Abstract 5787: Structure-function mapping of DHODH shows deviations in protein structure
Authors: Pokhriyal Ruchika , Evans Laura , Seo Hyuk-Soo , O'Neil Jillian K. , Rouaisnel Betty , Kamburov Atanas , Guenther Judith , Gradl Stefan , Arthanari Haribabu , Christian Sven , Yang Xiaoping , Dhe-Paganon Sirano , Losman Julie-Aurore 
Published: March 2024

Abstract
Dihydroorotate dehydrogenase (DHODH) is a CoQ-dependent enzyme that converts dihydroorotate to orotate in the de novo pyrimidine biosynthesis pathway. Multiple structures of DHODH in complex with various small molecule inhibitors have been solved that indicate that DHODH inhibitors bind in the CoQ binding tunnel of DHODH and act as competitive inhibitors of CoQ. We performed saturating mutagenesis screens to identify mutations in DHODH that confer resistance to brequinar and BAY2402234, two highly potent DHODH inhibitors. As expected, many of the resistance mutations localized to the drug/CoQ-binding tunnel of the enzyme. Among the top-scoring drug-binding-domain mutants in both the brequinar and BAY2402234 screens were substitutions A58H and A58T. In silico analysis predicted that A58H should impair DHODH binding to both brequinar and BAY2402234. However, A58T was predicted to only impair binding of DHODH to brequinar. We expressed the mutants in a DHODH inhibitor sensitive cell line and confirmed that they both confer resistance to brequinar and BAY2402234, and we then assessed the ability of the mutants to bind drug by cellular thermal shift assay (CETSA). We found that, as predicted, DHODH A58H did not bind brequinar or BAY2402234. However, to our surprise, we detected binding of both brequinar and BAY2402234 to DHODH A58T. We generated recombinant DHODH A58T and confirmed that the purified protein was enzymatically active and strongly drug-resistant by DCIP assay and then assessed binding of the mutant protein to brequinar and BAY2402234 by thermal shift assay (TSA). Consistent with our CETSA results, recombinant DHODH A58T bound both brequinar and BAY2402234 in vitro. These results suggested that DHODH A58T can simultaneously bind drug and CoQ. However, previously reported DHODH structures indicated that the drug/CoQ binding tunnel is quite narrow and rigid. To determine whether the structure of catalytically-active DHODH is more flexible than inhibitor-bound catalytically-inactive DHODH, we co-crystallized wild-type DHODH with decylubiquinone (DCU), a CoQ analog that can act as a DHODH co-substrate in vitro. We found that the structure of DCU-bound DHODH differs from previously published DHODH structures in two important ways: (1) there appears to be a second point of ingress into the active site of DHODH next to the CoQ tunnel that may be able to accommodate a highly non-polar molecule such as CoQ/DCU and (2) the ‘flap’ covering the DHO/orotate ingress/egress region of DHODH, which is ‘closed’ in inhibitor-bound DHODH, is ‘open’ in DCU-bound DHODH. We are currently working to solve the crystal structure of DHODH A58T bound to inhibitor and DCU to ask if DCU can access the active site of DHODH through an alternative hydrophobic tunnel and if DHODH A58T remains in the ‘open’ configuration when bound to drug.
Citation Format: Ruchika Pokhriyal, Laura Evans, Hyuk-Soo Seo, Jillian K. O'Neil, Betty Rouaisnel, Atanas Kamburov, Judith Guenther, Stefan Gradl, Haribabu Arthanari, Sven Christian, Xiaoping Yang, Sirano Dhe-Paganon, Julie-Aurore Losman. Structure-function mapping of DHODH shows deviations in protein structure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5787.