Proteolysis targeting chimeras (PROTAC®s) are chimeric small molecules harboring E3 ligase and target protein, which hijack the cellular machinery for ubiquitylation and degradation. Dihydroorotate dehydrogenase (DHODH) is the fourth enzyme implicated in the de novo pyrimidine nucleosides biosynthetic pathway. As a well-known expert around the world, Creative Biolabs pays attention to the knowledge on DHODHs with emphasis on their structural properties and provides ligands design services for DHODH and other enzymes to meet our clients’ requirements for developing specific PROTAC®s.
The overall structure of the human DHODH monomer holds two domains, a typical α/β barrel fold containing the active site and an α-helical domain forming the opening of a tunnel to the active site. This α/β barrel domain has a central barrel consisting of eight parallel β-strands (β1 to β8) surrounded by eight α-helices (α1 to α8). Two N-terminal antiparallel β-strands (βa and βb) are observed at the bottom of the barrel, while additional secondary structural components and loops organize a subdomain present on top of the barrel. The prosthetic group of flavin mononucleotide (FMN) resides between the top of the barrel and the subdomain.
Fig.1 Crystal structures of DHODHs. (Reis, 2017)
All DHODH structures have been determined by X-ray crystallography in the holo format or complex with substrates, products, or inhibitors, including class 1A, class 1B, and class 2. In class 1A homodimer, the monomers are linked by a non-crystallographic twofold axis. The class 1B heterotetramer comprises two dimers of two distinct proteins (PyrD and PyrK) and three distinct cofactors (FMN, FAD, and a 2Fe-2S cluster). In class 2 monomer, there’re two different portions that include a C-terminal domain containing an α/β barrel and an N-terminal helical domain. DHODH is a ubiquitous FMN flavoenzyme, which has attracted great interests from basic and clinical areas of research. This enzyme is broadly exploited as a promising drug target across oncology, rheumatology, and infection.
Bifunctional molecules of PROTAC®s are composed of a ligand to the E3 ubiquitin ligase, a ligand to the target protein, and a linker conjugating the two ligands. Once the ternary complex is formed, chemically induced cellular proteolysis will be initiated by the ubiquitin-proteasome system (UPS). In recent years, a series of rapid and efficient PROTAC®s with great pharmaceutical properties have been described. At Creative Biolabs, we’d like to take advantage of versatile PROTAC® platforms to provide sophisticated ligand design services for various metabolic enzymes based on the specific structure of these targets.
Fig.2 (A) Structures of brequinar and new probes, (B) Dose response curves from the DHODH assay, (C) IC50 values from brequinar and new probes tested in DHODH and MTT assay, (D) HCT-116 colonies treated with varying doses of brequinar probes. (Madak, 2017)
DHODHs from varying organisms have been separated and characterized previously, including eukaryotes and prokaryotes. The inhibition of DHODHs is capable of modulating pyrimidine biosynthesis. Effective inhibitors are potential candidates clinically useful in the treatment of cancers, immune disorders, infectious diseases, and parasitic diseases. For example, brequinar is a strong DHODH inhibitor that has been evaluated in clinical trials as an attractive treatment for cancer. In order to study the therapeutic effects of DHODH and cause an intracellular knockdown, a DHODH PROTAC® has been developed based on its ligand brequinar. Here, our teams focus on the substantial difference in structural, kinetic, dynamic, and functional properties of DHODH enzymes. We have combined in vitro, in vivo, and in silico approaches for the screening of inhibitors and offer optimized ligand design services by linking these inhibitors to DHODH-targeting PROTAC®s. Furthermore, there’re several forms of the ligand can be developed, such as peptides, antibodies, and numerous small molecules.
With extensive expertise in drug development, Creative Biolabs takes efforts to improve the current understanding of structural characteristics of DHODHs and devotes to capturing the new progress of novel modalities of PROTAC®s. Notably, we offer a series of advanced PROTAC® techniques for ideal ligand design during the process of drug discovery. As well, our experts have determined the many different types of metabolic enzymes in terms of sequence, structure, and biochemistry, and provide comprehensive ligand screening services for these targets. For more details, please don’t hesitate to contact us.
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