The identification of a proper lead compound for fructose 1,6-bisphosphatase (FBPase) is a critical step in the process of developing novel therapeutics against type-2 diabetes. Herein, we have successfully generated a library of allosteric inhibitors against FBPase as potential anti-diabetic drugs, of which, the lead compound 1b was identified through utilizing a virtual high-throughput screening (vHTS) system, which we have developed. The thiazole-based core structure was synthesized via the condensation of α-bromo-keotnes with thioureas and substituents on the two aryl rings were varied. 4c was found to inhibit pig kidney FBPase approximately 5-fold better than 1b. In addition, we have also identified 10b, a tight binding fragment, which can be use for fragment-based drug design purposes.

The enzyme aspartate transcarbamoylase (ATCase, EC 2.1.3.2 of Escherichia coli), which catalyzes the committed step of pyrimidine biosynthesis, is allosterically regulated by all four ribonucleoside triphosphates (NTPs) in a nonlinear manner. Here, we dissect this regulation using the recently developed approach of random sampling-high-dimensional model representation (RS-HDMR). ATCase activity was measured in vitro at 300 random NTP concentration combinations, each involving (consistent with in vivo conditions) all four NTPs being present. These data were then used to derive a RS-HDMR model of ATCase activity over the full four-dimensional NTP space. The model accounted for 90% of the variance in the experimental data. Its main elements were positive ATCase regulation by ATP and negative by CTP, with the negative effects of CTP dominating the positive ones of ATP when both regulators were abundant (i.e., a negative cooperative effect of ATP × CTP). Strong sensitivity to both ATP and CTP concentrations occurred in their physiological concentration ranges. UTP had only a slight effect, and GTP had almost none. These findings support a predominant role of CTP and ATP in ATCase regulation. The general approach provides a new paradigm for dissecting multifactorial regulation of biological molecules and processes.Many biochemical processes are sensitive to multiple signals, which may interact in a nonlinear manner. While such sensitivity often arises from complex reaction networks, even single enzymes can respond to multiple regulators. One such enzyme is aspartate transcarbamoylase (ATCase) (1), a complex in which activity and regulation are on distinct polypeptide chains (2). ATCase is composed of 12 polypeptides (6 c chains and 6 r chains), organized as two catalytic trimers bound to three regulatory dimers (3,4). It catalyzes the first committed step in the metabolic pathway for de novo pyrimidine biosynthesis: the condensation of aspartate with carbamoyl phosphate to yield carbamoyl aspartate. Binding of substrate to the enzyme is ordered, with carbamoyl phosphate binding before aspartate (5,6) and inducing local conformational changes leading to the formation of a viable aspartate-binding site (7). † Funding was provided by the NSF DDDAS program (CNS-0549181), the EPA STAR grant program, the DOD STTR program, the NSF CAREER award (MCB-0643859), the Beckman Foundation, the American Heart Association (0635188N), the NIH Center for Systems Biology at Princeton University (5 P50 GM071508), and NIH Grant GM26237. Both genetic and biochemical evidence support CTP, a primary end product of the pathway, being the key negative regulator of ATCase (a classical example of feedback inhibition) (8).Biochemical evidence also demonstrates that ATP substantially enhances enzyme activity (9), with this regulation serving to balance pyrimidine and purine concentrations. Crystal structures of the ATCase complex have been solved with ATP or CTP bound and confirm that ATP and CTP bind to and induce conformational changes in...

The mechanism of domain closure and the allosteric transition of Escherichia coli aspartate transcarbamoylase (ATCase) are investigated using L-Asn, in the presence of carbamoyl phosphate (CP), and N-phosphonacetyl-L-asparagine (PASN). ATCase was found to catalyze the carbamoylation of L-Asn with a K(m) of 122 mM and a maximal velocity 10-fold lower than observed with the natural substrate, L-Asp. As opposed to L-Asp, no cooperativity was observed with respect to L-Asn. Time-resolved small-angle X-ray scattering (SAXS) and fluorescence experiments revealed that the combination of CP and L-Asn did not convert the enzyme from the T to the R state. PASN was found to be a potent inhibitor of ATCase exhibiting a K(D) of 8.8 microM. SAXS experiments showed that PASN was able to convert the entire population of molecules to the R state. Analysis of the crystal structure of the enzyme in the presence of PASN revealed that the binding of PASN was similar to that of the R-state complex of ATCase with N-phosphonaceyl-L-aspartate, another potent inhibitor of the enzyme. The linking of CP and L-Asn into one molecule, PASN, correctly orients the asparagine moiety in the active site to induce domain closure and the allosteric transition. This entropic effect allows for the high affinity binding of PASN. However, the binding of L-Asn, in the presence of a saturating concentration of CP, does not induce the closure of the two domains of the catalytic chain, nor does the enzyme undergo the transition to the high-activity high- affinity R structure. These results imply that Arg229, which interacts with the beta-carboxylate of L-Asp, plays a critical role in the orientation of L-Asp in the active site and demonstrates the requirement of the beta-carboxylate of L-Asp in the mechanism of domain closure and the allosteric transition in E. coli ATCase.