Search bioRxiv⌕ Search

Biology subjects

SRINIVASAN, B.

Publications and source records attributed to SRINIVASAN, B..

2 recordsLinked to original sources
bioRxiv2022

Accelerating the Validation of Endogenous On-Target Engagement and In-cellulo Kinetic Assessment for Covalent Inhibitors of KRASG12C in Early Drug Discovery

Covalent inhibition is a valuable modality in drug-discovery due to its potential ability in decoupling pharmacokinetics from pharmacodynamics by prolonging the residence time of the drug on the target of interest. This increase in target occupancy is limited only by the rate of target turnover. However, a limitation in such studies is to translate the in-vitro inhibition assessment to the appropriate in-cellulo target engagement parameter by covalent probes. Estimation of such parameters is often impeded by the low-throughput nature of current label-free approaches. In this study, an ultra-performance liquid chromatography-multiple reaction monitoring (UPLC-MRM) mass spectrometry platform was utilised to develop a targeted proteomics workflow that can evaluate cellular on-target engagement of covalent molecules in an increased throughput manner. This workflow enabled a throughput increase of 5-10 fold when compared to traditional nanoLC-based proteomics studies. To demonstrate the applicability of the method, KRASG12C was used as a model system to interrogate the interaction of an irreversible covalent small-molecule, compound 25, both in-vitro and in-cellulo. Initial biochemical studies confirmed that the small-molecule forms an adduct with the targeted cysteine on the protein, as assessed at the level of both intact protein and on the target peptide. In-cellulo studies were carried out to quantify target engagement and selectivity assessment in heterozygous NCI-H358 cell line with both WT type and KRASG12C alleles. The workflow enabled evaluation of in-cellulo target engagement kinetics providing mechanistic insights into the irreversible mode of inhibition. In summary, the method has the potential for target agnostic application in the assessment of on-target engagement of covalent probes compatible with the high-throughput requirements of early drug discovery.

biochemistry↗
bioRxiv2022

Thermodynamic analysis of Zα domain-nucleic acid interactions

DNA/RNA molecules adopting the left-handed conformation (Z-form) have been attributed with immunogenic properties. However, their biological role and importance has been a topic of debate for many years. The discovery of Z-DNA/RNA binding domains (Z domains) in varied proteins that are involved in the innate immune response, such as the interferon inducible form of the RNA editing enzyme ADAR1 (p150), Z-DNA binding protein 1 (ZBP1), the fish kinase PKZ and the poxvirus inhibitor of interferon response E3L, indicates important roles of Z-DNA/RNA in immunity and self/non-self-discrimination. Such Z domain-containing proteins recognise left-handed Z-DNA/RNA in a conformation-specific manner. Recent studies have implicated these domains in virus recognition. Given these important emerging roles for the Z domains, it is pivotal to understand the mechanism of recognition of the Z-DNA/Z-RNA by these domains. To this end, we assessed the binding thermodynamics of Z domain from ORF112 and ADAR1 on T(CG)3 and T(CG)6 oligonucleotides which have high propensity to adopt the Z-conformation. Our study highlights important differences in the mode of binding by the two Z domains originating from different proteins. Site-directed mutagenesis was employed together with isothermal titration calorimetry to tease apart finer details of the binding thermodynamics. Our work advances the understanding on binding thermodynamics of Z domains to their cognate nucleic acid substrates and contributes to the efforts to gain a complete appreciation of this process.

biochemistry↗