Department of Chemistry
Indian Institute of Technology Bombay
Structural Biochemistry Lab
RESEARCH FOCUS
The core research interest of our group is to gain structural insights of protein in regulation and biosynthesis pathway. Along with crystallography and CryoEM we employ biochemical techniques like electromobility shift assays, isothermal calorimetry, surface plasma resonance (SPR), fluorescence spectroscopy to study protein-protein, protein-DNA/RNA, protein-ligand interactions and to understand mechanism of catalysis in certain enzyme systems.
Origins of antibiotic resistance:
RNA Methyltransferase:
TetR family proteins:
Owing to overuse and misuse of drugs, antibiotic resistance had become a significant threat to human health. One way to tackle this problem is to continuously synthesize and validate new antibiotics. However, this is an uphill task and invariably pathogenic strains tend to acquire resistance within months. Therefore, to address this issue we aim to reverse resistance by understanding the origins of resistance itself. In several cases pathogens which were earlier sensitive to standard drugs suddenly become resistant. In these cases lateral gene transfer results in acquisition of gene cassettes that renders normal pathogens resistant. Enzymes in these gene cassettes, such as methyltransferases, are capable of post-translationally modifying ribosome, the main target site of these antibiotics. We were able to decipher the mechanism of targeting of these ribosomal methyltransferase and have used a combination of structure, phylogenetic, biochemical and biophysical tools to understand the search algorithm adopted by these enzymes to methylate the ribosome with high degree of fidelity. This knowledge can now be used to explore design of new drugs that will stop pathogens from attaining superbug stature (JACS Communication 2019). Based on this initial work we are now exploring this path further and have been awarded DBT-Wellcome Trust Senior Fellowship to advance efforts on understanding the mechanism of drug resistance via this route. The overall aim is to develop combination therapy approaches such that the bugs become sensitive to existing antibiotics.
Tetracycline receptors (TetRs) are a class of transcription factors that bind to antibiotics and control their efflux. These receptors are prevalent in several multi drug resistant organisms and control antibiotic levels in the cell. Streptomyces that produce 70% of the commercially known antibiotics possess a high density of these TetRs. These organisms use them both as global and pathway regulators to regulate both antibiotic production and resistance pathways. Our group solved the structure of CprB a gamma butyralactone response regulator in complex with DNA from the antibiotic producer organism, Streptomyces coelicolor (NAR 2014). This structure was a breakthrough as it helped in providing a glimpse as to how the antibiotic producers protect themselves against the antibiotics they synthesize. This discovery showed that Streptomyces efflux pump regulators have close evolutionary links with TetRs found in pathogens such as the multi-drug resistant (MDR) organism Staphylococcus aureus. It is possible that the MDRs acquired it via lateral gene transfer and became superbugs. (NAR 2014, JPC 2014, BBA 2015, JSB 2017). Subsequently, her group solved structures of unique TetRs from Streptomyces fradiae, another antibiotic producer, that further helped decode the various architectures and mechanisms by which drug regulation occurs (JBC 2017). Based on the information harnessed here, developing efflux pump blockers can be envisioned that will help reverse resistance and re-sensitize the pathogens towards existing antibiotics.
Towards understanding new drug targets:
Understanding regulatory switches in PurL:
Purine salvage pathway is an essential pathway as it helps in synthesis of nucleobases that are precursors to genetic material in all cells. All organisms have stringent control on the enzymes that operate within these pathways. Here, we exploit this control to look for specific enzymes that can be used as potential drug targets. PurL that catalyzes the fourth step in the purine pathway is one such enzyme that exists as a complex of three proteins in gram positive and archea bacteria but is a single enzyme in humans and eubacteria. This aspect can be further exploited to design strategies that can target protein-protein interaction interfaces in a species specific manner. Our group has been trying to understand the working of this 140 kDa allosterically regulated bi-functional enzyme. Our recent work has unraveled an allosteric switch that helps in coordinating the reaction at the two 25 Å distal reaction centers. In addition, the tunnel that connects the two active sites which allows passage of the labile intermediate ammonia has also been elucidated (ACS Chemical Biology 2015, Science Advance 2020).
Nucleobase Deaminase:
In an effort to find new drug targets the Anand laboratory have chosen nucleobase deaminases as potential drugable targets. These enzymes are a group of essential enzymes that are involved in the nucleobase catabolic pathway and stringently regulate the concentration of the nucleobase derivative pool, which is paramount for nucleotide recycling. These enzymes show remarkable fidelity in function and interesting are evolutionary divergent between human and bacterial systems. Our structural studies on guanine deaminases has been vital in developing insights into their mechanism of action and the conformational changes that assist the reaction (Biochemistry 2013a, Biochemistry 2013b). Moreover, we have been able to develop initial inhibitor leads and show that guanine deaminases do not accept any variations in their substrates. These early studies lead us to discover a remarkable new enzyme, exclusively found in Mycobacterium, which confers innate resistance towards aza-scaffold of drugs. This work was published as a full article in the leading American Chemical Society journal (JACS 2017). Here, the X-ray structure of this novel enzyme and subsequent structures with substrate and their analogs aids in understanding how this enzyme only selectively deaminates mutagenic bases such as azacytosine etc. It was established that this enzyme scans for these mutagenic bases and eliminates them from the cell. This enzyme seems to have conferred Mycobacterium to develop innate immunity to aza-scaffold of drugs. Thus by targeting this newly discovered pathway one can sensitize these resilient organisms towards mutagenic base harboring drugs an ongoing theme in our laboratory.
Biosensor development against water pollution:
Supply of clean and fresh drinking water is a world-wide problem that has been plaguing this earth. Dr. Anand’s group has employed their structural biology expertise to address means to sense water quality levels in lakes, rivers and other water bodies. They focused on the group of pollutants belonging to the aromatic pollutant class. The acute environmental pollution by toxic contaminants such as phenol, benzene and other similar derivatives from oil, paper & tannery industries are of priority concern for sustainable environment as well as alarming for the society. Our lab is engaged to develop and optimize efficient biosensors for on-site xenobiotics monitoring in water and to develop subsequent bioremediation to control their levels. We solved the structure of the phenol sensor protein found in bacteria that has the extraordinary ability to sense this pollutant and degrade it (ACS Chemical Biology 2016). This further lead to use a combination of structure guided principles as well as evolutionary design to develop an array of optical biosensors to detect phenol and benzene derivatives, selectively down to ppb levels (ACS Sensors 2017, ACS Sensors 2018). Unlike the common belief that proteins are unstable, the sensors developed here are stable up to 70°C and have a long shelf. This remarkable robustness has enabled the development of a chip that can detect pollutants and hold great commercialization potential (Analytical Chemistry 2018, Patent 2018). They have collaborated with the chemical engineering group of Prof. Rajdeep Bandopadhya at IIT Bombay and have been able to extend this work to make a hand held device which was showcased at the Asia’s biggest technology festival TechFest 2020. Ongoing work in their laboratory is focused on development of oil based sensors and to extend this technology to electrochemical based sensing.