Mandal N, Stevens JA, Poma AB, Surpeta B, Sequeiros-Borja C, Thirunavukarasu AS, Marrink SJ, Brezovsky J, 2026: Unlocking high-throughput investigation of transport tunnels in enzymes using coarse-grained simulation methods. Journal of Chemical Theory and Computation 22: 135-150. full text dataset

Transport tunnels in enzymes with buried active sites are critical gatekeepers of enzymatic function, controlling substrate access, product release, and catalytic efficiency. Despite their importance, the transient nature of these tunnels makes them difficult to study using conventional simulation methods. In this study, we systematically evaluate three coarse-grained (CG) molecular dynamics approaches─Martini with Elastic network restraints, Martini with Go̅-model restraints, and SIRAH─for their ability to characterize tunnel structure and dynamics across diverse enzyme classes. Using haloalkane dehalogenase LinB and its engineered variants as model systems, we show that CG methods accurately reproduce the geometry of tunnel ensembles observed in all-atom (AA) simulations while providing notable computational speedups. The Martini-Go̅ model performed particularly well, capturing subtle mutation-induced changes in tunnel dynamics, such as the closure of a main tunnel and the de novo opening of a transient auxiliary tunnel in LinB variants. In contrast, Martini with Elastic network restraints was limited in capturing tunnel dynamics due to the structural bias introduced by the restraints. We further validated these findings across nine enzymes from the oxidoreductase, transferase, and hydrolase classes with diverse structural folds. Although all CG methods reliably identified functionally relevant tunnels and provided fairly accurate estimates of their ensemble geometry and key bottleneck residues, they differed in their ability to replicate tunnel dynamics, with tunnel occurrences and ranking showing moderate to good correspondence with AA results. This comprehensive evaluation highlights the strengths and weaknesses of CG simulations, establishing them as powerful tools for high-throughput analysis of enzyme tunnels, which enables more efficient enzyme engineering and drug design efforts targeting these critical structural features.

Nikulenkov F, Carbain B, Biswas R, Havel S, Prochazkova J, Sisakova A, Zacpalova M, Chavdarova M, Marini V, Vsiansky V, Weisova V, Slavikova K, Biradar D, Khirsariya P, Vitek M, Sedlak D, Bartunek P, Daniel L, Brezovsky J, Damborsky J, Paruch K, Krejci L, 2025: Discovery of new inhibitors of nuclease MRE11. European Journal of Medicinal Chemistry. DOI: 10.1016/j.ejmech.2024.117226.  full text

MRE11 nuclease is a central player in signaling and processing DNA damage, and in resolving stalled replication forks. Here, we describe the identification and characterization of new MRE11 inhibitors MU147 and MU1409. Both compounds inhibit MRE11 nuclease more specifically and effectively than the relatively weak state-of-the-art inhibitor mirin. They also abrogate double-strand break repair mechanisms that rely on MRE11 nuclease activity, without impairing ATM activation. Inhibition of MRE11 also impairs nascent strand degradation of stalled replication forks and selectively affects BRCA2-deficient cells. Herein, we illustrate that our newly discovered compounds MU147 and MU1409 can be used as chemical probes to further explore the biological role of MRE11 and support the potential clinical relevance of pharmacological inhibition of this nuclease.

Sethi A, Kumar J, Vemula D, Gadde D, Talla V, Qureshic IA, Alvala M, 2024: Sugar mimics and their probable binding sites: design and synthesis of thiazole linked coumarin-piperazine hybrids as galectin-1 inhibitors. RSC Advances 14: 36794-36803. full text

Sugar mimics are valuable tools in medicinal chemistry, offering the potential to overcome the limitations of carbohydrate inhibitors, such as poor pharmacokinetics and non-selectivity. In our continued efforts to develop heterocyclic galectin-1 inhibitors, we report the synthesis and characterization of thiazole-linked coumarin piperazine hybrids (10a–10i) as Gal-1 inhibitors. The compounds were characterized using ¹H NMR, ¹³C NMR and HRMS. Among the synthesized molecules, four compounds demonstrated significant inhibitory activity, with more than 50% inhibition observed at a concentration of 20 μM in a Gal-1 enzyme assay. Fluorescence spectroscopy was further utilized to elucidate the binding constant for the synthesized compounds. 10g exhibited the highest affinity for Gal-1, with a binding constant (K_a) of 9.8 × 10⁴ M⁻¹. To elucidate the mode of binding, we performed extensive computational analyses with 10g, including 1.2 μs all-atom molecular dynamics simulations coupled with a robust machine learning tool. Our findings indicate that 10g binds to the carbohydrate binding site of Gal-1, with the coumarin moiety playing a key role in binding interactions. Additionally, our study underscores the limitations of relying solely on docking scores for conformational selection and highlights the critical importance of performing multiple MD replicas to gain accurate insights.

Dhiman D, Sethi A, Sinha R, Biswas S, Franklin G, Mondal D, 2025: Bioinspired design of DNA in aqueous ionic liquid media for sustainable packaging of horseradish peroxidase under biotic stress. Chemical Communications. DOI:10.1039/D4CC05803H. full text

We show that a combination of DNA and ionic liquid significantly increases the stability and activity of HRP and achieves a 4.8-fold higher peroxidase activity than PBS buffer. Also, HRP retains 84% of its activity in IL+DNA compared to 24% in PBS against trypsin digestion. Molecular modeling and spectroscopic studies reveal a protective microenvironment.

Thirunavukarasu AS, Szleper K, Tanriver G, Marchlewski I, Mitusinska K, Gora A,^# Brezovsky J,^# 2024: Water migration through enzyme tunnels is sensitive to choice of explicit water model. Journal of Chemical Information and Modeling: DOI:10.1021/acs.jcim.4c01177 full text dataset-DhaA dataset-CYP2D6+AldO

The utilization of tunnels and water transport within enzymes is crucial for their catalytic function as water molecules can stabilize bound substrates and help with unbinding processes of products and inhibitors. Since the choice of water models for molecular dynamics simulations was shown to determine the accuracy of various calculated properties of the bulk solvent and solvated proteins, we have investigated if and to what extent water transport through the enzyme tunnels depends on the selection of the water model. Here, we focused on simulating enzymes with various well-defined tunnel geometries. In a systematic investigation using haloalkane dehalogenase as a model system, we focused on the well-established TIP3P, OPC, and TIP4P-Ew water models to explore their impact on the use of tunnels for water molecule transport. The TIP3P water model showed significantly faster migration, resulting in the transport of approximately 2.5 times more water molecules compared to that of the OPC and 1.7 times greater than that of the TIP4P-Ew. Finally, the transport was 1.4-fold more pronounced in TIP4P-Ew than in OPC. The increase in migration of TIP3P water molecules was mainly due to faster transit times through dehalogenase tunnels. We observed similar behavior in two different enzymes with buried active sites and different tunnel network topologies, i.e., alditol oxidase and cytochrome P450, indicating that our findings are likely not restricted to a particular enzyme family. Overall, this study showcases the critical importance of water models in comprehending the use of enzyme tunnels for small molecule transport. Given the significant role of water availability in various stages of the catalytic cycle and the solvation of substrates, products, and drugs, choosing an appropriate water model may be crucial for accurate simulations of complex enzymatic reactions, rational enzyme design, and predicting drug residence times.

Sethi A,* Agrawal N,* Brezovsky J, 2024: Impact of water models on the structure and dynamics of enzyme tunnels. Computational and Structural Biotechnology Journal DOI: 10.1016/j.csbj.2024.10.051. full text dataset

Protein hydration plays a vital role in many biological functions, and molecular dynamics simulations are frequently used to study it. However, the accuracy of these simulations is often sensitive to the water model used, a phenomenon particularly evident in intrinsically disordered proteins. Here, we investigated the extent to which the choice of water model alters the behavior of complex networks of tunnels within proteins. Tunnels are essential because they allow the exchange of substrates and products between buried enzyme active sites and the bulk solvent, directly affecting enzyme efficiency and selectivity, making the study of tunnels crucial for a holistic understanding of enzyme function at the molecular level. By performing simulations of haloalkane dehalogenase LinB and its two variants with engineered tunnels using TIP3P and OPC models, we investigated their effects on the overall tunnel topology. We also analyzed the properties of the primary tunnels, including their conformation, bottleneck dimensions, sampling efficiency, and the duration of tunnel openings. Our data demonstrate that all three proteins exhibited similar conformational behavior in both models but differed in the geometrical characteristics of their auxiliary tunnels, consistent with experimental observations. Interestingly, the results indicate that the stability of the open tunnels might be sensitive to the water model used. Because TIP3P can provide comparable data on the overall tunnel network, it is a valid choice when computational resources are limited or compatibility issues impede the use of OPC. However, OPC seems preferable for calculations requiring an accurate description of transport kinetics.

Carlos’s sci-art was just published as the front cover at JCIM journal. Great grand finale of cool project!