CoCoNuTs are a diverse subclass of Type IV restriction systems predicted to target RNA.

A comprehensive census of McrBC systems, among the most common forms of prokaryotic Type IV restriction systems, followed by phylogenetic analysis, reveals their enormous abundance in diverse prokaryotes and a plethora of genomic associations. We focus on a previously uncharacterized branch, which we denote coiled-coil nuclease tandems (CoCoNuTs) for their salient features: the presence of extensive coiled-coil structures and tandem nucleases. The CoCoNuTs alone show extraordinary variety, with three distinct types and multiple subtypes. All CoCoNuTs contain domains predicted to interact with translation system components, such as OB-folds resembling the SmpB protein that binds bacterial transfer-messenger RNA (tmRNA), YTH-like domains that might recognize methylated tmRNA, tRNA, or rRNA, and RNA-binding Hsp70 chaperone homologs, along with RNases, such as HEPN domains, all suggesting that the CoCoNuTs target RNA. Many CoCoNuTs might additionally target DNA, via McrC nuclease homologs. Additional restriction systems, such as Type I RM, BREX, and Druantia Type III, are frequently encoded in the same predicted superoperons. In many of these superoperons, CoCoNuTs are likely regulated by cyclic nucleotides, possibly, RNA fragments with cyclic termini, that bind associated CARF (CRISPR-Associated Rossmann Fold) domains. We hypothesize that the CoCoNuTs, together with the ancillary restriction factors, employ an echeloned defense strategy analogous to that of Type III CRISPR-Cas systems, in which an immune response eliminating virus DNA and/or RNA is launched first, but then, if it fails, an abortive infection response leading to PCD/dormancy via host RNA cleavage takes over.

All organisms, from animals to bacteria, are subject to genetic parasites, such as viruses and transposons. Genetic parasites are pieces of nucleic acids (DNA or RNA) that can use a cell's machinery to copy themselves at the expense of their hosts. This often leads to the host's demise, so organisms evolved many types of defense mechanisms. One of the most ancient and common forms of defense against viruses and transposons is the targeted restriction of nucleic acids, that is, deployment of host enzymes that can destroy or restrict nucleic acids containing specific sequence motifs or modifications. In bacteria, many of the restriction enzymes targeting parasitic genetic elements are formed by fusions of proteins from the so-called McrBC systems with a protein domain called EVE. EVE and other functionally similar domains are a part of proteins that recognize and bind modified bases in nucleic acids. Enzymes can use the ability of these specificity domains to bind modified bases to detect non-host nucleic acids. Bell et al. conducted a comprehensive computational search for McrBC systems and discovered a large and highly diverse branch of this family with unusual characteristic structural and functional domains. These features include regions that form long alpha-helices (coils) that coil with other alpha-helices (known as coiled-coils), as well as several distinct enzymatic domains that break down nucleic acids (known as nucleases). They call these systems CoCoNuTs (coiled-coiled nuclease tandems). All CoCoNuTs contain domains, including EVE-like ones, which are predicted to interact with components of the RNA-based systems responsible for producing proteins in the cell (translation), suggesting that the CoCoNuTs have an important impact on protein abundance and RNA metabolism. Bell et al.'s findings will be of interest to scientists working on prokaryotic immunity and virulence. Furthermore, similarities between CoCoNuTs and components of eukaryotic RNA-degrading systems suggest evolutionary connections between this diverse family of bacterial predicted RNA restriction systems and RNA regulatory pathways of eukaryotes. Further deciphering the mechanisms of CoCoNuTs could shed light on how certain pathways of RNA metabolism and regulation evolved, and how they may contribute to advances in biotechnology.

CoCoNuTs: A diverse subclass of Type IV restriction systems predicted to target RNA.

A comprehensive census of McrBC systems, among the most common forms of prokaryotic Type IV restriction systems, followed by phylogenetic analysis, reveals their enormous abundance in diverse prokaryotes and a plethora of genomic associations. We focus on a previously uncharacterized branch, which we denote CoCoNuTs (coiled-coil nuclease tandems) for their salient features: the presence of extensive coiled-coil structures and tandem nucleases. The CoCoNuTs alone show extraordinary variety, with 3 distinct types and multiple subtypes. All CoCoNuTs contain domains predicted to interact with translation system components, such as OB-folds resembling the SmpB protein that binds bacterial transfer-messenger RNA (tmRNA), YTH-like domains that might recognize methylated tmRNA, tRNA, or rRNA, and RNA-binding Hsp70 chaperone homologs, along with RNases, such as HEPN domains, all suggesting that the CoCoNuTs target RNA. Many CoCoNuTs might additionally target DNA, via McrC nuclease homologs. Additional restriction systems, such as Type I RM, BREX, and Druantia Type III, are frequently encoded in the same predicted superoperons. In many of these superoperons, CoCoNuTs are likely regulated by cyclic nucleotides, possibly, RNA fragments with cyclic termini, that bind associated CARF (CRISPR-Associated Rossmann Fold) domains. We hypothesize that the CoCoNuTs, together with the ancillary restriction factors, employ an echeloned defense strategy analogous to that of Type III CRISPR-Cas systems, in which an immune response eliminating virus DNA and/or RNA is launched first, but then, if it fails, an abortive infection response leading to PCD/dormancy via host RNA cleavage takes over.

A generic binding pocket for small molecule IKs activators at the extracellular inter-subunit interface of KCNQ1 and KCNE1 channel complexes.

The cardiac IKs ion channel comprises KCNQ1, calmodulin, and KCNE1 in a dodecameric complex which provides a repolarizing current reserve at higher heart rates and protects from arrhythmia syndromes that cause fainting and sudden death. Pharmacological activators of IKs are therefore of interest both scientifically and therapeutically for treatment of IKs loss-of-function disorders. One group of chemical activators are only active in the presence of the accessory KCNE1 subunit and here we investigate this phenomenon using molecular modeling techniques and mutagenesis scanning in mammalian cells. A generalized activator binding pocket is formed extracellularly by KCNE1, the domain-swapped S1 helices of one KCNQ1 subunit and the pore/turret region made up of two other KCNQ1 subunits. A few residues, including K41, A44 and Y46 in KCNE1, W323 in the KCNQ1 pore, and Y148 in the KCNQ1 S1 domain, appear critical for the binding of structurally diverse molecules, but in addition, molecular modeling studies suggest that induced fit by structurally different molecules underlies the generalized nature of the binding pocket. Activation of IKs is enhanced by stabilization of the KCNQ1-S1/KCNE1/pore complex, which ultimately slows deactivation of the current, and promotes outward current summation at higher pulse rates. Our results provide a mechanistic explanation of enhanced IKs currents by these activator compounds and provide a map for future design of more potent therapeutically useful molecules.

Effect of Colchicine Binding Site Inhibitors on the Tubulin Intersubunit Interaction.

Microtubules are dynamic, non-covalent polymers consisting of α- and ß-tubulin subunits that are involved in a wide range of intracellular processes. The polymerization and dynamics of microtubules are regulated by many factors, including small molecules that interact with different sites on the tubulin dimer. Colchicine binding site inhibitors (CBSIs) destabilize microtubules and inhibit tubulin polymerization, leading to cell cycle arrest. Because of their therapeutic potential, the molecular mechanism of CBSI function is an area of active research. Nevertheless, important details of this mechanism have yet to be resolved. In this study, we use atomistic molecular dynamics simulations to show that the binding of CBSIs to the tubulin heterodimer leads to the weakening of tubulin intersubunit interaction. Using atomistic molecular dynamics simulations and binding free energy calculations, we show that CBSIs act as protein-protein interaction inhibitors and destabilize interlinkage between α and ß subunits, which is crucial for longitudinal contacts in the microtubule lattice. Our results offer new insight into the mechanisms of microtubule polymerization inhibition by colchicine and its analogs.

Search for Origins of Anti-CRISPR Proteins by Structure Comparison.

Many bacterial and archaeal viruses encode anti-CRISPR proteins (Acrs) that specifically inhibit CRISPR-Cas systems via various mechanisms. The majority of the Acrs are small, non-enzymatic proteins that abrogate CRISPR activity by binding to Cas effector proteins. The Acrs evolve fast, due to the arms race with the respective CRISPR-Cas systems, which hampers the elucidation of their evolutionary origins by sequence comparison. We performed comprehensive structural modeling using AlphaFold2 for 3693 experimentally characterized and predicted Acrs, followed by a comparison to the protein structures in the Protein Data Bank database. After clustering the Acrs by sequence similarity, 363 high-quality structural models were obtained that accounted for 102 Acr families. Structure comparisons allowed the identification of homologs for 13 of these families that could be ancestors of the Acrs. Despite the limited extent of structural conservation, the inferred origins of Acrs show distinct trends, in particular, recruitment of toxins and antitoxins and SOS repair system components for the Acr function.

A Study of a Protein-Folding Machine: Transient Rotation of the Polypeptide Backbone Facilitates Rapid Folding of Protein Domains in All-Atom Molecular Dynamics Simulations.

Molecular dynamics simulations of protein folding typically consider the polypeptide chain at equilibrium and in isolation from the cellular components. We argue that in order to understand protein folding as it occurs in vivo, it should be modeled as an active, energy-dependent process, in which the cellular protein-folding machine directly manipulates the polypeptide. We conducted all-atom molecular dynamics simulations of four protein domains, whose folding from the extended state was augmented by the application of rotational force to the C-terminal amino acid, while the movement of the N-terminal amino acid was restrained. We have shown earlier that such a simple manipulation of peptide backbone facilitated the formation of native structures in diverse α-helical peptides. In this study, the simulation protocol was modified, to apply the backbone rotation and movement restriction only for a short time at the start of simulation. This transient application of a mechanical force to the peptide is sufficient to accelerate, by at least an order of magnitude, the folding of four protein domains from different structural classes to their native or native-like conformations. Our in silico experiments show that a compact stable fold may be attained more readily when the motions of the polypeptide are biased by external forces and constraints.

Mechanism of external K+ sensitivity of KCNQ1 channels.

KCNQ1 voltage-gated K+ channels are involved in a wide variety of fundamental physiological processes and exhibit the unique feature of being markedly inhibited by external K+. Despite the potential role of this regulatory mechanism in distinct physiological and pathological processes, its exact underpinnings are not well understood. In this study, using extensive mutagenesis, molecular dynamics simulations, and single-channel recordings, we delineate the molecular mechanism of KCNQ1 modulation by external K+. First, we demonstrate the involvement of the selectivity filter in the external K+ sensitivity of the channel. Then, we show that external K+ binds to the vacant outermost ion coordination site of the selectivity filter inducing a diminution in the unitary conductance of the channel. The larger reduction in the unitary conductance compared to whole-cell currents suggests an additional modulatory effect of external K+ on the channel. Further, we show that the external K+ sensitivity of the heteromeric KCNQ1/KCNE complexes depends on the type of associated KCNE subunits.

Structural and electrophysiological basis for the modulation of KCNQ1 channel currents by ML277.

The KCNQ1 ion channel plays critical physiological roles in electrical excitability and K+ recycling in organs including the heart, brain, and gut. Loss of function is relatively common and can cause sudden arrhythmic death, sudden infant death, epilepsy and deafness. Here, we report cryogenic electron microscopic (cryo-EM) structures of Xenopus KCNQ1 bound to Ca2+/Calmodulin, with and without the KCNQ1 channel activator, ML277. A single binding site for ML277 was identified, localized to a pocket lined by the S4-S5 linker, S5 and S6 helices of two separate subunits. Several pocket residues are not conserved in other KCNQ isoforms, explaining specificity. MD simulations and point mutations support this binding location for ML277 in open and closed channels and reveal that prevention of inactivation is an important component of the activator effect. Our work provides direction for therapeutic intervention targeting KCNQ1 loss of function pathologies including long QT interval syndrome and seizures.

Identification of non-classical hCA XII inhibitors using combination of computational approaches for drug design and discovery.

Human carbonic anhydrase XII (hCA XII) isozyme is of high therapeutic value as a pharmacological target and biomarker for different types of cancer. The hCA XII is one of the crucial effectors that regulates extracellular and intracellular pH and affects cancer cell proliferation, invasion, growth and metastasis. Despite the fact that interaction features of hCAs inhibitors with the catalytic site of the enzyme are well described, lack in the selectivity of the traditional hCA inhibitors based on the sulfonamide group or related motifs is an urgent issue. Moreover, drugs containing sulfanomides can cause sulfa allergies. Thus, identification of novel non-classical inhibitors of hCA XII is of high priority and is currently the subject of a vast field of study. This study was devoted to the identification of novel potential hCA XII inhibitors using comprehensive set of computational approaches for drug design discovery: generation and validation of structure- and ligand-based pharmacophore models, molecular docking, re-scoring of virtual screening results with MMGBSA, molecular dynamics simulations, etc. As the results of the study several compounds with alternative to classical inhibitors chemical scaffolds, in particular one of coumarins derivative, have been identified and are of high interest as potential non-classical hCA XII inhibitors.

Improving virtual screening results with MM/GBSA and MM/PBSA rescoring.

Virtual screening (VS) based on molecular docking is one of the most useful methods in computer-aided drug design. By allowing to identify computationally putative ligands binding to the proteins of interest, VS dramatically reduces the time and expense of the development of novel therapeutics. Among the limitations of the VS approaches is the low accuracy of scoring functions implemented in docking methods for assessing binding affinity. Many such scoring functions are developed for rapid, high-throughput evaluation of binding energy of multiple conformations generated by a searching algorithm. The methods for more rigorous calculation of binding affinity calculation are generally time-consuming. Even so, in many studies more accurate methods were used for rescoring of the final poses and false-positive hits evaluation. We performed VS for three benchmark sets and used energy minimization with MM/PB(GB)SA methods (molecular mechanics energies combined with the Poisson-Boltzmann or generalized Born and surface area) to rescore binding affinities. The comparison of the area under the curve (AUC), enrichment factor (EF), and Boltzmann-enhanced discrimination of receiver operating characteristics (BEDROC) showed essential improvements in the binding energy prediction after the rescoring. Finally, we provide a program for minimization and rescoring VS results based on freely available AmberTools. The code requires just the final binding poses of the ligand as the input and can be used with any docking program.

A new microtubule-stabilizing agent shows potent antiviral effects against African swine fever virus with no cytotoxicity.

African swine fever virus (ASFV) is the causal agent of a fatal disease of domestic swine for which no effective antiviral drugs are available. Recently, it has been shown that microtubule-targeting agents hamper the infection cycle of different viruses. In this study, we conducted in silico screening against the colchicine binding site (CBS) of tubulin and found three new compounds with anti-ASFV activity. The most promising antiviral compound (6b) reduced ASFV replication in a dose-dependent manner (IC50 = 19.5 µM) with no cellular (CC50 > 500 µM) and animal toxicity (up to 100 mg/kg). Results also revealed that compound 6b interfered with ASFV attachment, internalization and egress, with time-of-addition assays, showing that compound 6b has higher antiviral effects when added within 2-8 h post-infection. This compound significantly inhibited viral DNA replication and disrupted viral protein synthesis. Experiments with ASFV-infected porcine macrophages disclosed that antiviral effects of the compound 6b were similar to its effects in Vero cells. Tubulin polymerization assay and confocal microscopy demonstrated that compound 6b promoted tubulin polymerization, acting as a microtubule-stabilizing, rather than a destabilizing agent in cells. In conclusion, this work emphasizes the idea that microtubules can be targets for drug development against ASFV.

Energy-dependent protein folding: modeling how a protein folding machine may work.

Background: Proteins fold robustly and reproducibly in vivo, but many cannot fold in vitro in isolation from cellular components. Despite the remarkable progress that has been achieved by the artificial intelligence approaches in predicting the protein native conformations, the pathways that lead to such conformations, either in vitro or in vivo, remain largely unknown. The slow progress in recapitulating protein folding pathways in silico may be an indication of the fundamental deficiencies in our understanding of folding as it occurs in nature. Here we consider the possibility that protein folding in living cells may not be driven solely by the decrease in Gibbs free energy and propose that protein folding in vivo should be modeled as an active energy-dependent process. The mechanism of action of such a protein folding machine might include direct manipulation of the peptide backbone. Methods: To show the feasibility of a protein folding machine, we conducted molecular dynamics simulations that were augmented by the application of mechanical force to rotate the C-terminal amino acid while simultaneously limiting the N-terminal amino acid movements. Results: Remarkably, the addition of this simple manipulation of peptide backbones to the standard molecular dynamics simulation indeed facilitated the formation of native structures in five diverse alpha-helical peptides. Steric clashes that arise in the peptides due to the forced directional rotation resulted in the behavior of the peptide backbone no longer resembling a freely jointed chain. Conclusions: These simulations show the feasibility of a protein folding machine operating under the conditions when the movements of the polypeptide backbone are restricted by applying external forces and constraints. Further investigation is needed to see whether such an effect may play a role during co-translational protein folding in vivo and how it can be utilized to facilitate folding of proteins in artificial environments.

Synthesis, Antitumor Activity, and Docking Analysis of New Pyrido[3',2':4,5]furo(thieno)[3,2-d]pyrimidin-8-amines.

Continuing our research in the field of new heterocyclic compounds, herein we report on the synthesis and antitumor activity of new amino derivatives of pyrido[3',2':4,5](furo)thieno[3,2-d]pyrimidines as well as of two new heterocyclic systems: furo[2-e]imidazo[1,2-c]pyrimidine and furo[2,3-e]pyrimido[1,2-c]pyrimidine. Thus, by refluxing the 8-chloro derivatives of pyrido[3',2':4,5]thieno(furo)[3,2-d]pyrimidines with various amines, the relevant pyrido[3',2':4,5]thieno(furo)[3,2-d]pyrimidin-8-amines were obtained. Further, the cyclization of some amines under the action of phosphorus oxychloride led to the formation of new heterorings: imidazo[1,2-c]pyrimidine and pyrimido[1,2-c]pyrimidine. The possible antitumor activity of the newly synthesized compounds was evaluated in vitro. The biological tests evidenced that some of them showed pronounced antitumor activity. A study of the structure-activity relationships revealed that the compound activity depended mostly on the nature of the amine fragments. A docking analysis was also performed for the most active compounds.

In silico study of colchicine resistance molecular mechanisms caused by tubulin structural polymorphism.

Starting from 1972, colchicine is known as the most useful drug for prevention of familial Mediterranean fever attacks. However, some patients do not respond to colchicine treatment, even taken in high doses. Despite the fact, that different hypotheses have been proposed, the molecular mechanisms of colchicine resistance are not completely clear. It is generally known, that colchicine binds ß-tubulin and inhibits microtubules polymerization. The ß-tubulin gene has SNPs, which lead to amino acid substitutions, and some of them are located in colchicine binding site (CBS). We have assumed, that this SNPs can affect tubulin-colchicine interaction and might be the reason for colchicine resistance. With this in mind, we modeled 7 amino acid substitutions in CBS, performed molecular dynamics simulations of tubulin-colchicine complex and calculated binding energies for every amino acid substitution. Thus, our study shows, that two amino acid substitutions in the ß-tubulin, namely A248T and M257V, reduce binding energy for approximately 2-fold. Based on this, we assume, that these amino acid substitutions could be the reason for colchicine resistance. Thus, our study gives a new insight into colchicine resistance mechanism and provides information for designing colchicine alternatives, that could be effective for colchicine resistant patients.

Inhibition of African swine fever virus infection by genkwanin.

African swine fever virus (ASFV) is the causative agent of an economically important disease of pigs for which no effective vaccines or antiviral drugs are available. Recent outbreaks in EU countries and China have highlighted the critical role of antiviral research in combating this disease. We have previously shown that apigenin, a naturally occurring plant flavone, possesses significant anti-ASFV activity. However, apigenin is practically insoluble in highly polar solvents and it occurs typically in derivative forms in plants. Here we screened several commercially available apigenin derivatives for their ability to inhibit ASFV Ba71V strain in Vero cells. Among them, genkwanin showed significant inhibition of ASFV, reducing viral titer from 6.5 ±â€¯0.1 to 4.75 ±â€¯0.25 log TCID/ml in a dose-dependent manner (IC50 = 2.9 µM and SI = 205.2). Genkwanin reduced the levels of ASFV early and late proteins, as well as viral DNA synthesis. Our further experiments indicated that genkwanin is able to inhibit ASFV infection at entry and egress stages. Finally, genkwanin displayed potent antiviral activity against highly virulent ASFV isolate currently circulating in Europe and China, emphasizing its value as candidate for antiviral drug development.