Projects
Coarse grain models of Carboxylic acid reductases
Carboxylic acid reductases (CARs) offers a green alternative for synthesizing valuable aldehydes from carboxylic acids under mild conditions. However, the process faces challenges due to the need for NAD(P)H and ATP cofactors, requiring regeneration for efficiency. In this project, I am working to obtain coarse-grained models of CARs. Specifically, I will be working on optimizing to enhance CARs catalytic activity and stability, contributing to greener and more sustainable chemical synthesis processes.
Selective Binding of Oxytocin to Its Natural Receptor
A time limited project focused on uncovering the binding mechanism of the neuropeptide oxytocin (OXT) to its natural receptor (OXTR), a G-protein-coupled receptor. Collaboration with leading experimentalists, Prof. Dr. Mark Wheatley from Coventry University and Prof. Dr. Inga Neumann from the University of Regensburg, was pivotal. Building on previous contributions published in Nature's Molecular Psychiatry, the project involved modeling the active state structure of OXTR using the closely related Vasopressin receptor 2 (V2R) bound to Arginine Vasopressin (AVP). It was demonstrated that OXT and AVP share multiple molecular interactions with OXTR in the active state, with several of these interactions also present in the recently resolved active OXTR structure (PDB id: 7RYC). Further exploration through classical molecular dynamics simulations identified crucial OXTR residues that distinguish OXT from AVP, the latter of which, despite binding, fails to activate OXTR.
Substrate Access Mechanism in Membrane-Bound Phospholipase A
For my doctoral thesis, I worked on an exciting DFG-funded project related to membrane proteins at Collaborative Research Centre 1208. Collaborating with Prof. Dr. Karl-Erich Jaeger and Dr. Filip Kovacic at Forschungszentrum Jülich, I conducted free energy computations to understand the substrate access mechanism in the membrane-bound phospholipase PlaF from Pseudomonas aeruginosa. I investigated the glycerophospholipid (GPL) substrate access mechanism in PlaF by identifying tunnels connecting the deeply buried active site to the surface and simulating GPL access using Steered Molecular Dynamics simulations. The low-energy pathway obtained was further analyzed with Umbrella Sampling simulations to calculate free energy profiles, with my calculations indicating that the tunnel (TE) presented the lowest barrier for substrate access. To validate these findings, I proposed Tryptophan substitutions for each tunnel, revealing that the TE was most affected, reducing enzyme activity by ~70%. This project was completed using the computational resources at Jülich Supercomputing Centre, and a manuscript detailing the work was published in the Journal of Chemical Information and Modelling in 2021.
Exploring Nutrient Exchange in Plant-Fungi Symbiotic Relationships
This project was a part of the Mercator Module on Plant Biophysics - Membrane Biophysics, completed under the supervision of Prof. Ingo Dreyer from the University of TALCA, Chile. The course was divided into two parts: theoretical sessions on December 18 and 19, 2017, and a practical module from January 8 to 12, 2018. Key topics covered included the thermodynamic bases of membrane transport processes, ion channels, coupled transporters, pumps, experimental techniques for electrophysiology, mathematical descriptions of transport processes for computational modeling, and the dynamics of coupled transport processes. The practical module provided hands-on experience with the computational simulation of transport processes using the "Virtual Cell" tool. This course offered valuable insights and practical knowledge in the field of membrane biophysics. In the context of my broader research interests, I have focused on the nutrient exchange mechanisms in plant-fungi symbiotic relationships. Understanding these complex interactions can illuminate how plants and fungi communicate at a cellular level to exchange essential nutrients, ultimately benefiting both organisms and contributing to ecological balance and agricultural productivity.
Novel Small Molecules for Pcsk9-Mediated Dyslipidemia
I have also worked on a project focused on proprotein convertase, Pcsk9, identifying several inhibitors for Pcsk9-LDLR interactions. These interactions are known to increase bad cholesterol levels in the bloodstream. Inhibiting Pcsk9-LDLR interactions prevents the degradation of low-density lipid receptors (LDLR). Using structural information, I developed a pharmacophore model to identify potential molecules from chemical libraries. Molecular docking and simulation studies helped identify a few promising hits, which were experimentally validated to inhibit Pcsk9-LDLR interactions, reducing LDLR degradation. After refinement by chemists, these molecules underwent additional testing for toxicity, efficacy, and efficiency. In early 2024, we filed a patent for the most promising molecules with the Office of the Controller General of Patents, Designs & Trade Marks in India.
Cathepsin S as a Therapeutic Target: Insights into Selective Inhibition
Cathepsin S plays a crucial role in various physiological and pathological processes, particularly in antigen presentation, inflammation, and disease progression. This enzyme is implicated in several diseases. In autoimmune diseases, improper antigen presentation involving Cathepsin S can lead to autoimmune responses. In cancer, Cathepsin S facilitates tumor invasion and metastasis by degrading extracellular matrix proteins and modulating the tumor microenvironment. Its role in cardiovascular diseases includes degrading structural proteins, contributing to conditions such as atherosclerosis. Emerging research also suggests Cathepsin S's involvement in neuroinflammation and neurodegeneration. Therefore, given its critical roles, Cathepsin S is a promising therapeutic target. Inhibitors of Cathepsin S are being explored to treat various conditions. My research projects have focused on understanding and targeting Cathepsin S. In one project, we have demonstrated that the selective mechanism of action of the inhibitor RO5444101. Using molecular docking and molecular dynamics simulations, we gained insights into the molecular mechanism of RO5444101, explaining its selectivity for Cathepsin S over closely related cathepsins. Our study showed that the interaction of Cathepsin S with RO5444101 is more stable and involves more protein–molecule interactions compared to other cathepsins. In another project, we screened a library of compounds to identify potential inhibitors of Cathepsin S, focusing on the S2 pocket, which is structurally unique. Our efforts led to the identification of KM07987, a potent inhibitor with an IC50 of less than 5 μM. Molecular dynamics simulations supported our experimental findings, highlighting the importance of the S2 pocket in inhibitor interactions. This study provides a foundation for developing novel scaffolds for improved inhibition of Cathepsin S.
Vaccine Adjuvant Development Using TLR4-Derived Peptide
I also had the opportunity to collaborate on an exciting project focused on enhancing vaccine efficacy through the development of a novel adjuvant. The study aimed to stimulate specific and prolonged immune responses for long-term protection against infections and diseases. In our research, we investigated a human Toll-like receptor 4 (TLR4)-derived 20-residue peptide, known as TR-433. This peptide is present in the dimerization interface of the TLR4–myeloid differentiation protein-2 (MD2) complex. We discovered that TR-433 exhibits self-assembly properties and forms nanostructures. Importantly, both in vitro and in vivo experiments demonstrated that TR-433 is non-toxic. It induced pro-inflammatory responses in THP-1 monocytes and HEK293T cells transfected with TLR4/CD14/MD2, as well as in BALB/c mice. Our findings showed that TR-433, when combined with ovalbumin or the filarial antigen trehalose-6-phosphate phosphatase (TPP), significantly boosted IgG titers. This suggests that TR-433 has robust adjuvant capabilities, comparable to Freund’s complete adjuvant (FCA) and significantly higher than alum. Moreover, TR-433 preferentially activated type 1 helper T cell (Th1) responses over type 2 helper T cell (Th2) responses. This study is the first to report a short TLR4-derived peptide with both self-assembling and pro-inflammatory properties, demonstrating significant efficacy as an adjuvant capable of activating cellular responses in mice. Our results indicate that TR-433 holds substantial potential for development as a new adjuvant in therapeutic applications.
Plant-Insect Interaction and Plant Defense System
I collaborated on a research project focusing on the plant-insect interaction system, a widely studied model in ecology. This project extended the traditional predator-prey model by incorporating the molecular interactions behind the plant defense system and their ecological impacts. Our work built upon the molecular interactions described by Louis and Shah in 2014, developing a mathematical model to represent the molecular dependence and control within the plant-insect interaction system. We specifically investigated how insect infestation induces Botrytis Induced Kinase-1 (BIK1), which in turn inhibits Phyto Alexin Deficient-4 (PAD4) protein. This inhibition triggers the plant defense mechanism, leading to a decrease in plant immune potential and overall plant quality. By adapting these interactions mathematically, we demonstrated their influence on the plant-insect interaction system and hypothesized that inhibiting BIK1 could improve plant quality. Our research utilized computational modeling and all-atom molecular dynamics simulations to elucidate the Plant-Insect-PAD4-BIK1 interaction network. This allowed us to identify potential molecular mechanisms for enhancing plant health through BIK1 inhibition.
Unveiling Nature's Pest Defense: Power of Mustard Plant Proteins
This project focused on the isolation and characterization of a phytocystatin from Brassica juncea, commonly known as Indian mustard. The crop holds agricultural and economic importance as an oilseed crop. This study involved the purification of a 18.1 kDa phytocystatin using gel filtration chromatography, achieving a yield of 24.3% and purification fold of 204. The protein was visualized using 2D gel electrophoresis, confirming its specificity for cysteine proteinases. The isolated mustard cystatin demonstrated potent inhibitory activity against cathepsin B, highlighting its role in imparting pest resistance to the plant. Notably, the inhibitor exhibited high stability over a broad pH range (3–10) and retained significant inhibitory potential even at temperatures up to 70°C. Isothermal calorimetry studies revealed a 1:1 stoichiometry of interaction with papain, a model cysteine protease. Structural analysis using far-UV circular dichroism spectroscopy indicated the presence of 18.8% α-helical and 21% β-sheet secondary structural elements. Further characterization included determination of the protein's shape and size using Stokes radius and frictional coefficient measurements. In this collaboration, I supported the project with computational studies. With the help of homology modeling, molecular docking, and molecular dynamics (MD) simulations, we got insights into the molecular basis of papain inhibition by Brassica phytocystatin. The MD simulation highlighted the stable nature of the papain-phytocystatin complex over 100 nanoseconds. Overall, this study underscores the significance of mustard cystatin as a key member of the phytocystatin family, with implications for enhancing pest resistance in agricultural crops.
Exploring Coumestrol's Binding to Estrogen Receptors as Targets in Breast Cancer Research
I this project we aimed at understanding how coumestrol, a phytoestrogen, selectively binds to estrogen receptor (ER) subtypes and its implications in breast cancer treatment. Epidemiological studies have indicated that phytoestrogens, including coumestrol, may lower the risk of breast cancer by inducing apoptosis in cancer cells through ER-mediated mechanisms. Our study focused on elucidating the molecular binding mechanism of coumestrol to ERα and ERβ using advanced computational techniques. Molecular docking, access channel analysis, and molecular dynamics (MD) simulations were employed to investigate the stability and interactions of coumestrol with ERα and ERβ at the atomistic level. Key parameters such as hydrogen bonds, interaction energy, radius of gyration, solvent-accessible surface area, root mean square deviation (RMSD), RMS fluctuation, and secondary structure elements were analyzed during the MD simulations. Our findings revealed that coumestrol exhibits a higher affinity and stability when bound to ERα compared to ERβ. Specifically, interactions with ERα resulted in strong substrate binding and increased structural stability of the ERα complex, whereas interactions with ERβ led to destabilization of the ERβ structure. Principal component analysis further supported these observations, highlighting the dynamic differences between coumestrol-bound ERα and ERβ complexes. By leveraging computational modeling, our findings provide valuable insights for future drug development strategies targeting ER-mediated pathways in breast cancer treatment.
Schistosomiasis: Molecular Modeling and Screening of SmCL1 as a Therapeutic Target
In this research project we have focused on addressing the urgent need for new treatments against schistosomiasis, a prevalent and debilitating disease in developing countries. With praziquantel as the sole treatment option, the emergence of drug resistance underscores the importance of discovering alternative therapies. Our study targeted Cathepsin SmCL1, a crucial enzyme in Schistosoma mansoni responsible for digesting host proteins essential for parasite growth and development. Using a homology modeling approach, we generated a theoretical three-dimensional structure of SmCL1 to facilitate structure-based drug design. Employing a variety of in silico techniques, including virtual screening and molecular dynamics (MD) simulations, we screened a library of non-peptide inhibitors against SmCL1. The results, validated by receiver operating characteristic (ROC) curve analysis and MD simulations of protein-ligand interactions, identified Simalikalactone-D as a promising lead compound. The pharmacophore model derived from Simalikalactone-D offers a framework for future screenings to identify potential drug candidates. This study represents the first virtual screening of non-peptide inhibitors targeting SmCL1 of S. mansoni, highlighting their therapeutic potential in combating schistosomiasis. Our findings contribute significantly to the ongoing efforts to develop effective antischistosomal therapies and pave the way for further optimization and development of these promising lead compounds.