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!

Mandal N, Surpeta B, Brezovsky J, 2024: Reinforcing Tunnel Network Exploration in Proteins using Gaussian Accelerated Molecular Dynamics. Journal of Chemical Information and Modeling, DOI: 10.1021/acs.jcim.4c00966. full text dataset dataset-trajectories

Tunnels are structural conduits in biomolecules responsible for transporting chemical compounds and solvent molecules from the active site. They have been shown to be present in a wide variety of enzymes across all functional and structural classes. However, the study of such pathways is experimentally challenging, because they are typically transient. Computational methods, such as molecular dynamics (MD) simulations, have been successfully proposed to explore tunnels. Conventional MD (cMD) provides structural details to characterize tunnels but suffers from sampling limitations to capture rare tunnel openings on longer time scales. Therefore, in this study, we explored the potential of Gaussian accelerated MD (GaMD) simulations to improve the exploration of complex tunnel networks in enzymes. We used the haloalkane dehalogenase LinB and its two variants with engineered transport pathways, which are not only well-known for their application potential but have also been extensively studied experimentally and computationally regarding their tunnel networks and their importance in multistep catalytic reactions. Our study demonstrates that GaMD efficiently improves tunnel sampling and allows the identification of all known tunnels for LinB and its two mutants. Furthermore, the improved sampling provided insight into a previously unknown transient side tunnel (ST). The extensive conformational landscape explored by GaMD simulations allowed us to investigate in detail the mechanism of ST opening. We determined variant-specific dynamic properties of ST opening, which were previously inaccessible due to limited sampling of cMD. Our comprehensive analysis supports multiple indicators of the functional relevance of the ST, emphasizing its potential significance beyond structural considerations. In conclusion, our research proves that the GaMD method can overcome the sampling limitations of cMD for the effective study of tunnels in enzymes, providing further means for identifying rare tunnels in enzymes with the potential for drug development, precision medicine, and rational protein engineering.