Discovery of Novel Allosteric Effectors Based on the Predicted Allosteric Sites for Escherichia coli D-3-Phosphoglycerate Dehydrogenase

Abstract

D-3-phosphoglycerate dehydrogenase (PGDH) from Escherichia coli catalyzes the first critical step in serine biosynthesis, and can be allosterically inhibited by serine. In a previous study, we developed a computational method for allosteric site prediction using a coarse-grained two-state Gō Model and perturbation. Two potential allosteric sites were predicted for E. coli PGDH, one close to the active site and the nucleotide binding site (Site I) and the other near the regulatory domain (Site II). In the present study, we discovered allosteric inhibitors and activators based on site I, using a high-throughput virtual screen, and followed by using surface plasmon resonance (SPR) to eliminate false positives. Compounds 1 and 2 demonstrated a low-concentration activation and high-concentration inhibition phenomenon, with IC50 values of 34.8 and 58.0 µM in enzymatic bioassays, respectively, comparable to that of the endogenous allosteric effector, L-serine. For its activation activity, compound 2 exhibited an AC50 value of 34.7 nM. The novel allosteric site discovered in PGDH was L-serine- and substrate-independent. Enzyme kinetics studies showed that these compounds influenced Km, kcat, and kcat/Km. We have also performed structure-activity relationship studies to discover high potency allosteric effectors. Compound 2-2, an analog of compound 2, showed the best in vitro activity with an IC50 of 22.3 µM. Compounds targeting this site can be used as new chemical probes to study metabolic regulation in E. coli. Our study not only identified a novel allosteric site and effectors for PGDH, but also provided a general strategy for designing new regulators for metabolic enzymes.

Rational Design of Selective Allosteric Inhibitors of PHGDH and Serine Synthesis with Anti-tumor Activity

“…PHGDH was immobilized on a CM5 sensor chip by using standard amine-coupling at 25°C with 1× running buffer PBS-P (GE Healthcare), as described previously (Wang et al, 2014). For details, see the Supplemental Experimental Procedures.…”

“…Computational methods for rational design of allosteric effectors were emerging (Wagner et al, 2016; Ma et al, 2016) and a number of successful application examples have been reported. For example, using the two-state Go model based allosteric site prediction method that we developed (Qi et al, 2012), we obtained novel allosteric inhibitors for Escherichia coli ( E. coli ) phosphoglycerate dehydrogenase (Wang et al, 2014). Novel enzymes activators were also found using combined computational and experimental approach (Meng et al, 2016), providing an alternative way to control disease-related molecular networks (Pei et al, 2014).…”

Engineered control of enzyme structural dynamics and function

“…These results and many others (e.g., Refs. ) have suggested that surface‐exposed network residues may serve as druggable interfaces for allosteric modulators.…”

Allosteric sites can be identified based on the residue-residue interaction energy difference

“…In other cases, the effect is dependent on the interaction between the allosteric ligand and protein. Modifications of the allosteric ligand may change the effect from activation to inhibition, and sometimes the effect is even concentration dependent …”