Dihydroorotate dehydrogenase (DHODH) is the key enzyme in de novo biosynthesis of pyrimidine in both prokaryotes and eukaryotes. The de novo pathway of pyrimidine biosynthesis is essential in cancer cells proliferation. Leflunomide is an approved DHODH inhibitor that has been widely used for the treatment of arthritis. Similarly, brequinar sodium is another DHODH inhibitor that showed anti-tumour effect in MC38 colon carcinoma cells when used in combination with fluorouracil. Despite the potential role of DHODH inhibitors in cancer therapy, their mechanisms of action remain obscure and await further elucidation. Here, we evaluated the effect of DHODH inhibitors on the production of ATP and ROS in sensitive and non-sensitive breast cancer cells. Subsequently, the effects of DHODH inhibitors on cell cycle as well as on signalling molecules such as p53, p65 and STAT6 were evaluated in sensitive T-47D and non-sensitive MDAMB-436 cells. The correlations between DHODH protein expression, proliferation speed and sensitivity to DHODH inhibitors were also investigated in a panel of cancer cell lines. DHODH inhibitors-sensitive T-47D and MDAMB-231 cells appeared to preserve ROS production closely to endogenous ROS level whereas the opposite was observed in non-sensitive MDAMB-436 and W3.006 cells. In addition, we observed approximately 90% of intracellular ATP depletion in highly sensitive T-47D and MDAMB-231 cells compared to non-sensitive MDAMB-436 cells. There was significant over-expression of p53, p65 and STAT6 signalling molecules in sensitive cells which may be involved in mediating the S-phase arrest in cell cycle progression. The current study suggests that DHODH inhibitors are most effective in cells that express high levels of DHODH enzyme. The inhibition of cell proliferation by these inhibitors appears to be accompanied by ROS production as well as ATP depletion. The increase in expression of signalling molecules observed may be due to pyrimidine depletion which subsequently leads to cell cycle arrest at S-phase.
Various clinical symptoms are caused by dengue virus ranging from mild fever to severe hemorrhagic fever while there is no successful anti-dengue therapeutics available. Among different strategies towards identifying and developing anti-dengue therapeutics, testing anti-dengue properties of known drugs could represent an efficient strategy for which information of its medical approval, toxicity and side effects is readily available. In this study, we evaluated the antiviral activity of some medical compounds towards dengue NS2B-NS3 protease (DENV2 NS2B-NS3pro) as a target to inhibit dengue virus replication. Mefenamic acid, a non-steroid anti inflammatory drug and doxycycline, a derivative antibiotic of tetracycline both showed significant inhibition potential against DENV2 NS2B-NS3pro Ki values 32 ± 2 μM and 55 ± 5 μM respectively. The effective cytotoxic concentrations of 50% (CC50) against Vero cells were evaluated for mefenamic acid (150 ± 5 μM) and doxycycline (125 ± 4 μM). Concentrations lower than CC50 were used to test the inhibition potential of these compounds against DENV2 replication in Vero cells. The results showed significant reduction in viral load after applying mefenamic acid and doxycyline in concentration dependent manner. Mefenamic acid reduced viral RNA at EC50 of 32 ± 4 μM whilst doxycycline EC50 was 40 ± 3 μM. Mefenamic acid showed higher selectivity against dengue virus replication in vitro compared to doxycycline. These findings underline the need for further experimental and clinical studies on these drugs utilizing its anti-dengue and anti-inflammatory activities to attenuate the clinical symptoms of dengue infection.