Original 2-(3-Alkoxy-1H-pyrazol-1-yl)azines Inhibitors of Human Dihydroorotate Dehydrogenase (DHODH)

Abstract

Following our discovery of human dihydroorotate dehydrogenase (DHODH) inhibition by 2-(3-alkoxy-1H-pyrazol-1-yl)pyrimidine derivatives as well as 2-(4-benzyl-3-ethoxy-5-methyl-1H-pyrazol-1-yl)-5-methylpyridine, we describe here the syntheses and evaluation of an array of azine-bearing analogues. As in our previous report, the structure–activity study of this series of human DHODH inhibitors was based on a phenotypic assay measuring measles virus replication. Among other inhibitors, this round of syntheses and biological evaluation iteration led to the highly active 5-cyclopropyl-2-(4-(2,6-difluorophenoxy)-3-isopropoxy-5-methyl-1H-pyrazol-1-yl)-3-fluoropyridine. Inhibition of DHODH by this compound was confirmed in an array of in vitro assays, including enzymatic tests and cell-based assays for viral replication and cellular growth. This molecule was found to be more active than the known inhibitors of DHODH, brequinar and teriflunomide, thus opening perspectives for its use as a tool or for the design of an original series of immunosuppressive agent. Moreover, because other series of inhibitors of human DHODH have been found to also affect Plasmodium falciparum DHODH, all the compounds were assayed for their effect on P. falciparum growth. However, the modest in vitro inhibition solely observed for two compounds did not correlate with their inhibition of P. falciparum DHODH.

Original Chemical Series of Pyrimidine Biosynthesis Inhibitors That Boost the Antiviral Interferon Response

“…Most importantly, we characterized in detail the interactions between this new series of pyrimidine biosynthesis inhibitors, which were identified in an ISRE-luciferase screen, and the innate antiviral response. It has been previously shown that pyrimidine biosynthesis inhibitors are weak stimulators of ISGs when applied alone ( 8 , 13 – 18 ), but these drugs boost the expression of ISGs in response to either extracellular or intracellular stimuli, such as IFN-β, RIG-I ligands, or viruses ( 8 , 12 , 13 , 15 , 18 ). Besides, pyrimidine biosynthesis inhibitors do not induce any type of IFNs when applied alone, and virus growth inhibition by these drugs seems to occur independent of IFN-α/β synthesis ( 8 , 15 , 16 ), despite some recent report showing no antiviral effects in Vero cells which are deleted for the IFN-α/β gene cluster ( 18 ).…”

“…The T7 transcription start is located 36 nucleotides upstream of the Xba1 restriction site, but runoff transcription in vitro is known for producing at a high yield longer 5′-triphosphate RNA molecules that include copy-back sequences and fold into stem-loop structures ( 33 ). Such ssRNA molecules were previously shown to efficiently stimulate the interferon response by the RIG-I/MAVS pathway when transfected in HEK-293 or STING-37 cells ( 8 , 12 , 22 , 34 ). For each production lot, pools of in vitro transcripts were purified with a filtering membrane (SEQ96 sequencing reaction cleanup; Millipore), analyzed on a Bioanalyzer RNA Nano kit (Agilent), and qualified for their immunostimulatory properties on STING-37 reporter cells.…”

“…In the presence of pyrimidine biosynthesis inhibitors, cellular pools of pyrimidines collapse, and the lack of pyrimidine is usually considered to be directly responsible for the inhibition of viral growth. However, it was also reported that inhibiting de novo pyrimidine biosynthesis stimulates the innate immune response, in particular the transcription of some interferon-stimulated genes (ISGs) independently of interferons (IFNs) and the canonical JAK-STAT pathway ( 8 , 12 – 18 ). In addition, the antiviral activity of pyrimidine biosynthesis inhibitors was found to be strictly dependent on cellular gene transcription and nuclear export machinery and required interferon regulatory factor 1 (IRF1), a key transcription factor driving the expression of antiviral genes, including ISGs ( 8 ).…”

Replication of Equine arteritis virus is efficiently suppressed by purine and pyrimidine biosynthesis inhibitors

“…Ribavirin and uridine were from Sigma-Aldrich. IPPA17-A04 and GAC50 were previously described and kindly provided by Dr. Yves Janin (Institut Pasteur, Paris, France) and Dr. Daniel Dauzonne (Institut Curie, Paris, France), respectively 25-27 . Ribavirin was dissolved in PBS buffer and uridine in sterile water, whereas IPPA17-A04 and GAC50 were dissolved in DMSO.…”

“…Besides ribavirin, we also tested two newly designed BSA that target the pyrimidine biosynthesis pathway, namely GAC50 and IPPA17-A04. These two molecules are inhibitors of dihydroorotate dehydrogenase (DHODH), the fourth and rate-limiting enzyme of the de novo pyrimidine biosynthesis pathway, and were shown to impair the replication of many different positive-strand and negative-strand RNA viruses 25-27 . In this study, we have shown, for the first time, that both ribavirin and pyrimidine biosynthesis inhibitors inhibit EAV in equine dermal cell cultures.…”