Identification of Dihydroorotate Dehydrogenase Inhibitors Using the Cell Painting Assay

Abstract

Profiling approaches have been increasingly employed for the characterization of disease-relevant phenotypes or compound perturbation as they provide a broad, unbiased view on impaired cellular states. We report that morphological profiling using the cell painting assay (CPA) can detect modulators of de novo pyrimidine biosynthesis and of dihydroorotate dehydrogenase (DHODH) in particular. The CPA can differentiate between impairment of pyrimidine and folate metabolism, which both affect cellular nucleotide pools. The identified morphological signature is shared by inhibitors of DHODH and the functionally tightly coupled complex III of the mitochondrial respiratory chain as well as by UMP synthase, which is downstream of DHODH. The CPA appears to be particularly suited for the detection of DHODH inhibitors at the site of their action in cells. As DHODH is a validated therapeutic target, the CPA will enable unbiased identification of DHODH inhibitors and inhibitors of de novo pyrimidine biosynthesis for biological research and drug discovery.

Illuminating Dark Chemical Matter Using the Cell Painting Assay

“…CPA identified DCM compound 13 as a potential inhibitor of de novo pyrimidine biosynthesis as it displayed 84% similarity to the pyrimidine synthesis cluster (Figure A–C). As recently reported, this cluster contains modulators of enzymes in pyrimidine biosynthesis like dihydroorotate dehydrogenase (DHODH) and UMP synthase (UMPS) as well as inhibitors of mitochondrial complex III …”

Scaffold Remodelling of Diazaspirotricycles Enables Synthesis of Diverse sp3‐Rich Compounds With Distinct Phenotypic Effects

“…The fingerprints for the compounds with the highest induction value from each of these clusters ( 25 , 15 b , 23 and 20 ) are displayed as heatmap profiles in Figure 3 (Panel C, left). Crucially, pairwise comparison, based on Pearson correlation, revealed that the morphological fingerprints for these compounds have low biosimilarity (Panel C, right) (fingerprints are typically considered similar if biosimilarity >75 % [27] ). These results suggest that our top‐down synthetic approach can deliver sets of screening compounds are functionally (as well as structurally) diverse [28]…”

Collective Synthesis of Sarpagine and Macroline Alkaloid‐Inspired Compounds

“…High similarities to bioactivity cluster profiles, i. e. >80 %, suggest similar modes of action and thereby facilitate the annotation of uncharacterized compounds without prior knowledge of the top biosimilar reference compounds. So far, 12 bioactivity cluster profiles have been defined that include biological targets related to AKT/PI3K/MTOR, Aurora kinases, BET, DNA synthesis, HDAC, HSP90, Na + /K + ATPases, [36] lysosomotropism/cholesterol homeostasis regulation (LCH), protein synthesis, pyrimidine biosynthesis, [37] tubulin, and uncoupling of the mitochondrial proton gradient.…”