The Open Form Inducer Approach for Structure-Based Drug Design

Abstract

Many open form (OF) structures of drug targets were obtained a posteriori by analysis of co-crystals with inhibitors. Therefore, obtaining the OF structure of a drug target a priori will accelerate development of potent inhibitors. In addition to its small active site, Trypanosoma cruzi dihydroorotate dehydrogenase (TcDHODH) is fully functional in its monomeric form, making drug design approaches targeting the active site and protein-protein interactions unrealistic. Therefore, a novel a priori approach was developed to determination the TcDHODH active site in OF. This approach consists of generating an "OF inducer" (predicted in silico) to bind the target and cause steric repulsion with flexible regions proximal to the active site that force it open. We provide the first proof-of-concept of this approach by predicting and crystallizing TcDHODH in complex with an OF inducer, thereby obtaining the OF a priori with its subsequent use in designing potent and selective inhibitors. Fourteen co-crystal structures of TcDHODH with the designed inhibitors are presented herein. This approach has potential to encourage drug design against diseases where the molecular targets are such difficult proteins possessing small AS volume. This approach can be extended to study open/close conformation of proteins in general, the identification of allosteric pockets and inhibitors for other drug targets where conventional drug design approaches are not applicable, as well as the effective exploitation of the increasing number of protein structures deposited in Protein Data Bank.

Novel Characteristics of Mitochondrial Electron Transport Chain from Eimeria tenella

“…It is an enzyme involved in glycerol metabolism as well as gluconeogenesis [32,33]. Finally, DHODH catalyzes the oxidation of dihydroorotate into orotate with concomitant reduction of ubiquinone [34]. DHODH is the rate-limiting step in the pyrimidine de novo biosynthesis pathway and is required to generate UMP, the building block of DNA and RNA necessary to sustain life [35,36].…”

Selective Cytotoxicity of Dihydroorotate Dehydrogenase Inhibitors to Human Cancer Cells Under Hypoxia and Nutrient-Deprived Conditions

“…Recombinant human DHODH was purified, crystallized, and assayed as previously described ( Inaoka et al, 2016 ) without modification. Bovine mitochondrial fractions were prepared as previously described ( Kita et al, 1988 ) and NADH dehydrogenase (complex I), succinate:quinone reductase (complex II), quinol oxidase (complexes III–IV), NADH-cytochrome c reductase (complexes I–III), and succinate-cytochrome c (complexes II–III) activities were assayed following an established method ( Takamiya et al, 1986 ; Miyadera et al, 2001 ; Matsumoto et al, 2008 ; Kido et al, 2010 ; Nihashi et al, 2017 ).…”

“…All data sets were processed and scaled using HKL-2000 ( Otwinowski and Minor, 1997 ). The structure of the HsDHODH- 9 complex was solved by molecular replacement using the refined protein coordinates of HsDHODH-mii-4-087 complex (PDB code 3W7R) ( Inaoka et al, 2016 ) as a search model. All other structures of HsDHODH inhibitor complexes were solved by molecular replacement using the refined protein coordinates of HsDHODH- 9 complex (PDB code 5ZF4) as a search model.…”

“…Family 1 DHODHs are cytosolic enzymes and further sub-classified as family 1A and 1B according to the ability to use fumarate ( Inaoka et al, 2008 ; Kubota et al, 2018 ) or NAD + ( Jensen and Bjornberg, 1998 ) as electron acceptor, respectively. DHODHs from trypanosomatid parasites belong to family 1A and have been suggested to be drug targets to combat Chagas disease ( Inaoka et al, 2016 , 2017 ), African trypanosomiasis ( Arakaki et al, 2008 ; Kubota et al, 2018 ), and leishmaniasis ( Pinheiro et al, 2013 ). Family 2 DHODHs are membrane-bound enzymes and use quinone pool as its acceptor ( Rawls et al, 2000 ) and thus, contribute to the formation of the electrochemical gradient through complexes III and IV activities ( Löffler et al, 1997 ; Rawls et al, 2000 ).…”

Discovery of trypanocidal coumarins with dual inhibition of both the glycerol kinase and alternative oxidase of Trypanosoma brucei brucei

“…A key objective of our study was to discover TbgGK inhibitors that could replace glycerol for combination therapy with AF. Therefore, the effect of the TbgGK inhibitors identified above on the trypanocidal potency of AF was evaluated by the addition of 10 mM each of the secondary hit compounds (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) improved the potency of AF. We found that such compounds shifted the EC 50 of AF to ,0.01 nM ( Table 1).…”