium methods are no longer valid. Here, we made several recommendations of how to design the optimal experiments and apply special mathematical calculation approaches to quantitatively evaluate the accurate affinity and BK as the examples of two tight binding inhibitors against the xanthine oxidase (XO), as well as compared the differences in the results calculated from the different data analytical methods and analyzed the influence of these differences on the XO engagement in human. Analysis of the results displayed that the accurate apparent dissociation constant (Ki*,app) was 0.2 ± 0.06 nM for topiroxotstat and was 0.45 ± 0.2 nM for febuxostat; that on-rate (kon) was (4.3 ± 1.1) × 106 M−1s−1 for topiroxotstat and was(133.3 ± 3.5) × 106 M−1s−1 for febuxostat, and off-rate (koff) was (1.0±0.2) × 10−5 s−1 for topiroxotstat and was ≤ 0.16 × 10−5 s−1for febuxostat. Moreover, there were significant differences in the Ki*,app and koff values estimated using the appropriate specialized methods for tight binding inhibition versus classical steady state equilibrium methods, with the substantial differences of 14-fold and 32-fold reduction for topiroxostat, respectively, and of 9.6-fold and ≥ 213-fold reduction for febuxostat, while the kon values remain the moderate differences for the two inhibitors. The obvious greater AUC of XO engagement time courses and longer durations of above 70% engagement by the appropriate specialized methods for tight binding inhibition were observed that the results display the differences of 70.1% and 88%, respectively for topiroxostat and of 38.1% and 35.0%, respectively for febuxostat in human liver cell than by classical steady state equilibrium methods. Again, our studies provide several valuable recommendations of the optimal experiment protocols and appropriate analytical approaches for accurately quantitatively assessing the affinity and BK parameters as well as demonstrate the ability of our recommended methods to generate reliable data for tight binding inhibitors against XO.

Arginine substitution by alanine at the P1 position increases the selectivity of CmPI-II, a non-classical Kazal inhibitor

CmPI-II is a Kazal-type tight-binding inhibitor isolated from the Caribbean snail Cenchritis muricatus. This inhibitor has an unusual specificity in the Kazal family, as it can inhibit subtilisin A (SUBTA), elastases and trypsin. An alanine in CmPI-II P1 site could avoid trypsin inhibition while improving/maintaining SUBTA and elastases inhibition. Thus, an alanine mutant of this position (rCmPI-II R12A) was obtained by site-directed mutagenesis. The gene cmpiR12A was expressed in P. pastoris KM71H yeast. The recombinant protein (rCmPI-II R12A) was purified by the combination of two ionic exchange chromatography (1:cationic, 2 anionic) followed by and size exclusion chromatography. The N-terminal sequence obtained as well as the experimental molecular weight allowed verifying the identity of the recombinant protein, while the correct folding was confirmed by CD experiments. rCmPI-II R12A shows a slightly increase in potency against SUBTA and elastases. The alanine substitution at P1 site on CmPI-II abolishes the trypsin inhibition, confirming the relevance of an arginine residue at P1 site in CmPI-II for trypsin inhibition and leading to a molecule with more potentialities in biomedicine.

Structure and Function of Human Cytochrome P450 8B1

Human Cytochrome P450 8B1 (CYP8B1) is involved in the conversion of cholesterol to bile acids. It specifically hydroxylates the steroid ring system at C12 to produce the bile acid cholic acid. As such, CYP8B1 activity controls the ratio of cholic acid over another bile acid called chenodeoxycholic acid. The ratio of these two bile acids controls the overall hydrophobicity of the bile pool and the signaling through the farnesoid X receptor (FXR). Recent studies knocking out CYP8B1 implicated this enzyme as a good drug target for nonalcoholic fatty liver disease and type 2 diabetes. However, there are no selective inhibitors known for this enzyme, which restricts the further study of this enzyme and potential disease treatment. There are also no structures of CYP8B1 to guide the development of such inhibitors. Herein the CYP8B1 protein was generated recombinantly in E. coli from a synthetic engineered gene, purified to homogeneity, and characterized. The resulting protein was catalytically active and displayed normal spectral features. Changes in the absorbance spectra were used to evaluate the binding of multiple ligands. The tight-binding non-selective CYP8B1 inhibitor tioconazole was identified and then used to solve the first available X-ray structure of CYP8B1. This structure reveals the (S)-tioconazole imidazole nitrogen forming a coordinate covalent bond to the CYP8B1 heme iron, consistent with enzyme inhibition. The ligand is close towards the B’ helix due to the spatial hinderance of W281 and N286 on the opposite side of the active site. The availability of the CYP8B1 active site architecture should aid structure-based design of inhibitors that selectively bind CYP8B1 vs. other closely related P450 enzymes. Selective inhibitors should promote a better understanding of the role of CYP8B1 inhibition in these normal physiology and disease states and provide a possible treatment for nonalcoholic fatty liver disease and type 2 diabetes.

Cystatin M/E (Cystatin 6): A Janus-Faced Cysteine Protease Inhibitor with Both Tumor-Suppressing and Tumor-Promoting Functions.

Simple SummaryCystatin M/E is a low molecular mass protein and a potent inhibitor of proteolytic enzymes (cathepsins B, L, V, and legumain). Cystatin M/E participates in epidermal homeostasis and its deregulation is associated with several skin diseases. Cystatin M/E was initially identified as a tumor suppressor in breast cancer. Its gene is epigenetically downregulated and its methylation status holds a prognostic significance. Moreover, it may also serve as a biomarker for clinical diagnosis. This tumor-suppressing role was documented in cutaneous carcinoma, melanoma, lung, cervical, brain, prostate, gastric and renal cancers. Conversely, increased levels of cystatin M/E in triple-negative breast cancer tissues correlate with a higher risk of metastasis and a lower disease-free survival rate. Beside its orthodox tumor-suppressing role, cystatin M/E may operate as a tumor-promoting effector as reported for thyroid, oral and pancreatic cancer and hepatocellular carcinoma. Given its seemingly contradictory results, in-depth analysis of the regulatory mechanisms of the expression and activity of cystatin M/E during tumorigenesis have to be expanded. Likewise, the possible involvement of cystatin M/E in signaling pathways, beside its protease inhibitor function, remains to be scrutinized.Alongside its contribution in maintaining skin homeostasis and its probable involvement in fetal and placental development, cystatin M/E (also known as cystatin 6) was first described as a tumor suppressor of breast cancer. This review aims to provide an update on cystatin M/E with particular attention paid to its role during tumorigenesis. Cystatin M/E, which is related to type 2 cystatins, displays the unique property of being a dual tight-binding inhibitor of both legumain (also known as asparagine endopeptidase) and cysteine cathepsins L, V and B, while its expression level is epigenetically regulated via the methylation of the CST6 promoter region. The tumor-suppressing role of cystatin M/E was further reported in melanoma, cervical, brain, prostate, gastric and renal cancers, and cystatin M/E was proposed as a biomarker of prognostic significance. Contrariwise, cystatin M/E could have an antagonistic function, acting as a tumor promoter (e.g., oral, pancreatic cancer, thyroid and hepatocellular carcinoma). Taking into account these apparently divergent functions, there is an urgent need to decipher the molecular and cellular regulatory mechanisms of the expression and activity of cystatin M/E associated with the safeguarding homeostasis of the proteolytic balance as well as its imbalance in cancer.

Limited proteolysis by acrosin affects sperm-binding and mechanical resilience of the mouse zona pellucida

The encounter of oocyte and sperm is the key event initiating embryonic development in mammals. Crucial functions of this existential interaction are determined by proteolytic enzymes, such as acrosin, carried in the sperm head acrosome, and ovastacin, stored in the oocyte cortical granules. Ovastacin is released upon fertilisation to cleave the zona pellucida, a glycoprotein matrix surrounding the oocyte. This limited proteolysis hardens the oocyte envelope, and thereby provides a definitive block against polyspermy and protects the developing embryo. On the other hand, acrosin, the renowned and most abundant acrosomal protease, has been thought to enable sperm to penetrate the oocyte envelope. Depending on the species, proteolytic cleavage of the zona pellucida by acrosin is either essential or conducive for fertilisation. However, the specific target cleavage sites and the resulting physiological consequences of this proteolysis remained obscure. Here, we treated native mouse zonae pellucidae with active acrosin and identified two cleavage sites in zona pellucida protein 1 (ZP1), five in ZP2 and one in ZP3 by mass spectrometry. Several of these sites are highly conserved in mammals. Remarkably, limited proteolysis by acrosin leads to zona pellucida remodelling rather than degradation. Thus, acrosin affects both sperm binding and mechanical resilience of the zona pellucida, as assessed by microscopy and nanoindentation measurements, respectively. Furthermore, we ascertained potential regulatory effects of acrosin, via activation of latent pro-ovastacin and inactivation of fetuin-B, a tight binding inhibitor of ovastacin. These results offer novel insights into the complex proteolytic network modifying the extracellular matrix of the mouse oocyte, which might apply also to other species.

A Long Residence Time Enoyl-Reductase Inhibitor Explores an Extended Binding Region with Isoenzyme-Dependent Tautomer Adaptation and Differential Substrate-Binding Loop Closure.

The enoyl-acyl carrier protein (ACP) reductase (ENR) is a key enzyme within the bacterial fatty-acid synthesis pathway. It has been demonstrated that small-molecule inhibitors carrying the diphenylether (DPE) scaffold bear a great potential for the development of highly specific and effective drugs against this enzyme class. Interestingly, different substitution patterns of the DPE scaffold have been shown to lead to varying effects on the kinetic and thermodynamic behavior toward ENRs from different organisms. Here, we investigated the effect of a 4'-pyridone substituent in the context of the slow tight-binding inhibitor SKTS1 on the inhibition of the Staphylococcus aureus enoyl-ACP-reductase saFabI and the closely related isoenzyme from Mycobacterium tuberculosis, InhA, and explored a new interaction site of DPE inhibitors within the substrate-binding pocket. Using high-resolution crystal structures of both complexes in combination with molecular dynamics (MD) simulations, kinetic measurements, and quantum mechanical (QM) calculations, we provide evidence that the 4'-pyridone substituent adopts different tautomeric forms when bound to the two ENRs. We furthermore elucidate the structural determinants leading to significant differences in the residence time of SKTS1 on both enzymes.