Page 57 - Biennial Report 2018-20 Jun 2021

new drug targets and designing and testing new drug molecules. The Mtb dihydrodipicolinate
synthase (Mtb-DapA) is a potential drug target. Mtb-DapA catalyzes the first committed step in
the Lysine biosynthesis pathway which is aldol condensation of pyruvate and L-aspartate-beta-
semialdehyde (ASA) to yield dihydrodipicolinate. There is an urgent need for integrating
computational and experimental approaches in search for new inhibitors for M. tuberculosis.
The overall goal is to reduce both time and cost. A coupled enzymatic activity assay using His-
tagged purified recombinant proteins has been developed. The assay uses
spectrophotometrically measurable components, the NADPH producing reducing electrons in
the reaction. In principle, the structural analogues of both substrates could be tested for
inhibition potential. The previous work from the lab tested the inhibitory effect of pyruvate
analogues on recombinant Mtb-DapA using a coupled assay with recombinant
dihydrodipicolinate reductase (Mtb-rDapB). Alpha-keto pimelic acid (a-KPA) showed maximum
inhibition of 88% and IC50 of 21 µM in presence of pyruvate (500 µM) and ASA (400 µM).
Competition experiments with pyruvate and ASA revealed competition of a-KPA with pyruvate.
A new scoring function based on H-bonds simulation of the protein target was developed by Dr.
Vladimir Ivanisenko (collaborator) that operates after learning from a training set, which has
potential Mtb DapA inhibitors and their IC50 values. These data and also some other inhibitor-
based study will be used for developing a model for Mtb DapA inhibitor design.

The structure of pyruvate plays an important role in binding to the active site of Mtb-DHDPS.
pyruvate analogues and Fluorine derivatives of pyruvate as potential leads for Mtb-DHDPS
inhibitors are being screened biochemically. Fluorine can be used as a substitute for lone pairs
of electrons, hydrogen atoms, the methyl group because of its polar properties and small size. It
can also mimic the function of carbonyl, carbinol and nitrile moieties. So, it can enhance the
potency of inhibitors. Some pyruvate analogue and fluorinated compound were shortlisted from
literature with solubility values: 2-ketobutyric acid (119mg/ml), α-hydroxypyruvic acid, 2-
ketoglutaric acid (541.5mg/ml), 4,4,4-trifluoro butyric acid (3.4mg/ml), 5,5,5-trifluro pentanoic
acid (5mg/ml) and 2,3,3,3-tetraflouro propanoic acid. Based on the observed results, some
compounds were prioritized for biochemical assay. In the prioritization step, solubility of the
compound and availability was look into. The product analogues currently in the testing phase
are: 6-hydroxymethyl-pyridine-2-carboxylic acid (0.75mg/ml), 2-Picolinic acid (31mg/ml),
Chelidonic acid, 2,6-Pyridinedicarboxylic acid (0.048mg/ml) and Isophthalic acid (120mg/ml).
Biochemical validation was performed using coupled-enzymatic activity assay. A fixed
concentration of pyruvate (500uM), ASA (400uM) and varied concentrations of compounds was
used. Coupled enzymatic assay with 2-Ketobutyric acid, -hydroxypyruvic acid, 2- picolinic acid
and 2-ketoglutaric acid were performed. The fluorescence based thermal shift assay was used
for screening of identified ligands, based on the principle that ligand binding alters thermal
stability of protein. This method allowed to monitor the protein denaturation upon heating using
fluorescence-based dye (Sypro orange), which binds to the hydrophobic part of proteins during
protein denaturation and emits fluorescence at 580.nm. The changes in fluorescence were
monitored and a thermal melting curve using real time PCR was generated. The midpoint of the
melting curve was designated as the melting temperature Tm corresponding to 50% of protein
being denatured. The binding of pyruvate and screened compound to Mtb-rDapA were checked
by performing a thermal shift assay, in which the assay mixture was prepared containing 20uM
of Mtb_DapA, 15X Sypro orange dye, 50mM sodium phosphate buffer pH 7.4 and ligand in range
of 0.2mM to 1mM, the thermal scanning was started from 20. to 95. at a heating rate of 0.5./min.
A control thermal shift assay was also performed.

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