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  • The resolution cocrystal structure of ACL in complex with

    2022-11-18

    The 2.10Å resolution cocrystal structure of ACL in complex with citrate indicates that the carboxyl (COH) groups of citrate forms a network of H-bonds with hydrophilic residues surrounding the ACL citrate binding domain (CBD) (A). The - carboxyl group forms a salt bridge with the guanidine moiety of Arg379. Important H-bonds are present between the middle carboxyl group of citrate and the amino L-Adrenaline backbone of Asn346 and Phe347, as well as the side chains of Arg379 and Thr348. In addition, the hydroxyl (OH) group of the citrate participates in H-bond interactions with Gly309 and Ala310. These amino acids compose the tight and highly hydrophilic CBD (A and B). It is also worth noting that the - carboxyl group protrudes to surrounding solvents and does not form interactions with the ACL enzyme, rendering it free to participate in subsequent reactions to form acetyl-CoA. The synthetic inhibitor SB-201076 () lacks this third carboxyl group and attains potent inhibitory activity. While directly targeting this polar and charged CBD by competitive inhibitors including SB-201076 () and (−)-HCA () is unlikely to yield inhibitors with adequate cellular permeability, we became interested in targeting what appeared to be an allosteric hydrophobic domain directly adjacent to the CBD. This putative allosteric domain, as indicated by the red colored oval dish in B, forms the entrance of the buried CBD. We envisioned that targeting this hydrophobic domain, in addition to the CBD, by drug-like small molecules might provide a viable strategy to inhibit ACL activity while achieving cellular permeability. We therefore sought to identify novel ACL inhibitors by performing docking analyses of small molecule compound library to the CBD and the adjacent hydrophobic domain. It has been well documented that the various docking algorithms that rely on empirical scoring functions performed differently depending upon the biological targets as well as the ligand library,, , , , , which is likely due to the bias inherent to the dataset and targets deployed to curve fit the algorithms. While improvements in the scoring functions are expected as new approaches are adopted, consensus docking using two different docking algorithms can increase hit rate of a vHTS campaigns., , , We applied both the Autodock Vina and Glamdock protocols for docking studies to minimize biases inherent to empirical scoring functions. These two docking algorithms use different scoring functions. In addition, Autodock Vina and Glamdock were optimized against different sets of known targets using the PDBbind and the sc-PDB databases, respectively. Docking of large numbers of chemical structures has been performed routinely in vHTS., Yet, large scale vHTS still demands high computing power to perform the molecular docking in a time efficient manner, as well as significant time for post screening data mining. This could be particularly challenging for our approach, as we desired to use two docking protocols with the aim of increasing hit rate. We therefore chose to screen a fused compound library containing a furan carboxylate moiety. The reason to choose a furoic acid library for screening was based on a 1985 publication by Hall et al. indicating that at high concentrations 2- and 3-furoic acids suppressed ACL enzymatic activity in rodent liver. The authors showed that after mice were treated with furoic acids for 16days, the ACL activity in liver homogenates was significantly inhibited. Similarly, significant inhibition of ACL activity was observed when they treated rodent liver homogenates with 50–200μM furoic acids . While the authors did not delineate the mechanism of action by the furoic acids, we suspected that they were capable of mimicking the citrate and competing for binding to the CBD of the ACL enzyme. Indeed, our docking analyses as detailed below suggest both furoic acids can occupy the CBD of the ACL protein. To prepare for vHTS campaign aimed to identify novel ACL inhibitors, we first confirmed that both docking protocols Vina and Glamdock could recapitulate the binding pose from the citrate-ACL cocrystal structure. The ACL crystal structure (PDB: ) and small molecules were prepared for docking using Autodocktools (v1.5.6). After removal of citrate from the co-crystal structure complex, we docked citrate back to the CBD of ACL structure. The receptor was treated as a fully rigid structure L-Adrenaline in this instance and all subsequent dockings with both Autodock Vina and Glamdock. In addition, rotational freedom was allowed around all rotatable bonds for each small molecule for Autodock Vina and Glamdock. The lowest energy docking pose of citrate generated by Autodock Vina (C) was in excellent agreements with the conformation of the citrate and its interactions with key amino acid residuals presented in the cocrystal structure. We next performed docking of the citrate-competitive inhibitor (−)-HCA), as well as 2-/3-furoic acids (D–F). Common to all three ligands is the formation of the salt bridge between the carboxylate and Arg379. The lowest energy docking pose of (−)-HCA generated by Autodock Vina (D) indicated extensive H-bond interactions, many of which are similar to that between citrate and ACL such as Asn346, Gly309 and Ala310. Unlike the - carboxylate of citrate that is free from interactions with the ACL protein, the third carboxylic acid group of (−)-HCA forms H-bond with Asn346. It is tempting to speculate that this interaction transforms (−)-HCA to an inhibitor, as the carboxylate is no longer available to participate in reactions to produce acetyl-CoA. Similarly, docking of 2- and 3-furoic acids to the CBD indicates that the carboxylic acid moiety forms H-bond interactions with G309, and the furan ring may participate in hydrophobic interactions with the side chains of Phe347 and Ala345 (E and F). Glamdock provided similar docking results for citrate, as well as the furoic acids (not shown).