Variants in the pancreatic CUB and zona pellucida-like domains 1 (CUZD1) gene in early-onset chronic pancreatitis - A possible new susceptibility gene.

OBJECTIVE: Non-alcoholic chronic pancreatitis (NACP) frequently develops in the setting of genetic susceptibility associated with alterations in genes that are highly expressed in the pancreas. However, the genetic basis of NACP remains unresolved in a significant number of patients warranting a search for further risk genes. DESIGN: We analyzed CUZD1, which encodes the CUB and zona pellucida-like domains 1 protein that is found in high levels in pancreatic acinar cells. We sequenced the coding region in 1163 European patients and 2018 European controls. In addition, we analyzed 297 patients and 1070 controls from Japan. We analyzed secretion of wild-type and mutant CUZD1 from transfected cells using Western blotting. RESULTS: In the European cohort, we detected 30 non-synonymous variants. Using different prediction tools (SIFT, CADD, PROVEAN, PredictSNP) or the combination of these tools, we found accumulation of predicted deleterious variants in patients (p-value range 0.002-0.013; OR range 3.1-5.2). No association was found in the Japanese cohort, in which 13 non-synonymous variants were detected. Functional studies revealed >50% reduced secretion of 7 variants, however, these variants were not significantly enriched in European CP patients. CONCLUSION: Our data indicate that CUZD1 might be a novel susceptibility gene for NACP. How these variants predispose to pancreatitis remains to be elucidated.

Calmodulin as Ca2+-Dependent Interactor of FTO Dioxygenase.

FTO is an N6-methyladenosine demethylase removing methyl groups from nucleic acids. Several studies indicate the creation of FTO complexes with other proteins. Here, we looked for regulatory proteins recognizing parts of the FTO dioxygenase region. In the Calmodulin (CaM) Target Database, we found the FTO C-domain potentially binding CaM, and we proved this finding experimentally. The interaction was Ca2+-dependent but independent on FTO phosphorylation. We found that FTO-CaM interaction essentially influences calcium-binding loops in CaM, indicating the presence of two peptide populations-exchanging as CaM alone and differently, suggesting that only one part of CaM interacts with FTO, and the other one reminds free. The modeling of FTO-CaM interaction showed its stable structure when the half of the CaM molecule saturated with Ca2+ interacts with the FTO C-domain, whereas the other part is disconnected. The presented data indicate calmodulin as a new FTO interactor and support engagement of the FTO protein in calcium signaling pathways.

Insight into the Binding and Hydrolytic Preferences of hNudt16 Based on Nucleotide Diphosphate Substrates.

Nudt16 is a member of the NUDIX family of hydrolases that show specificity towards substrates consisting of a nucleoside diphosphate linked to another moiety X. Several substrates for hNudt16 and various possible biological functions have been reported. However, some of these reports contradict each other and studies comparing the substrate specificity of the hNudt16 protein are limited. Therefore, we quantitatively compared the affinity of hNudt16 towards a set of previously published substrates, as well as identified novel potential substrates. Here, we show that hNudt16 has the highest affinity towards IDP and GppG, with Kd below 100 nM. Other tested ligands exhibited a weaker affinity of several orders of magnitude. Among the investigated compounds, only IDP, GppG, m7GppG, AppA, dpCoA, and NADH were hydrolyzed by hNudt16 with a strong substrate preference for inosine or guanosine containing compounds. A new identified substrate for hNudt16, GppG, which binds the enzyme with an affinity comparable to that of IDP, suggests another potential regulatory role of this protein. Molecular docking of hNudt16-ligand binding inside the hNudt16 pocket revealed two binding modes for representative substrates. Nucleobase stabilization by Π stacking interactions with His24 has been associated with strong binding of hNudt16 substrates.

Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase.

The FTO protein is involved in a wide range of physiological processes, including adipogenesis and osteogenesis. This two-domain protein belongs to the AlkB family of 2-oxoglutarate (2-OG)- and Fe(II)-dependent dioxygenases, displaying N6-methyladenosine (N6-meA) demethylase activity. The aim of the study was to characterize the relationships between the structure and activity of FTO. The effect of cofactors (Fe2+/Mn2+ and 2-OG), Ca2+ that do not bind at the catalytic site, and protein concentration on FTO properties expressed in either E. coli (ECFTO) or baculovirus (BESFTO) system were determined using biophysical methods (DSF, MST, SAXS) and biochemical techniques (size-exclusion chromatography, enzymatic assay). We found that BESFTO carries three phosphoserines (S184, S256, S260), while there were no such modifications in ECFTO. The S256D mutation mimicking the S256 phosphorylation moderately decreased FTO catalytic activity. In the presence of Ca2+, a slight stabilization of the FTO structure was observed, accompanied by a decrease in catalytic activity. Size exclusion chromatography and MST data confirmed the ability of FTO from both expression systems to form homodimers. The MST-determined dissociation constant of the FTO homodimer was consistent with their in vivo formation in human cells. Finally, a low-resolution structure of the FTO homodimer was built based on SAXS data.

Synthesis of Novel Halogenated Heterocycles Based on o-Phenylenediamine and Their Interactions with the Catalytic Subunit of Protein Kinase CK2.

Protein kinase CK2 is a highly pleiotropic protein kinase capable of phosphorylating hundreds of protein substrates. It is involved in numerous cellular functions, including cell viability, apoptosis, cell proliferation and survival, angiogenesis, or ER-stress response. As CK2 activity is found perturbed in many pathological states, including cancers, it becomes an attractive target for the pharma. A large number of low-mass ATP-competitive inhibitors have already been developed, the majority of them halogenated. We tested the binding of six series of halogenated heterocyclic ligands derived from the commercially available 4,5-dihalo-benzene-1,2-diamines. These ligand series were selected to enable the separation of the scaffold effect from the hydrophobic interactions attributed directly to the presence of halogen atoms. In silico molecular docking was initially applied to test the capability of each ligand for binding at the ATP-binding site of CK2. HPLC-derived ligand hydrophobicity data are compared with the binding affinity assessed by low-volume differential scanning fluorimetry (nanoDSF). We identified three promising ligand scaffolds, two of which have not yet been described as CK2 inhibitors but may lead to potent CK2 kinase inhibitors. The inhibitory activity against CK2α and toxicity against four reference cell lines have been determined for eight compounds identified as the most promising in nanoDSF assay.

Native Structure-Based Peptides as Potential Protein-Protein Interaction Inhibitors of SARS-CoV-2 Spike Protein and Human ACE2 Receptor.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a positive-strand RNA virus that causes severe respiratory syndrome in humans, which is now referred to as coronavirus disease 2019 (COVID-19). Since December 2019, the new pathogen has rapidly spread globally, with over 65 million cases reported to the beginning of December 2020, including over 1.5 million deaths. Unfortunately, currently, there is no specific and effective treatment for COVID-19. As SARS-CoV-2 relies on its spike proteins (S) to bind to a host cell-surface receptor angiotensin-converting enzyme-2(ACE2), and this interaction is proved to be responsible for entering a virus into host cells, it makes an ideal target for antiviral drug development. In this work, we design three very short peptides based on the ACE2 sequence/structure fragments, which may effectively bind to the receptor-binding domain (RBD) of S protein and may, in turn, disrupt the important virus-host protein-protein interactions, blocking early steps of SARS-CoV-2 infection. Two of our peptides bind to virus protein with affinity in nanomolar range, and as very short peptides have great potential for drug development.

Diketopiperazine-Based, Flexible Tadalafil Analogues: Synthesis, Crystal Structures and Biological Activity Profile.

Phosphodiesterase 5 (PDE5) is one of the most extensively studied phosphodiesterases that is highly specific for cyclic-GMP hydrolysis. PDE5 became a target for drug development based on its efficacy for treatment of erectile dysfunction. In the present study, we synthesized four novel analogues of the phosphodiesterase type 5 (PDE5) inhibitor-tadalafil, which differs in (i) ligand flexibility (rigid structure of tadalafil vs. conformational flexibility of newly synthesized compounds), (ii) stereochemistry associated with applied amino acid building blocks, and (iii) substitution with bromine atom in the piperonyl moiety. For both the intermediate and final compounds as well as for the parent molecule, we have established the crystal structures and performed a detailed analysis of their structural features. The initial screening of the cytotoxic effect on 16 different human cancer and non-cancer derived cell lines revealed that in most cases, the parent compound exhibited a stronger cytotoxic effect than new derivatives, except for two cell lines: HEK 293T (derived from a normal embryonic kidney, that expresses a mutant version of SV40 large T antigen) and MCF7 (breast adenocarcinoma). Two independent studies on the inhibition of PDE5 activity, based on both pure enzyme assay and modulation of the release of nitric oxide from platelets under the influence of tadalafil and its analogues revealed that, unlike a reference compound that showed strong PDE5 inhibitory activity, the newly obtained compounds did not have a noticeable effect on PDE5 activity in the range of concentrations tested. Finally, we performed an investigation of the toxicological effect of synthesized compounds on Caenorhabditis elegans in the highest applied concentration of 6a,b and 7a,b (160 µM) and did not find any effect that would suggest disturbance to the life cycle of Caenorhabditis elegans. The lack of toxicity observed in Caenorhabditis elegans and enhanced, strengthened selectivity and activity toward the MCF7 cell line made 7a,b good leading structures for further structure activity optimization and makes 7a,b a reasonable starting point for the search of new, selective cytotoxic agents.

The halogenation of natural flavonoids, baicalein and chrysin, enhances their affinity to human protein kinase CK2.

A series of halogenated derivatives of natural flavonoids: baicalein and chrysin were designed and investigated as possible ligands for the catalytic subunit of tumor-associated human kinase CK2. Thermal shift assay method, in silico modeling, and high-performance liquid chromatography-derived hydrophobicity together with IC50 values determined in biochemical assay were used to explain the ligand affinity to the catalytic subunit of human protein kinase CK2. Obtained results revealed that substitution of baicalein and chrysin with halogen atom increases their binding affinity to hCK2α, and for 8-chlorochrysin the observed effect is even stronger than for the reference CK2 inhibitor-4,5,6,7-tetrabromo-1H-benzotriazole. The cytotoxic activities of the baicalein and chrysin derivatives in the in vitro model have been evaluated for MV4-11 (human biphenotypic B myelomonocytic leukemia), A549 (human lung adenocarcinoma), LoVo (human colon cancer), and MCF-7 (human breast cancer) as well as on the nontumorigenic human breast epithelial MCF-10A cell lines. Among the baicalein derivatives, the strongest cytotoxic effect was observed for 8-bromobaicalein, which exhibited the highest activity against breast cancer cell line MCF-7 (IC50 10 ± 3 µM). In the chrysin series, the strongest cytotoxic effect was observed for unsubstituted chrysin, which exhibited the highest activity against leukemic cell line MV4-11 (IC50 10 ± 4 µM).

Effect of histidine protonation state on ligand binding at the ATP-binding site of human protein kinase CK2.

Histidine residues contribute to numerous molecular interactions, owing to their structure with the ionizable aromatic side chain with pKa close to the physiological pH. Herein, we studied how the two histidine residues, His115 and His160 of the catalytic subunit of human protein kinase CK2, affect the binding of the halogenated heterocyclic ligands at the ATP-binding site. Thermodynamic studies on the interaction between five variants of hCK2α (WT protein and four histidine mutants) and three ionizable bromo-benzotriazoles and their conditionally non-ionizable benzimidazole counterparts were performed with nanoDSF, MST, and ITC. The results allowed us to identify the contribution of interactions involving the particular histidine residues to ligand binding. We showed that despite the well-documented hydrogen bonding/salt bridge formation dragging the anionic ligands towards Lys68, the protonated His160 also contributes to the binding of such ligands by long-range electrostatic interactions. Simultaneously, His 115 indirectly affects ligand binding, placing the hinge region in open/closed conformations.

The pursuit of new alternative ways to eradicate Helicobacter pylori continues: Detailed characterization of interactions in the adenylosuccinate synthetase active site.

Purine nucleotide synthesis is realised only through the salvage pathway in pathogenic bacterium Helicobacter pylori. Therefore, the enzymes of this pathway, among them also the adenylosuccinate synthetase (AdSS), present potential new drug targets. This paper describes characterization of His6-tagged AdSS from H. pylori. Thorough analysis of 3D-structures of fully ligated AdSS (in a complex with guanosine diphosphate, 6-phosphoryl-inosine monophosphate, hadacidin and Mg2+) and AdSS in a complex with inosine monophosphate (IMP) only, enabled identification of active site interactions crucial for ligand binding and enzyme activity. Combination of experimental and molecular dynamics (MD) simulations data, particularly emphasized the importance of hydrogen bond Arg135-IMP for enzyme dimerization and active site formation. The synergistic effect of substrates (IMP and guanosine triphosphate) binding was suggested by MD simulations. Several flexible elements of the structure (loops) are stabilized by the presence of IMP alone, however loops comprising residues 287-293 and 40-44 occupy different positions in two solved H. pylori AdSS structures. MD simulations discovered the hydrogen bond network that stabilizes the closed conformation of the residues 40-50 loop, only in the presence of IMP. Presented findings provide a solid basis for the design of new AdSS inhibitors as potential drugs against H. pylori.

Improvement of native structure-based peptides as efficient inhibitors of protein-protein interactions of SARS-CoV-2 spike protein and human ACE2.

New pathogens responsible for novel human disease outbreaks in the last two decades are mainly the respiratory system viruses. Not different was the last pandemic episode, caused by infection of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). One of the extensively explored targets, in the recent scientific literature, as a possible way for rapid development of COVID-19 specific drug(s) is the interaction between the receptor-binding domain of the virus' spike (S) glycoprotein and human receptor angiotensin-converting enzyme 2 (hACE2). This protein-protein recognition process is involved in the early stages of the SARS-CoV-2 life cycle leading to the host cell membrane penetration. Thus, disrupting this interaction may block or significantly reduce the infection caused by the novel pathogen. Previously we have designed (by in silico structure-based analysis) three very short peptides having sequences inspirited by hACE2 native fragments, which effectively bind to the SARS-CoV-2 S protein and block its interaction with the human receptor. In continuation of the above mentioned studies, here we presented an application of molecular modeling approach resulting in improved binding affinity of the previously proposed ligand and its enhanced ability to inhibit meaningful host-virus protein-protein interaction. The new optimized hexapeptide binds to the virus protein with affinity one magnitude higher than the initial ligand and, as a very short peptide, has also great potential for further drug development. The peptide-based strategy is rapid and cost-effective for developing and optimizing efficient protein-protein interactions disruptors and may be successfully applied to discover antiviral candidates against other future emerging human viral infections.

Competition between electrostatic interactions and halogen bonding in the protein-ligand system: structural and thermodynamic studies of 5,6-dibromobenzotriazole-hCK2α complexes.

CK2 is a member of the CMGC group of eukaryotic protein kinases and a cancer drug target. It can be efficiently inhibited by halogenated benzotriazoles and benzimidazoles. Depending on the scaffold, substitution pattern, and pH, these compounds are either neutral or anionic. Their binding poses are dictated by a hydrophobic effect (desolvation) and a tug of war between a salt bridge/hydrogen bond (to K68) and halogen bonding (to E114 and V116 backbone oxygens). Here, we test the idea that binding poses might be controllable by pH for ligands with near-neutral pKa, using the conditionally anionic 5,6-DBBt and constitutively anionic TBBt as our models. We characterize the binding by low-volume Differential Scanning Fluorimetry (nanoDSF), Isothermal Calorimetry (ITC), Hydrogen/Deuterium eXchange (HDX), and X-ray crystallography (MX). The data indicate that the ligand pose away from the hinge dominates for the entire tested pH range (5.5-8.5). The insensitivity of the binding mode to pH is attributed to the perturbation of ligand pKa upon binding that keeps it anionic in the ligand binding pocket at all tested pH values. However, a minor population of the ligand, detectable only by HDX, shifts towards the hinge in acidic conditions. Our findings demonstrate that electrostatic (ionic) interactions predominate over halogen bonding.

5,6-diiodo-1H-benzotriazole: new TBBt analogue that minutely affects mitochondrial activity.

4,5,6,7-Tetrabromo-1H-benzotriazole is widely used as the reference ATP-competitive inhibitor of protein kinase CK2. Herein, we study its new analogs: 5,6-diiodo- and 5,6-diiodo-4,7-dibromo-1H-benzotriazole. We used biophysical (MST, ITC) and biochemical (enzymatic assay) methods to describe the interactions of halogenated benzotriazoles with the catalytic subunit of human protein kinase CK2 (hCK2α). To trace the biological activity, we measured their cytotoxicity against four reference cancer cell lines and the effect on the mitochondrial inner membrane potential. The results obtained lead to the conclusion that iodinated compounds are an attractive alternative to brominated ones. One of them retains the cytotoxicity against selected cancer cell lines of the reference TBBt with a smaller side effect on mitochondrial activity. Both iodinated compounds are candidate leaders in the further development of CK2 inhibitors.

Halogen Atoms in the Protein-Ligand System. Structural and Thermodynamic Studies of the Binding of Bromobenzotriazoles by the Catalytic Subunit of Human Protein Kinase CK2.

Binding of a family of brominated benzotriazoles to the catalytic subunit of human protein kinase CK2 (hCK2α) was used as a model system to assess the contribution of halogen bonding to protein-ligand interaction. CK2 is a constitutively active pleiotropic serine/threonine protein kinase that belongs to the CMGC group of eukaryotic protein kinases (EPKs). Due to the addiction of some cancer cells, CK2 is an attractive and well-characterized drug target. Halogenated benzotriazoles act as ATP-competitive inhibitors with unexpectedly good selectivity for CK2 over other EPKs. We have characterized the interaction of bromobenzotriazoles with hCK2α by X-ray crystallography, low-volume differential scanning fluorimetry, and isothermal titration calorimetry. Properties of free ligands in solution were additionally characterized by volumetric and RT-HPLC measurements. Thermodynamic data indicate that the affinity increases with bromo substitution, with greater contributions from 5- and 6-substituents than 4- and 7-substituents. Except for 4,7-disubstituted compounds, the bromobenzotriazoles adopt a canonical pose with the triazole close to lysine 68, which precludes halogen bonding. More highly substituted benzotriazoles adopt many additional noncanonical poses, presumably driven by a large hydrophobic contribution to binding. Some noncanonical ligand orientations allow the formation of halogen bonds with the hinge region. Consistent with a predominantly hydrophobic interaction, the isobaric heat capacity decreases upon ligand binding, the more so the higher the substitution.

Single tryptophan Y160W mutant of homooligomeric E. coli purine nucleoside phosphorylase implies that dimers forming the hexamer are functionally not equivalent.

E. coli purine nucleoside phosphorylase is a homohexamer, which structure, in the apo form, can be described as a trimer of dimers. Earlier studies suggested that ligand binding and kinetic properties are well described by two binding constants and two sets of kinetic constants. However, most of the crystal structures of this enzyme complexes with ligands do not hold the three-fold symmetry, but only two-fold symmetry, as one of the three dimers is different (both active sites in the open conformation) from the other two (one active site in the open and one in the closed conformation). Our recent detailed studies conducted over broad ligand concentration range suggest that protein-ligand complex formation in solution actually deviates from the two-binding-site model. To reveal the details of interactions present in the hexameric molecule we have engineered a single tryptophan Y160W mutant, responding with substantial intrinsic fluorescence change upon ligand binding. By observing various physical properties of the protein and its various complexes with substrate and substrate analogues we have shown that indeed three-binding-site model is necessary to properly describe binding of ligands by both the wild type enzyme and the Y160W mutant. Thus we have pointed out that a symmetrical dimer with both active sites in the open conformation is not forced to adopt this conformation by interactions in the crystal, but most probably the dimers forming the hexamer in solution are not equivalent as well. This, in turn, implies that an allosteric cooperation occurs not only within a dimer, but also among all three dimers forming a hexameric molecule.

Rational drug-design approach supported with thermodynamic studies - a peptide leader for the efficient bi-substrate inhibitor of protein kinase CK2.

Numerous inhibitors of protein kinases act on the basis of competition, targeting the ATP binding site. In this work, we present a procedure of rational design of a bi-substrate inhibitor, complemented with biophysical assays. The inhibitors of this type are commonly engineered by combining ligands carrying an ATP-like part with a peptide or peptide-mimicking fragment that determines specificity. Approach presented in this paper led to generation of a specific system for independent screening for efficient ligands and peptides, by means of thermodynamic measurements, that assessed the ability of the identified ligand and peptide to combine into a bi-substrate inhibitor. The catalytic subunit of human protein kinase CK2 was used as the model target. Peptide sequence was optimized using peptide libraries [KGDE]-[DE]-[ST]-[DE]3-4-NH2, originated from the consensus CK2 sequence. We identified KESEEE-NH2 peptide as the most promising one, whose binding affinity is substantially higher than that of the reference RRRDDDSDDD peptide. We assessed its potency to form an efficient bi-substrate inhibitor using tetrabromobenzotriazole (TBBt) as the model ATP-competitive inhibitor. The formation of ternary complex was monitored using Differential Scanning Fluorimetry (DSF), Microscale Thermophoresis (MST) and Isothermal Titration Calorimetry (ITC).