Current projects
May 2022 - Present (The Structural Genomics Consortium/Goethe University, Frankfurt, Germany)
1) General responsibilities
Currently, I am engaged in multiple projects at the Structural Genomics Consortium, Frankfurt, providing structural biology support that include:
• Crystallization and structure determination of protein-ligand complexes
• Crystallographic fragment screening at Xchem, DLS, UK
• Protein purification for crystallography and compound screening (e.g., DNA encoded libraries)
• General crystallography support to projects in the lab
2) Targeting Transcriptional AddiCTion In Cancer (TACTIC)
The project aims to target the historically "undruggable" proteome involved in cancer development. My focus in this project is AAA+ class of chromatin remodelers, e.g., ATAD2. I am trying to establish expression and purification protocols to generate decent yield, so that we can screen for binders, especially for the AAA domain. There are ligands already known for the bromodomainsm, which are being explored to develop PROTACs.
3) LigandAI
LigandAI is a major five-year international public-private research partnership dedicated to advancing early-stage drug discovery by applying artificial intelligence and machine learning to protein-ligand interactions. My responsibility includes purifying human proteins for molecule screening through ASMS and DEL libraries. Once the screening is done, I am involved in hit validation, especially structure determination.
4) Next-gen degrader development
Recent successes in developing small-molecule degraders that act through the ubiquitin system have spurred efforts to extend this technology to other mechanisms, including the autophagosomal-lysosomal pathway. Therefore, reports of autophagosome tethering compounds (ATTECs) ‒ based on the target recruitment to LC3/GABARAP, a family of membrane-bound proteins that tether autophagy receptors to the autophagosome ‒ have received considerable attention from the drug development community. I am exploring the druggability of LC3/GABARAP by high-throughput crystallographic fragment screening at Xchem. The data obtained thus far revealed that most fragments bound to the HP2, but not the HP1 pocket of the LC3-interacting region (LIR) docking site, suggesting favorable druggability of this binding pocket. The screening also revealed two new binding sites. These locations can be exploited for future ATTEC development.
Additionally, I am also exploring the application of room-temperature crystallography for ligand/fragment screening. The LC3 system is under testing in collaboration with VMXi and Xchem at the Diamond Light Source, UK, and it might be expanded to other proteins in the future.
5) Development of inhibitors/probes for less studied kinases (dark kinases)
I have just started with a project involving studies of dark kinases. Dark kinases are understudied or poorly characterized protein kinases with unknown biological functions, despite being part of critical signaling pathways. Research into these kinases can reveal new therapeutic targets. Interesting part (structurally speaking) for me here is that they do not possess canonical kinase fold. Hence, they are also interesting from the mechanistic point of view. I am working on determining apo- and ligand-bound structures for these kinases, thereby trying to understand the mechanisms behind their functions and helping with structure aided molecule design.
6) Collaborations
I am also involved in multiple collaborative projects with labs in our institute and elsewhere.
Past Projects
2018-2021 (The Florida State University, Tallahassee, Florida, USA)
1) Human DNA polymerase beta (hPolβ)
My experience with structure and a brief stint with enzyme kinetics invigorated my interest in understanding the atomic level details of enzymatic reactions. When I joined Dr. Suo’s lab in September 2018, I started working on hPolβ. This enzyme plays a key role in the base excision repair (BER) pathway in humans. It has two active sites, one for polymerase activity and the other for dRP lyase activity. Although this enzyme has been studied quite extensively, several questions were unanswered regarding its mechanism of catalysis. Three questions that we specifically tried to answer in this study are, the dRP lyase mechanism, the sequence of events within the two active sites that lead to the completion of the reaction, and the role of three metal ions in the polymerase active site. We also hypothesized a revised version of the BER pathway.
My job was to visualize the reaction in crystallo. For this purpose, we had to cross-link DNA substrate with hPolβ using two additional enzymes (Uracil DNA glycosylase and human apurinic/apyrimidinic endonuclease 1). The initial protocol for the cross-linking was standardized by Dr. Sahska Daskalova at Dr. Sidney M. Hecht’s laboratory at Arizona State University. However, the hPolβ-DNA complex thus obtained was ~50-60% cross-linked, not homogeneous enough for crystallization. I picked up clues from their protocol and refined it till I was able to produce enough protein with more than 90% cross-linking. Single nucleotide incorporation kinetics was performed by Dr. Walter J. Zahurancik to do a comparison between cross-linked and non-cross-linked complexes. Three of the pre-reaction structures were determined by Dr. Andrew J. Reed. I was also able to standardize the in crystallo reaction protocol and was able to capture polymerase reaction at 30%, 50%, 70%, and 100% stages along with some post-chemistry structures with longer incubation times. Our results from the structures and kinetics supported our hypothesis and we were able to present an improved version of the BER pathway and hPolβ-dRP lyase mechanism.
In our day-to-day life, we are exposed to several DNA-damaging factors, like, pollution, sunlight, etc. BER pathway is critical in keeping our DNA, and subsequently, our body healthy. Any deviations in it can lead to cancers. Understanding the BER pathway and the enzymes involved therein is, therefore, crucial. Our current study is another stepping stone in this direction.
2) Additional projects
Other than hPolβ, I am also involved in different stages of multiple projects. One such project is based on DNA polymerase IV (Dpo4) from Saccharolobus solfataricus. It is a Y-family polymerase that the organism uses to promote the replication of damaged DNA through lesion bypass. We were trying to understand how this enzyme differentiates between a dNTP and rNTP. I have solved structures of five complexes. The data for these complexes were collected by Dr. Vineet Gaur, a former postdoc from our lab.
2012-2018 (CSIR-IMTech, Chandigarh, India)
1) Hypothetical proteins
Over the years the availability of genome-sequence data has rapidly increased. The availability of a wide range of bioinformatics tools has made it possible to identify the existence of proteins based on the sequence. However, it is increasingly getting difficult to assign a possible function to every protein sequence thus generated due to a lack of similarity to any protein with a known function. Structural biology provides a viable method to tackle this problem.
My research focused on one such hypothetical protein, Rv2229c, from Mycobacterium tuberculosis. Rv2229c spiked my interest as it was designated as essential for M. tuberculosis in several publications. My work started with general bioinformatics analysis of the sequence like secondary structure prediction, domain identification, operon information, and interacting partners. Since there were no established protocols for this protein, I had to standardize the protein purification procedure, followed by general characterization using circular dichroism (CD) and mass spectrometry. I was also able to confirm the presence of bound zinc in the protein. To get an idea of the actual function of this protein we had to obtain a crystal structure. I tried crystallization of Rv2229c using various crystallization screens (both commercial and self-made), temperatures, buffers, crystallization methods, and concentrations but failed after repeated attempts.
The failure to crystallize led me to look into close homologs of this protein. I intentionally avoided proteins with really close identities assuming they may also fail crystallization. We selected close homologs (60-75 % identity) from four organisms viz., M. smegmatis, M. xenopi, M. avium subsp. avium, and M. parascrofulaceum. The only protein that crystallized as MSMEG_4306 from M. smegmatis. We were able to determine its structure by single-wavelength anomalous dispersion using Zinc as the anomalous scatterer (Zn-SAD).
A comparison of the structure with those in Protein Data Bank (PDB) yielded two proteins, HP0958 from H. pylori and CT398 from C. trachomatis. Both these proteins have been characterized. Based on a similar structural organization containing a long coiled-coil helix at the N-terminal end and a Zinc-ribbon domain at the C-terminal end, we hypothesized that MSMEG_4306 (and consequently Rv2229c) could also hold functions similar to those of HP0958 and CT398. It means that the N-terminal domain could be involved in protein-protein interactions (possibly some RNA polymerase) and the C-terminal domain may also be involved in protein-protein and protein-RNA interactions. We also hypothesized that MSMEG_4306 may be involved in some sort of secretion system. I could not take this project further owing to the completion of my PhD term. In the future, this work can be extended to establish this protein as a potential drug target. However, at present, this project has added to the ever-growing knowledge base of M. tuberculosis.
2) 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS)
A second project that I was involved in was related to DHBPS from Vibrio cholerae. DHBPS is a part of the riboflavin biosynthesis pathway, which is essential for the survival of organisms. This project was started by Dr. Zeya Ul Islam. He worked on the structures of the protein and its complexes with ligand (ribulose-5-phosphate) and inhibitor (4PEH). I was responsible for refining and submitting structure coordinates to PDB for some of the structures and determining the kinetic constants for the inhibitor.
Overall, we were able to propose a mechanism for the conversion of ribulose-5-phosphate to 3,4-dihydroxy-2-butanone-4-phosphate. We were also able to establish that 4PEH inhibits the DHBPS activity by competitive inhibition with a Ki value of 100±10 μM. In the future, this molecule can be used as a lead to design novel antibiotics against pathogens.
Peer Review
October 2018 - September 2022 (Faculty Opinions)
I was an Associate Faculty Member for Structural Biology and reviewed manuscripts for Faculty Opinions.
November 2022 - November 2023 (MDPI group of journals)
Tech-marketing
February 2012 - May 2012 (Idea Brahma Consulting Pvt. Ltd.)
Marketing Executive
My responsibilities included presenting company products along with demonstration to prospective customers, mostly at B2B level. During this short stay with company I have also presented the company products at jury round of Aegis Graham Bell Awards 2012, where one of the products won the award for "Innovation in mHealth".