Enzyme Upregulation by Computer-Aided Molecular Design

Our research program for enzyme activation is comprised of computational and experimental components. The computational component builds upon publications in the Proceedings of the National Academy of Sciences (PNAS) on biophysical modeling and optimization of enzymatic catalysis by Dr. Raj Chakrabarti (Founder of CAT). The computational component is further complemented with an experimental ultrahigh-throughput screening component based on DNA-Encoded Libraries (DELs). State-of-the-art machine learning algorithms are applied to this big data to guide the Darwinian evolutionary process towards optimal drug leads faster and more efficiently.

Two primary applications are pursued: a) enzyme active site design for catalysis on unnatural substrates; b) small molecule induced enzyme activation. Both problems involve modeling and optimization of the effects of design variables on protein structure and function. With respect to application b), allosteric activation is currently the only known means by which small molecule drugs can enhance the activity of enzymes. However, the vast majority of potential drug targets are not amenable to allosteric activation due to the lack of an allosteric site. It is known that the ability to upregulate a variety of such enzymatic targets could enable the treatment of a host of associated diseases.

Our research program for non-allosteric activation of enzymes through the modulation of active site structure, includes the activation of the so-called sirtuin enzymes, which have been shown to play a central role in age-related conditions, such as diabetes, neurodegenerative disorders, and cardiovascular diseases. Among the 7 mammallian sirtuin enzymes SIRT1-7, only SIRT1 has an allosteric site. In the absence of nonallosteric activators, to date sirtuin activation has been pursued pharmaceutically for SIRT1 (by Sirtris Pharmaceuticals, now GSK) by allosteric activation and for SIRT3 by nutraceutical NAD supplements which are not drugs (by Elysium Health). Using the above integrated drug discovery platform, we have discovered novel hits for activation of SIRT3 – the major mitochondrial sirtuin – that are more potent and activate under a much wider range of physiologically relevant conditions than the small number of hits that were previously identified in the literature. The SIRT1 activators from Sirtris, for instance, only work on a small number of substrates. Moreover, our platform may be used to identify activators of multiple other SIRT enzymes and for much wider variety of substrates. We experimentally characterized the mode of action for novel sirtuin activators identified using ultrahigh-throughput computational and experimental screening techniques, uncovering for the first time the mechanism whereby they can activate enzymes without allosteric sites. In particular, these compounds were shown to increase up to two-fold the catalytic efficiency of SIRT3 and its deacetylation rate under the essential cellular cofactor nicotinamide adenine dinucleotide (NAD)+ depletion conditions characteristic of old age. The compounds have the potential to rejuvenate the activity of the major mitochondrial sirtuin to levels observed in youth.

Selected Publications:

• Sequence optimization and designability of enzyme active sites. Raj Chakrabarti, Alexander M. Klibanov and Richard A. Friesner. Proc. Natl. Acad. Sci. USA 102 (34) 12035-12040 (2005)
• Computational prediction of native protein ligand-binding and enzyme active site sequences. Raj Chakrabarti, Alexander M. Klibanov and Richard A. Friesner. Proc. Natl. Acad. Sci. USA 102 (29) 10153-10158 (2005)
• Mechanism of inhibition of the human sirtuin enzyme SIRT3 by nicotinamide: computational and experimental studies. Xiangying Guan, Ping Lin, Eric Knoll and Raj Chakrabarti. PLOS ONE 9 (9): e107729 (2014)
• Molecular system identification for enzyme directed evolution and design. Xiangying Guan and Raj Chakrabarti. J Chem Phys 147 124106 (2017)
• Biophysical characterization of hit compounds for mechanism-based enzyme activation. Xiangying Guan, Alok Upadhyay, Sudipto Munshi and Raj Chakrabarti. PLOS ONE 13 (3): e0194175 (2018)
• Discovery of novel compounds as potent activators of Sirt3. Célina Reverdy, Gaetan Gitton, Xiangying Guan, Indranil Adhya, Rama Krishna Dumpati, Samir Roy, Santu Chall, Anisha Ghosh, Gauthier Errasti, Thomas Delacroix and Raj Chakrabarti. Bioorganic and Medicinal Chemistry 73 116999 (2022)
• Computationally Driven Discovery and Characterization of SIRT3-Activating Compounds that Fully Recover Catalytic Activity under NAD+ Depletion. Xiangying Guan, Rama Krishna Dumpati, Sudipto Munshi, Santu Chall, Rahul Bose, Ali Rahnamoun, Celina Reverdy, Gauthier Errasti, Thomas Delacroix, Anisha Ghosh, and Raj Chakrabarti. Phys. Rev. X 14 041019 (2024).