Influenza is still a chronic global health threat, inducing a sustained search for effective antiviral therapeutics. Computational methods have played a pivotal role in developing small molecule therapeutics. In this study, we applied a combined in silico and in vitro approach to explore the potential anti-influenza activity of cyproheptadine, a clinically used histamine H1 receptor antagonist. Virtual screening based on the average quasivalence number (AQVN) and electron-ion interaction potential (EIIP) descriptors suggests similarities between cyproheptadine and several established anti-influenza agents. The subsequent ligand-based pharmacophore screening of a focused H1 antagonist library was aligned with the bioinformatics prediction, and further experimental in vitro evaluation of cyproheptadine demonstrated its anti-influenza activity. These findings provide proof of concept for cyproheptadine's in silico-predicted antiviral potential and underscore the value of integrating computational predictions with experimental validation. The results of the current study provide a preliminary proof of concept for the predicted anti-influenza potential based on computational analysis and emphasize the utility of integrating in silico screening with experimental validation in the early stages of drug repurposing efforts.

Water-based pharmacophore modeling is an emerging approach in inhibitor design that leverages the dynamics of explicit water molecules within ligand-free, water-filled binding sites to derive 3D pharmacophores for virtual screening. In this study, we assess the potential of this strategy through a case study targeting the ATP binding sites of Fyn and Lyn protein kinases─members of the Src family that have been less explored in anticancer drug discovery compared to other family members. Molecular dynamics simulations of multiple kinase structures were used to generate and validate several water-derived pharmacophores, which were subsequently employed to screen chemically diverse libraries of compounds. Two active compounds were identified in biochemical assays: a flavonoid-like molecule with low-micromolar inhibitory activity and a weaker inhibitor from the library of nature-inspired synthetic compounds. Structural analysis via molecular docking and simulations revealed that key predicted interactions, particularly with the hinge region and the ATP binding pocket, were retained in the bound states of these hits. However, interactions with more flexible regions, such as the N-terminal lobe and activation loop, were less consistently captured. These findings outline both the strengths and challenges of using water-based pharmacophores: while effective at modeling conserved core interactions, they may miss peripheral contacts governed by protein flexibility. Incorporating ligand information where available may help address this challenge. Overall, water-based pharmacophore modeling presents a promising ligand-independent strategy for identifying novel chemotypes and exploring undercharged chemical and conformational space in kinases as well as other therapeutically relevant targets.

The inherent complexity of Alzheimer's disease (AD) and failed clinical trials have spiked the interest in multifunctional ligands that target at least two key disease-associated macromolecules in AD pathology. Here we present a focused series of pleiotropic N-carbamoylazole prodrugs with dual mechanism of action. Pseudo-irreversible inhibition of the first therapeutic target, human butyrylcholinesterase (hBChE), enhances cholinergic transmission, and thereby provides symptomatic treatment, same as the standard therapeutics in use for AD. Simultaneously, this step also functions as a metabolic activation that liberates a nanomolar selective α 2-adrenergic antagonist atipamezole, which blocks pathological amyloid β (Aβ)-induced and noradrenaline-dependent activation of GSK3β that ultimately leads to hyperphosphorylation of tau, thus achieving a disease-modifying effect. Lead compound 8 demonstrated long-term pseudo-irreversible hBChE inhibition, metabolic activation in human plasma, blood-brain barrier permeability, and p.o. bioavailability in mice. Multi-day in vivo treatment with 8 in an Aβ-induced AD murine model revealed a significant alleviation of cognitive deficit that was comparable to rivastigmine, the current drug of choice for AD therapy. Furthermore, decreased GSK3β activation and lowered tau phosphorylation were observed in APP/PS1 mice. This surpasses the symptomatic-only treatment with cholinesterase inhibitors, as it directly blocks an essential pathological cascade in AD. Therefore, these multifunctional α 2-adrenergic antagonists-butyrylcholinesterase inhibitors, exemplified by lead compound 8, present an innovative, small molecule-based, disease-modifying approach to treatment of AD.

Type IIA DNA topoisomerases are molecular nanomachines that alter DNA topology during essential cellular processes. The final steps of their catalytic cycle, after translocation of the transported (T-) segment into the C-gate, are still not fully understood. Here, we performed all-atom molecular dynamics simulations of several conformational states of Saccharomyces cerevisiae topoisomerase IIA, each with a T-segment inserted into the C-gate. Bound ATP and ADP nucleotides allosterically modulated the N-gate dynamics, likely stabilizing the dimer and preventing premature dissociation. The T-segment was asymmetrically bound and stabilized within the C-gate by positively charged residues, and this gate remained structurally rigid, highlighting its role as a retention site. The positioning of the T-segment in the C-gate allosterically influenced the G-segment to a straighter geometry that favors religation and release. Our simulations support coordinated release of DNA segments and point to a potentially important role for dynamic communication between the gates in the mechanism. These results provide new insights into the late stages of the catalytic cycle and highlight the intertwined roles of nucleotide binding, DNA topology and coupled protein domain dynamics in regulating this important enzyme.

A handful of carbamate warheads is utilised in chemical biology to target serine hydrolases. The following case study on cholinesterases is the first comprehensive structure- reactivity exploration of the carbamoyl warhead, rather than one-target-oriented structure- activity study, with in-depth profiling of diverse halogen, chalcogen, and nitrogen-based leaving groups (nucleofuges) that can tune warhead reactivity. With computational tools we correlated the experimentally observed reactivities with steric and electronic factors of the investigated warheads. QM/MM simulations considering the enzymatic environment explained how substitution of carbon for nitrogen in the leaving groups of compounds 26 and 28 through resonance stabilisation, inductive bond polarization, and acidity amplification lowered the reaction barrier and increased the reaction rate >360 million times, making compound 28 a covalent inhibitor. Our findings underline the complexity of covalent inhibition and demonstrate that multiple complementary methods are required to interpret and predict covalent behaviour. Additionally, even though carbamates typically act as slow substrates, we were able to slow down decarbamoylation to a point where inhibition became de facto irreversible. The most interesting O -isoxazol-3-yl carbamate warhead was further profiled against the wider human proteome and showed low off-target reactivity, making it useful in further drug discovery. By establishing structure- reactivity principles for carbamoyl warhead, this study provides a generalisable framework for the development of selective covalent inhibitors and activity-based probes across diverse targets. ### Competing Interest Statement The authors have declared no competing interest. Slovenian Research and Innovation Agency, P1-0208, P1-0012, P4-0127, P4-0053, P1-0230, bilateral travel grant BI-US/22-24-004

DNA topoisomerases (topo) are essential enzymes that maintain the integrity of the genome by regulating the topological state of DNA during replication, transcription, recombination and repair [...]

Molecular dynamics (MD) simulations are a key computational chemistry technique that provide dynamic insight into the underlying atomic-level processes in the system under study. These insights not only improve our understanding of the molecular world, but also aid in the design of experiments and targeted interventions. Currently, MD is associated with several limitations, the most important of which are: insufficient sampling, inadequate accuracy of the atomistic models, and challenges with proper analysis and interpretation of the obtained trajectories. Although numerous efforts have been made to address these limitations, more effective solutions are still needed. The recent development of artificial intelligence, particularly machine learning (ML), offers exciting opportunities to address the challenges of MD. In this review we aim to familiarize readers with the basics of MD while highlighting its limitations. The main focus is on exploring the integration of deep learning with MD simulations. The advancements made by ML are systematically outlined, including the development of ML-based force fields, techniques for improved conformational space sampling, and innovative methods for trajectory analysis. Additionally, the challenges and implications associated with the integration of ML and artificial intelligence are discussed. While the potential of ML-MD fusion is clearly established, further applications are needed to confirm its superiority over traditional methods. This comprehensive overview of the new perspectives of MD, which ML has opened up, serves as a gentle introduction to the exciting phase of MD development.

Understanding how membrane composition influences the dynamics and function of transmembrane proteins is crucial for the comprehensive elucidation of cellular signaling mechanisms and the development of targeted therapeutics. In this study, we employed all-atom molecular dynamics simulations to investigate the impact of different membrane compositions on the conformational dynamics of the NKG2A/CD94/HLA-E immune receptor complex, a key negative regulator of natural killer cell cytotoxic activity. Our results reveal significant variations in the behavior of the immune complex structure across five different membrane compositions, which include POPC, POPA, DPPC, and DLPC phospholipids, and a mixed POPC/cholesterol system. These variations are particularly evident in the intracellular domain of NKG2A, manifested as changes in mobility, tyrosine exposure, and interdomain communication. Additionally, we found that a large concentration of negative charge at the surface of the POPA-based membrane greatly increased the number of contacts with lipid molecules and significantly decreased the exposure of intracellular NKG2A ITIM regions to water molecules, thus likely halting the signal transduction process. Furthermore, the DPPC model with a membrane possessing a high transition temperature in a gel-like state became curved, affecting the exposure of one ITIM region. The decreased membrane thickness in the DPLC model caused a significant transmembrane domain tilt, altering the linker protrusion angle and potentially disrupting the hydrogen bonding network in the extracellular domain. Overall, our findings highlight the importance of considering membrane composition in the analysis of transmembrane protein dynamics and in the exploration of novel strategies for the external modulation of their signaling pathways.

Type IIA DNA topoisomerases are molecular nanomachines responsible for controlling topological states of DNA molecules. Here, we explore the dynamic landscape of yeast topoisomerase IIA during key stages of its catalytic cycle, focusing in particular on the events preceding the passage of the T-segment. To this end, we generated six configurations of fully catalytic yeast topo IIA, strategically inserted a T-segment into the N-gate in relevant configurations and performed all-atom simulations.The essential motion of topo IIA protein dimer was characterized by rotational gyrating-like movement together with sliding motion within the DNA-gate. Both appear to be inherent properties of the enzyme and an inbuilt feature that allows passage of the T-segment through the cleaved G-segment. Coupled dynamics of the N-gate and DNA-gate residues may be particularly important for controlled and smooth passage of the T-segment and consequently the prevention of DNA double-strand breaks. QTK loop residue Lys367, which interacts with ATP and ADP molecules, is involved in regulating the size and stability of the N-gate. The unveiled features of the simulated configurations provide insights into the catalytic cycle of type IIA topoisomerases and elucidate the molecular choreography governing their ability to modulate the topological states of DNA topology.

The molecular nanomachine, human DNA topoisomerase IIα, plays a crucial role in replication, transcription, and recombination by catalyzing topological changes in the DNA, rendering it an optimal target for cancer chemotherapy. Current clinical topoisomerase II poisons often cause secondary tumors as side effects due to the accumulation of double-strand breaks in the DNA, spurring the development of catalytic inhibitors. Here, we used a dynamic pharmacophore approach to develop catalytic inhibitors targeting the ATP binding site of human DNA topoisomerase IIα. Our screening of a library of nature-inspired compounds led to the discovery of a class of 3-(imidazol-2-yl) morpholines as potent catalytic inhibitors that bind to the ATPase domain. Further experimental and computational studies identified hit compound 17, which exhibited selectivity against the human DNA topoisomerase IIα versus human protein kinases, cytotoxicity against several human cancer cells, and did not induce DNA double-strand breaks, making it distinct from clinical topoisomerase II poisons. This study integrates an innovative natural product-inspired chemistry and successful implementation of a molecular design strategy that incorporates a dynamic component of ligand-target molecular recognition, with comprehensive experimental characterization leading to hit compounds with potential impact on the development of more efficient chemotherapies.

The 4,6-substituted-1,3,5-triazin-2(1H)-ones are promising inhibitors of human DNA topoisomerase IIα. To further develop this chemical class targeting the enzyme´s ATP binding site, the triazin-2(1H)-one substitution position 6 was optimized. Inspired by binding of preclinical substituted 9H-purine derivative, bicyclic substituents were incorporated at position 6 and the utility of this modification was validated by a combination of molecular simulations, dynamic pharmacophores, and free energy calculations. Considering also predictions of Deepfrag, a software developed for structure-based lead optimization based on deep learning, compounds with both bicyclic and monocyclic substitutions were synthesized and investigated for their inhibitory activity. The SAR data showed that the bicyclic substituted compounds exhibited good inhibition of topo IIα, comparable to their mono-substituted counterparts. Further evaluation on a panel of human protein kinases showed selectivity for the inhibition of topo IIα. Mechanistic studies indicated that the compounds acted predominantly as catalytic inhibitors, with some exhibiting topo IIα poison effects at higher concentrations. Integration of STD NMR experiments and molecular simulations, provided insights into the binding model and highlighted the importance of the Asn120 interaction and hydrophobic interactions with substituents at positions 4 and 6. In addition, NCI-60 screening demonstrated cytotoxicity of the compounds with bicyclic substituents and identified sensitive human cancer cell lines, underlining the translational relevance of our findings for further preclinical development of this class of compounds. The study highlights the synergy between simulation and AI-based approaches in efficiently guiding molecular design for drug optimization, which has implications for further preclinical development of this class of compounds.

Hypoxia-inducible factor-1α (HIF-1α) constitutes the principal mediator of cellular adaptation to hypoxia in humans. The HIF-1α protein level and activity are tightly regulated by the ubiquitin E3 ligase von Hippel-Lindau (VHL). Here, we performed a structure-guided and bioactivity-driven design of new VHL inhibitors. Our iterative and combinatorial strategy focused on chemical variability at the phenylene unit and encompassed further points of diversity. The exploitation of tailored phenylene fragments and the stereoselective installation of the benzylic methyl group provided potent VHL ligands. Three high-resolution structures of VHL-ligand complexes were determined, and bioactive conformations of these ligands were explored. The most potent inhibitor (30) exhibited dissociation constants lower than 40 nM, independently determined by fluorescence polarization and surface plasmon resonance and an enhanced cellular potency, as evidenced by its superior ability to induce HIF-1α transcriptional activity. Our work is anticipated to inspire future efforts toward HIF-1α stabilizers and new ligands for proteolysis-targeting chimera (PROTAC) degraders.

Natural killer (NK) cells play an important role in the innate immune response against tumors and various pathogens such as viruses and bacteria. Their function is controlled by a wide array of activating and inhibitory receptors, which are expressed on their cell surface. Among them is a dimeric NKG2A/CD94 inhibitory transmembrane (TM) receptor which specifically binds to the non-classical MHC I molecule HLA-E, which is often overexpressed on the surface of senescent and tumor cells. Using the Alphafold 2 artificial intelligence system, we constructed the missing segments of the NKG2A/CD94 receptor and generated its complete 3D structure comprising extracellular (EC), TM, and intracellular regions, which served as a starting point for the multi-microsecond all-atom molecular dynamics simulations of the receptor with and without the bound HLA-E ligand and its nonameric peptide. The simulated models revealed that an intricate interplay of events is taking place between the EC and TM regions ultimately affecting the intracellular immunoreceptor tyrosine-based inhibition motif (ITIM) regions that host the point at which the signal is transmitted further down the inhibitory signaling cascade. Signal transduction through the lipid bilayer was also coupled with the changes in the relative orientation of the NKG2A/CD94 TM helices in response to linker reorganization, mediated by fine-tuned interactions in the EC region of the receptor, taking place after HLA-E binding. This research provides atomistic details of the cells' protection mechanism against NK cells and broadens the knowledge regarding the TM signaling of ITIM-bearing receptors.

Hypoxia-inducible factor-1alpha (HIF-1alpha) constitutes the principal mediator of cellular adaptation to hypoxia in humans. Hydroxylation of proline residues on HIF-1alpha is recognized by the E3 ligase von Hippel-Lindau (VHL), leading to ubiquitin-dependent proteasomal degradation of HIF-1alpha. In this study, we performed a structure-guided and bioactivity-driven design of new VHL inhibitors with improved cellular activity at blocking HIF-1alpha degradation. With the prototypical ligand VH298 as starting point, our iterative and combinatorial optimization strategy focused on introducing chemical variability into the phenylene unit of the VHL ligand structure and encompassed further points of diversity. The exploitation of tailored phenylene fragments combined with the stereoselective installation of the (S)-configured methyl group at the benzylic position provided VHL ligands with superior binding affinity compared to VH298. Three high-resolution structures of VHL in complex with three new ligands were determined and bioactive conformations of these ligands were explored. The most potent inhibitor (compound 30) exhibited dissociation constants (Kd) lower than 40 nM, independently determined by fluorescence polarization (FP) and surface plasmon resonance (SPR). The improved binding affinity to VHL was accompanied by enhanced cellular potency of 30, considerably exceeding the stabilization of HIF-1alpha by established VHL inhibitors. Our work is anticipated to inspire future efforts towards HIF-1alpha stabilizers and our optimized ligands to serve as an excellent starting point for proteolysis-targeting chimeras (PROTACs) hijacking VHL.

Type IIA DNA topoisomerases are complex molecular nanomachines that manage topological states of the DNA molecule in the cell and play a crucial role in cellular processes such as cell division and transcription. They are also established targets of cancer chemotherapy. Starting from the available crystal structure of a fully catalytic topoisomerase IIA homodimer from Saccharomyces cerevisiae, we constructed three states of this molecular motor primarily changing the configurations of the DNA segment bound in the DNA gate and performed μs-long all-atom molecular simulations. A comprehensive analysis revealed a sliding motion within the DNA gate and a teamwork between the N-gate and DNA gate that may be associated with the necessary molecular events that allow passage of the T-segment of DNA. The observed movement of the ATPase dimer relative to the DNA domain was reflected in different interaction patterns between the K-loops of the transducer domain and the B-A-B form of the bound DNA. Based on the obtained results, we mapped simulated configurations to the structures in the proposed catalytic cycle through which type IIA topoisomerases exert their function and discussed the possible transition events. The results extend our understanding of the mechanism of action of type IIA topoisomerases and provide an atomistic interpretation of some of the observed features of these molecular motors.

Hypoxia-inducible factor-1α (HIF-1α) constitutes the principal mediator of cellular adaptation to hypoxia in humans. HIF-1α protein level and activity is tightly regulated by the ubiquitin E3 ligase von Hippel-Lindau (VHL). Here, we performed a structure-guided and bioactivity-driven design of new VHL inhibitors. Our iterative and combinatorial strategy focused on chemical variability at the phenylene unit and encompassed further points of diversity. The exploitation of tailored phenylene fragments and the stereoselective installation of the benzylic methyl group provided potent VHL ligands. Three high-resolution structures of VHL-ligand complexes were determined and bioactive conformations of these ligands were explored. The most potent inhibitor (30) exhibited dissociation constants lower than 40 nM, independently determined by fluorescence polarization and surface plasmon resonance and an enhanced cellular potency, as evidenced by its superior ability to induce HIF-1α transcriptional activity. Our work is anticipated to inspire future efforts towards HIF-1α stabilizers and new ligands for proteolysis-targeting chimeras.

Phenotypic screening of α-substituted thiocarbohydrazones revealed promising activity of 1,5-bis(salicylidene)thiocarbohydrazide against leukemia and breast cancer cells. Supplementary cell-based studies indicated an impairment of DNA replication via the ROS-independent pathway. The structural similarity of α-substituted thiocarbohydrazone to previously published thiosemicarbazone catalytic inhibitors targeting the ATP-binding site of human DNA topoisomerase IIα prompted us to investigate the inhibition activity on this target. Thiocarbohydrazone acted as a catalytic inhibitor and did not intercalate the DNA molecule, which validated their engagement with this cancer target. A comprehensive computational assessment of molecular recognition for a selected thiosemicarbazone and thiocarbohydrazone provided useful information for further optimization of this discovered lead compound for chemotherapeutic anticancer drug discovery.

Alongside reversible butyrylcholinesterase inhibitors, a plethora of covalent butyrylcholinesterase inhibitors have been reported in the literature, typically pseudo-irreversible carbamates. For these latter, however, most cases lack full confirmation of their covalent mode of action. Additionally, the available reports regarding the structure–activity relationships of the O-arylcarbamate warhead are incomplete. Therefore, a follow-up on a series of pseudo-irreversible covalent carbamate human butyrylcholinesterase inhibitors and the structure–activity relationships of the N-dialkyl O-arylcarbamate warhead are presented in this study. The covalent mechanism of binding was tested by IC50 time-dependency profiles, and sequentially and increasingly confirmed by kinetic analysis, whole protein LC-MS, and crystallographic analysis. Computational studies provided valuable insights into steric constraints and identified problematic, bulky carbamate warheads that cannot reach and carbamoylate the catalytic Ser198. Quantum mechanical calculations provided further evidence that steric effects appear to be a key factor in determining the covalent binding behaviour of these carbamate cholinesterase inhibitors and their duration of action. Additionally, the introduction of a clickable terminal alkyne moiety into one of the carbamate N-substituents and in situ derivatisation with azide-containing fluorophore enabled fluorescent labelling of plasma human butyrylcholinesterase. This proof-of-concept study highlights the potential of this novel approach and for these compounds to be further developed as clickable molecular probes for investigating tissue localisation and activity of cholinesterases.

Human DNA topoisomerase IIα is a biological nanomachine that regulates the topological changes of the DNA molecule and is considered a prime target for anticancer drugs. Despite intensive research, many atomic details about its mechanism of action remain unknown. We investigated the ATPase domain, a segment of the human DNA topoisomerase IIα, using all-atom molecular simulations, multiscale quantum mechanics/molecular mechanics (QM/MM) calculations, and a point mutation study. The results suggested that the binding of ATP affects the overall dynamics of the ATPase dimer. Reaction modeling revealed that ATP hydrolysis favors the dissociative substrate-assisted reaction mechanism with the catalytic Glu87 serving to properly position and polarize the lytic water molecule. The point mutation study complemented our computational results, demonstrating that Lys378, part of the important QTK loop, acts as a stabilizing residue. The work aims to pave the way to a deeper understanding of these important molecular motors and to advance the development of new therapeutics.