Pyrimidines are essential for the cell survival and proliferation of living parasitic organisms, such as Helicobacter pylori, Plasmodium falciparum and Schistosoma mansoni, that are able to impact ...upon human health. Pyrimidine building blocks, in human cells, are synthesised via both de novo biosynthesis and salvage pathways, the latter of which is an effective way of recycling pre-existing nucleotides. As many parasitic organisms lack pyrimidine salvage pathways for pyrimidine nucleotides, blocking de novo biosynthesis is seen as an effective therapeutic means to selectively target the parasite without effecting the human host. Dihydroorotate dehydrogenase (DHODH), which is involved in the de novo biosynthesis of pyrimidines, is a validated target for anti-infective drug research. Recent advances in the DHODH microorganism field are discussed herein, as is the potential for the development of DHODH-targeted therapeutics.
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•Blocking biosynthesis of pyrimidines is a target for some parasitic diseases.•Dihydroorotate dehydrogenase is involved in the de novo biosynthesis of pyrimidines.•DHODH as a potential therapeutic target for many microorganisms is here discussed.
Human dihydroorotate dehydrogenase (hDHODH) is an enzyme belonging to a flavin mononucleotide (FMN)-dependent family involved in de novo pyrimidine biosynthesis, a key biological pathway for highly ...proliferating cancer cells and pathogens. In fact, hDHODH proved to be a promising therapeutic target for the treatment of acute myelogenous leukemia, multiple myeloma, and viral and bacterial infections; therefore, the identification of novel hDHODH ligands represents a hot topic in medicinal chemistry. In this work, we reported a virtual screening study for the identification of new promising hDHODH inhibitors. A pharmacophore-based approach combined with a consensus docking analysis and molecular dynamics simulations was applied to screen a large database of commercial compounds. The whole virtual screening protocol allowed for the identification of a novel compound that is endowed with promising inhibitory activity against hDHODH and is structurally different from known ligands. These results validated the reliability of the in silico workflow and provided a valuable starting point for hit-to-lead and future lead optimization studies aimed at the development of new potent hDHODH inhibitors.
The assignment of structure by tandem mass spectrometry (MS/MS) relies on the interpretation of the fragmentation behavior of gas‐phase ions. Mass spectra were acquired for a series of heterocyclic ...mimetics of acidic amino acids and a related series of nitrile amino acids. All amino acids were readily protonated or deprotonated by electrospray ionization (ESI), and distinctive fragmentation processes were observed when the ions were subjected to collision‐induced dissociation (CID). The deprotonated heterocycles showed bond cleavages of the 3‐hydroxyfurazan ring with formation of oxoisocyanate and the complementary deprotonated nitrile amino acid. Further fragmentation of the deprotonated nitrile amino acids was greatly dependent on the length of the alkyl nitrile side chain. Competing losses of CO2 versus HCN occurred from α‐cyanoglycinate (shortest chain), whereas water was lost from 2‐amino‐5‐cyanopentanoate (longest chain). Interestingly, loss of acrylonitrile by a McLafferty‐type fragmentation process was detected for 2‐amino‐4‐cyanobutanoate, and several competing processes were observed for β‐cyanoalanate. In one process, cyanide ion was formed either by consecutive losses of ammonia, carbon dioxide, and acetylene or by a one‐step decarboxylative elimination. In another, complementary ions were obtained from β‐cyanoalanate by loss of acetonitrile or HN=CHCO2H. Fragmentation of the protonated 3‐hydroxyfurazan and nitrile amino acids resulted in the cumulative loss (H2O + CO), a loss that is commonly observed for protonated aliphatic α‐amino acids. Overall, the distinct fragmentation behavior of the multifunctional 3‐hydroxyfurazan amino acids correlated with the charged site, whereas fragmentations of the deprotonated nitrile amino acids showed cooperative interactions between the nitrile and the carboxylate groups.
The identification of different compound series with corresponding structure–activity relationship (SAR) progression for a given target is referred to as SAR transfer, which is of interest in lead ...optimization. If difficulties are encountered during multiproperty optimization, the SAR transfer concept can be applied attempting to replace a lead compound with another candidate. For a systematic assessment of SAR transfer, computational approaches are required. So far, SAR transfer has been investigated at the level of compounds and analogue series. Herein, we introduce a new computational method for structure-guided exploration of SAR transfer. The approach relies on a three-dimensional molecular fragmentation and recombination scheme and the identification of analogues of crystallographic ligands. On the basis of spatially aligned X-ray ligands, alternative substituents and compound cores are identified, enabling the detection of multiple SAR transfer events. Application of the methodology across different targets identified SAR transfer events with high frequency.
Mycobacterium tuberculosis (MTB) is the etiologic agent of tuberculosis (TB), an ancient disease which causes 1.5 million deaths worldwide. Dihydroorotate dehydrogenase (DHODH) is a key enzyme of the ...MTB de novo pyrimidine biosynthesis pathway, and it is essential for MTB growth in vitro, hence representing a promising drug target. We present: (i) the biochemical characterization of the full‐length MTB DHODH, including the analysis of the kinetic parameters, and (ii) the previously unreleased crystal structure of the protein that allowed us to rationally screen our in‐house chemical library and identify the first selective inhibitor of mycobacterial DHODH. The inhibitor has fluorescence properties, potentially instrumental to in cellulo imaging studies, and exhibits an IC50 value of 43 μm, paving the way to hit‐to‐lead process.
Dihydroorotate dehydrogenase (DHODH) is a key enzyme of the pyrimidine biosynthesis pathway in Mycobacterium tuberculosis. Annotated as an essential gene for mycobacterial growth, it represents a potential drug target. Here, we present the crystal structure of the full‐length DHODH along with its biochemical characterization. Structural analysis has driven the screening for the identification of the first inhibitor of mycobacterial DHODH.
Emergence of drug resistance and adverse effects often affect the efficacy of nucleoside analogues in the therapy of Herpes simplex type 1 (HSV-1) and type 2 (HSV-2) infections. Host-targeting ...antivirals could therefore be considered as an alternative or complementary strategy in the management of HSV infections. To contribute to this advancement, here we report on the ability of a new generation inhibitor of a key cellular enzyme of de novo pyrimidine biosynthesis, the dihydroorotate dehydrogenase (DHODH), to inhibit HSV-1 and HSV-2 in vitro replication, with a potency comparable to that of the reference drug acyclovir. Analysis of the HSV replication cycle in MEDS433-treated cells revealed that it prevented the accumulation of viral genomes and reduced late gene expression, thus suggesting an impairment at a stage prior to viral DNA replication consistent with the ability of MEDS433 to inhibit DHODH activity. In fact, the anti-HSV activity of MEDS433 was abrogated by the addition of exogenous uridine or of the product of DHODH, the orotate, thus confirming DHODH as the MEDS433 specific target in HSV-infected cells. A combination of MEDS433 with dipyridamole (DPY), an inhibitor of the pyrimidine salvage pathway, was then observed to be effective in inhibiting HSV replication even in the presence of exogenous uridine, thus mimicking in vivo conditions. Finally, when combined with acyclovir and DPY in checkerboard experiments, MEDS433 exhibited highly synergistic antiviral activity. Taken together, these findings suggest that MEDS433 is a promising candidate as either single agent or in combination regimens with existing direct-acting anti-HSV drugs to develop new strategies for treatment of HSV infections.
•We characterize the anti-HSV activity of the new DHODH inhibitor MEDS433 that targets the de novo pyrimidine biosynthesis.•MEDS433 impairs the in vitro replication of HSV-1 and HSV-2 in the nanomolar range.•The anti-HSV activity of MEDS433 is reversed by uracil and orotic acid confirming that it derives from inhibition of DHODH.•The simultaneous treatment with MEDS433 and acyclovir potentiates the anti-HSV activity of the combination.•MEDS433 differs from current anti-HSV drugs, and may represents a promising candidate for new antiviral strategies.
Human dihydroorotate dehydrogenase (hDHODH) catalyzes the rate-limiting step in de novo pyrimidine biosynthesis, the conversion of dihydroorotate to orotate. hDHODH has recently been found to be ...associated with acute myelogenous leukemia, a disease for which the standard of intensive care has not changed over decades. This work presents a novel class of hDHODH inhibitors, which are based on an unusual carboxylic group bioisostere 2-hydroxypyrazolo1,5-apyridine, that has been designed starting from brequinar, one of the most potent hDHODH inhibitors. A combination of structure-based and ligand-based strategies produced compound 4, which shows brequinar-like hDHODH potency in vitro and is superior in terms of cytotoxicity and immunosuppression. Compound 4 also restores myeloid differentiation in leukemia cell lines at concentrations that are one log digit lower than those achieved in experiments with brequinar. This Article reports the design, synthesis, SAR, X-ray crystallography, biological assays, and physicochemical characterization of the new class of hDHODH inhibitors.
Human dihydroorotate dehydrogenase (hDHODH, EC 1.3.5.2), a flavindependent mitochondrial enzyme involved in de novo pyrimidine biosynthesis, is a validated therapeutic target for the treatment of ...autoimmune diseases, such as rheumatoid arthritis and multiple sclerosis. However, human DHODH inhibitors have also been investigated as treatment for cancer, parasite infections (i.e. malaria) and viruses as well as in the agrochemicals industry.
An overview of current knowledge of hDHODH inhibitors and their potential uses in diseases where hDHODH is involved.
This review focuses on recent advances in the development and application of hDHODH inhibitors, specifically covering the patent field, starting from a brief description of enzyme topography and of the strategies usually followed in designing its selective inhibitors.
The most important and well-described novelty is the fact that the discovery, in the autumn of 2016, that hDHODH inhibitors are able to induce in vivo myeloid differentiation has led to the possibility of developing novel hDHODH based treatments for Acute Myelogenous Leukemia (AML).
The review will describe a variety of specific inhibitor classes and conclude on recent and future therapeutic perspectives for this target.
Human respiratory syncytial virus (RSV) is an important cause of acute lower respiratory infections, for which no effective drugs are currently available. The development of new effective anti-RSV ...agents is therefore an urgent priority, and Host-Targeting Antivirals (HTAs) can be considered to target RSV infections. As a contribution to this antiviral avenue, we have characterized the molecular mechanisms of the anti-RSV activity of MEDS433, a new inhibitor of human dihydroorotate dehydrogenase (hDHODH), a key cellular enzyme of de novo pyrimidine biosynthesis. MEDS433 was found to exert a potent antiviral activity against RSV-A and RSV-B in the one-digit nanomolar range. Analysis of the RSV replication cycle in MEDS433-treated cells, revealed that the hDHODH inhibitor suppressed the synthesis of viral genome, consistently with its ability to specifically target hDHODH enzymatic activity. Then, the capability of MEDS433 to induce the expression of antiviral proteins encoded by Interferon-Stimulated Genes (ISGs) was identified as a second mechanism of its antiviral activity against RSV. Indeed, MEDS433 stimulated secretion of IFN-β and IFN-λ1 that, in turn, induced the expression of some ISG antiviral proteins, such as IFI6, IFITM1 and IRF7. Singly expression of these ISG proteins reduced RSV-A replication, thus likely contributing to the overall anti-RSV activity of MEDS433. Lastly, MEDS433 proved to be effective against RSV-A replication even in a primary human small airway epithelial cell model. Taken as a whole, these observations provide new insights for further development of MEDS433, as a promising candidate to develop new strategies for treatment of RSV infections.
•We examine the mechanisms of the anti-RSV activity of the new hDHODH inhibitor MEDS433 that targets pyrimidine biosynthesis.•MEDS433 inhibits the replication of RSV-A and RSV-B in the one-digit nanomolar range by impairing virus genome synthesis.•The anti-RSV activity of MEDS433 is reversed by uracil and orotic acid confirming that it derives from inhibition of hDHODH.•MEDS433 induces the expression of antiviral proteins encoded by Interferon Stimulated Genes able to reduce RSV replication.•The simultaneous treatment with MEDS433 and dipyridamole or ribavirin potentiates the anti-RSV activity of the combinations.
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•Triazolopyrimidines, isoxazolopyrimidines and pyrrole-based (DSM compounds) PfDHODH inhibitors emerged as antimalarial agents.•This review paper highlighted all the synthetic ...approaches used for the synthesis of DSM compounds.•Synthetic schemes along with various catalysts and chemical reagents used for the synthesis of DSM compounds are depicted here.•SAR studies are discussed with the effects of various substitutions at different positions in DSM compounds.•This review highlighted the optimization of DSM compounds with improved potency, selectivity, and metabolic stability.
One of the deadliest infectious diseases, malaria, still has a significant impact on global morbidity and mortality. Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) catalyzes the fourth step in de novo pyrimidine nucleotide biosynthesis and has been clinically validated as an innovative and promising target for the development of novel targeted antimalarial drugs. PfDHODH inhibitors have the potential to significantly slow down parasite growth at the blood and liver stages. Several PfDHODH inhibitors based on various scaffolds have been explored over the past two decades. Among them, triazolopyrimidines, isoxazolopyrimidines, and pyrrole-based derivatives known as DSM compounds showed tremendous potential as novel antimalarial agents, and one of the triazolopyrimidine-based compounds (DSM265) was able to reach phase IIa clinical trials. DSM compounds were synthesized as PfDHODH inhibitors with various substitutions based on structure-guided medicinal chemistry approaches and further optimised as well. For the first time, this review provides an overview of all the synthetic approaches used for the synthesis, alternative synthetic routes, and novel strategies involving various catalysts and chemical reagents that have been used to synthesize DSM compounds. We have also summarized SAR study of all these PfDHODH inhibitors. In an attempt to assist readers, scientists, and researchers involved in the development of new PfDHODH inhibitors as antimalarials, this review provides accessibility of all synthetic techniques and SAR studies of the most promising triazolopyrimidines, isoxazolopyrimidines, and pyrrole-based PfDHODH inhibitors.
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