Structural and Functional Analyses of Inhibition of Human Dihydroorotate Dehydrogenase by Antiviral Furocoumavirin.

Natural products are important sources of seed compounds for drug discovery. However, it has become difficult in recent years to discover new compounds with valuable pharmacological activities. On the other hand, among the vast number of natural products that have been isolated so far, a considerable number of compounds with specific biological activities are thought to be overlooked in screening that uses biological activity as an index. Therefore, it is conceivable that such overlooked useful compounds may be found by screening compound libraries that have been amassed previously through specific assays. Previously, NPD723, a member of the Natural Products Depository library comprised of a mixture of natural and non-natural products developed at RIKEN, and its metabolite H-006 were found to inhibit growth of various cancer cells at low nanomolar half-maximal inhibitory concentration. Subsequent analysis revealed that H-006 strongly inhibited human dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme in the de novo pyrimidine biosynthetic pathway. Here, we elucidated the crystal structure of the DHODH-flavin mononucleotide-orotic acid-H-006 complex at 1.7 Å resolution to determine that furocoumavirin, the S-enantiomer of H-006, was the actual inhibitor. The overall mode of interaction of furocoumavirin with the inhibitor binding pocket was similar to that described for previously reported tight-binding inhibitors. However, the structural information together with kinetic characterizations of site-specific mutants identified key unique features that are considered to contribute to the sub-nanomolar inhibition of DHODH by furocoumavirin. Our finding identified new chemical features that could improve the design of human DHODH inhibitors.

In Silico Comparison of Bioactive Compounds Characterized from Azadirachta indica with an FDA-Approved Drug against Schistosomal Agents: New Insight into Schistosomiasis Treatment.

The burden of human schistosomiasis, a known but neglected tropical disease in Sub-Saharan Africa, has been worrisome in recent years. It is becoming increasingly difficult to tackle schistosomiasis with praziquantel, a drug known to be effective against all Schistosoma species, due to reports of reduced efficacy and resistance. Therefore, this study seeks to investigate the antischistosomal potential of phytochemicals from Azadirachta indica against proteins that have been implicated as druggable targets for the treatment of schistosomiasis using computational techniques. In this study, sixty-three (63) previously isolated and characterized phytochemicals from A. indica were identified from the literature and retrieved from the PubChem database. In silico screening was conducted to assess the inhibitory potential of these phytochemicals against three receptors (Schistosoma mansoni Thioredoxin glutathione reductase, dihydroorotate dehydrogenase, and Arginase) that may serve as therapeutic targets for schistosomiasis treatment. Molecular docking, ADMET prediction, ligand interaction, MMGBSA, and molecular dynamics simulation of the hit compounds were conducted using the Schrodinger molecular drug discovery suite. The results show that Andrographolide possesses a satisfactory pharmacokinetic profile, does not violate the Lipinski rule of five, binds with favourable affinity with the receptors, and interacts with key amino acids at the active site. Importantly, its interaction with dihydroorotate dehydrogenase, an enzyme responsible for the catalysis of the de novo pyrimidine nucleotide biosynthetic pathway rate-limiting step, shows a glide score and MMGBSA of -10.19 and -45.75 Kcal/mol, respectively. In addition, the MD simulation shows its stability at the active site of the receptor. Overall, this study revealed that Andrographolide from Azadirachta indica could serve as a potential lead compound for the development of an anti-schistosomal drug.

Elucidation of the Catalytic Sequence of Dihydroorotate Dehydrogenase B from Lactoccocus lactis: Evidence for Accumulation of a Flavin Bisemiquinone State in Catalysis.

The physiological role of dihydroorotate dehydrogenase (DHOD) enzymes is to catalyze the oxidation of dihydroorotate to orotate in pyrimidine biosynthesis. DHOD enzymes are structurally diverse existing as both soluble and membrane-associated forms. The Family 1 enzymes are soluble and act either as conventional single subunit flavin-dependent dehydrogenases known as Class 1A (DHODA) or as unusual heterodimeric enzymes known as Class 1B (DHODB). DHODBs possess two active sites separated by ∼20 Å, each with a noncovalently bound flavin cofactor. NAD is thought to interact at the FAD containing site, and the pyrimidine substrate is known to bind at the FMN containing site. At the approximate center of the protein is a single Fe2S2 center that is assumed to act as a conduit, facilitating one-electron transfers between the flavins. We present anaerobic transient state analysis of a DHODB enzyme from Lactoccocus lactis. The data presented primarily report the exothermic reaction that reduces orotate to dihydroorotate. The reductive half reaction reveals rapid two-electron reduction that is followed by the accumulation of a four-electron reduced state when NADH is added in excess, suggesting that the initial two electrons acquired reside on the FMN cofactor. Concomitant with the first reduction is the accumulation of a long-wavelength absorption feature consistent with the blue form of a flavin semiquinone. Spectral deconvolution and fitting to a model that includes reversibility for the second electron transfer reveals equilibrium accumulation of a flavin bisemiquinone state that has features of both red and blue semiquinones. Single turnover reactions with limiting NADH and excess orotate reveal that the flavin bisemiquinone accumulates with reduction of the enzyme by NADH and decays with reduction of the pyrimidine substrate, establishing the bisemiquinone as a fractional state of the two-electron reduced intermediate observed.

Discovery of a new class of potent pyrrolo[3,4-c]quinoline-1,3-diones based inhibitors of human dihydroorotate dehydrogenase: Synthesis, pharmacological and toxicological evaluation.

Twenty N-substituted pyrrolo[3,4-c]quinoline-1,3-diones 3a-t were synthesized by a cyclization reaction of Pfitzinger's quinoline ester precursor with the selected aromatic, heteroaromatic and aliphatic amines. The structures of all derivatives were confirmed by IR, 1H NMR, 13C NMR and HRMS spectra, while their purity was determined using HPLC techniques. Almost all compounds were identified as a new class ofpotent inhibitors against hDHODH among which 3a and 3t were the most active ones with the same IC50 values of 0.11 µM, about seven times better than reference drug leflunomide. These two derivatives also exhibited very low cytotoxic effects toward healthy HaCaT cells and the optimal lipophilic properties with logP value of 1.12 and 2.07 respectively, obtained experimentally at physiological pH. We further evaluated the comparative differences in toxicological impact of the three most active compounds 3a, 3n and 3t and reference drug leflunomide. The rats were divided into five groups and were treated intraperitoneally, control group (group I) with a single dose of leflunomide (20 mg/kg) group II and the other three groups, III, IV and V were treated with 3a, 3n and 3t (20 mg/kg bw) separately. The investigation was performed in liver, kidney and blood by examining serum biochemical parameters and parameters of oxidative stress.

DHCR7 links cholesterol synthesis with neuronal development and axonal integrity.

The DHCR7 enzyme converts 7-DHC into cholesterol. Mutations in DHCR7 can block cholesterol production, leading to abnormal accumulation of 7-DHC and causing Smith-Lemli-Opitz syndrome (SLOS). SLOS is an autosomal recessive disorder characterized by multiple malformations, including microcephaly, intellectual disability, behavior reminiscent of autism, sleep disturbances, and attention-deficit/hyperactivity disorder (ADHD)-like hyperactivity. Although 7-DHC affects neuronal differentiation in ex vivo experiments, the precise mechanism of SLOS remains unclear. We generated Dhcr7 deficient (dhcr7-/-) zebrafish that exhibited key features of SLOS, including microcephaly, decreased neural stem cell pools, and behavioral phenotypes similar to those of ADHD-like hyperactivity. These zebrafish demonstrated compromised myelination, synaptic anomalies, and neurotransmitter imbalances. The axons of the dhcr7-/- zebrafish showed increased lysosomes and attenuated autophagy, suggesting that autophagy-related neuronal homeostasis is disrupted.

Inhibition of 7-dehydrocholesterol reductase prevents hepatic ferroptosis under an active state of sterol synthesis.

Recent evidence indicates ferroptosis is implicated in the pathophysiology of various liver diseases; however, the organ-specific regulation mechanism is poorly understood. Here, we demonstrate 7-dehydrocholesterol reductase (DHCR7), the terminal enzyme of cholesterol biosynthesis, as a regulator of ferroptosis in hepatocytes. Genetic and pharmacological inhibition (with AY9944) of DHCR7 suppress ferroptosis in human hepatocellular carcinoma Huh-7 cells. DHCR7 inhibition increases its substrate, 7-dehydrocholesterol (7-DHC). Furthermore, exogenous 7-DHC supplementation using hydroxypropyl ß-cyclodextrin suppresses ferroptosis. A 7-DHC-derived oxysterol metabolite, 3ß,5α-dihydroxycholest-7-en-6-one (DHCEO), is increased by the ferroptosis-inducer RSL-3 in DHCR7-deficient cells, suggesting that the ferroptosis-suppressive effect of DHCR7 inhibition is associated with the oxidation of 7-DHC. Electron spin resonance analysis reveals that 7-DHC functions as a radical trapping agent, thus protecting cells from ferroptosis. We further show that AY9944 inhibits hepatic ischemia-reperfusion injury, and genetic ablation of Dhcr7 prevents acetaminophen-induced acute liver failure in mice. These findings provide new insights into the regulatory mechanism of liver ferroptosis and suggest a potential therapeutic option for ferroptosis-related liver diseases.

Identification and validation of DHCR7 as a diagnostic biomarker involved in the proliferation and mitochondrial function of breast cancer.

BACKGROUND: Energy metabolism has a complex intersection with pathogenesis and development of breast cancer (BC). This allows for the possibility of identifying energy-metabolism-related genes (EMRGs) as novel prognostic biomarkers for BC. 7-dehydrocholesterol reductase (DHCR7) is a key enzyme of cholesterol biosynthesis involved in many cancers, and in this paper, we investigate the effects of DHCR7 on the proliferation and mitochondrial function of BC. METHODS: EMRGs were identified from the Gene Expression Omnibus (GEO) and MSigDB databases using bioinformatics methods. Key EMRGs of BC were then identified and validated by functional enrichment analysis, interaction analysis, weighted gene co-expression network analysis (WGCNA), least absolute shrinkage and selection operator (LASSO) regression, Cox analysis, and immune infiltration. Western blot, qRT-PCR, immunohistochemistry (IHC), MTT assay, colony formation assay and flow cytometry assay were then used to analyze DHCR7 expression and its biological effects on BC cells. RESULTS: We identified 31 EMRGs in BC. These 31 EMRGs and related transcription factors (TFs), miRNAs, and drugs were enriched in glycerophospholipid metabolism, glycoprotein metabolic process, breast cancer, and cell cycle. Crucially, DHCR7 was a key EMRG in BC identified and validated by WGCNA, LASSO regression and receiver operating characteristic (ROC) curve analysis. High DHCR7 expression was significantly associated with tumor immune infiltration level, pathological M, and poor prognosis in BC. In addition, DHCR7 knockdown inhibited cell proliferation, induced apoptosis and affected mitochondrial function in BC cells. CONCLUSIONS: DHCR7 was found to be a key EMRG up-regulated in BC cells. This study is the first to our knowledge to report that DHCR7 acts as an oncogene in BC, which might become a novel therapeutic target for BC patients.

Fluorination of a conserved tyrosine in POR offers new clues for proton transfer.

Reduction of the 17,18-double bond in the D-ring during chlorophyll biosynthesis is catalyzed by the rare, naturally occurring photoenzyme protochlorophyllide oxidoreductase (POR). A conserved tyrosine residue has been suggested to donate a proton to C18 of the substrate in the past decades. Taylor and colleagues scrutinized the model with a powerful tool that utilized a modified genetic code to introduce fluorinated tyrosine analogues into POR. The presented results show that the suggested catalytically critical tyrosine is unlikely to participate in the reaction chemistry but is required for substrate binding, and instead, a cysteine residue preceding the lid helix is proposed to have the role of proton donor.

DHCR24 Insufficiency Promotes Vascular Endothelial Cell Senescence and Endothelial Dysfunction via Inhibition of Caveolin-1/ERK Signaling.

Endothelial cells (ECs) senescence is critical for vascular dysfunction, which leads to age-related disease. DHCR24, a 3ß-hydroxysterol δ 24 reductase with multiple functions other than enzymatic activity, has been involved in age-related disease. However, little is known about the relationship between DHCR24 and vascular ECs senescence. We revealed that DHCR24 expression is chronologically decreased in senescent human umbilical vein endothelial cells (HUVECs) and the aortas of aged mice. ECs senescence in endothelium-specific DHCR24 knockout mice was characterized by increased P16 and senescence-associated secretory phenotype, decreased SIRT1 and cell proliferation, impaired endothelium-dependent relaxation, and elevated blood pressure. In vitro, DHCR24 knockdown in young HUVECs resulted in a similar senescence phenotype. DHCR24 deficiency impaired endothelial migration and tube formation and reduced nitric oxide (NO) levels. DHCR24 suppression also inhibited the caveolin-1/ERK signaling, probably responsible for increased reactive oxygen species production and decreased eNOS/NO. Conversely, DHCR24 overexpression enhanced this signaling pathway, blunted the senescence phenotype, and improved cellular function in senescent cells, effectively blocked by the ERK inhibitor U0126. Moreover, desmosterol accumulation induced by DHCR24 deficiency promoted HUVECs senescence and inhibited caveolin-1/ERK signaling. Our findings demonstrate that DHCR24 is essential in ECs senescence.

Neuroprotective Roles of the Biliverdin Reductase-A/Bilirubin Axis in the Brain.

Biliverdin reductase-A (BVRA) is a multi-functional enzyme with a multitude of important roles in physiologic redox homeostasis. Classically, BVRA is well known for converting the heme metabolite biliverdin to bilirubin, which is a potent antioxidant in both the periphery and the brain. However, BVRA additionally participates in many neuroprotective signaling cascades in the brain that preserve cognition. Here, we review the neuroprotective roles of BVRA and bilirubin in the brain, which together constitute a BVRA/bilirubin axis that influences healthy aging and cognitive function.

Discovery of Nanomolar Inhibitors for Human Dihydroorotate Dehydrogenase Using Structure-Based Drug Discovery Methods.

We used a structure-based drug discovery approach to identify novel inhibitors of human dihydroorotate dehydrogenase (DHODH), which is a therapeutic target for treating cancer and autoimmune and inflammatory diseases. In the case of acute myeloid leukemia, no previously discovered DHODH inhibitors have yet succeeded in this clinical application. Thus, there remains a strong need for new inhibitors that could be used as alternatives to the current standard-of-care. Our goal was to identify novel inhibitors of DHODH. We implemented prefiltering steps to omit PAINS and Lipinski violators at the earliest stages of this project. This enriched compounds in the data set that had a higher potential of favorable oral druggability. Guided by Glide SP docking scores, we found 20 structurally unique compounds from the ChemBridge EXPRESS-pick library that inhibited DHODH with IC50, DHODH values between 91 nM and 2.7 µM. Ten of these compounds reduced MOLM-13 cell viability with IC50, MOLM-13 values between 2.3 and 50.6 µM. Compound 16 (IC50, DHODH = 91 nM) inhibited DHODH more potently than the known DHODH inhibitor, teriflunomide (IC50, DHODH = 130 nM), during biochemical characterizations and presented a promising scaffold for future hit-to-lead optimization efforts. Compound 17 (IC50, MOLM-13 = 2.3 µM) was most successful at reducing survival in MOLM-13 cell lines compared with our other hits. The discovered compounds represent excellent starting points for the development and optimization of novel DHODH inhibitors.

Structural insights into the Smirnoff-Wheeler pathway for vitamin C production in the Amazon fruit camu-camu.

l-Ascorbic acid (AsA, vitamin C) is a pivotal dietary nutrient with multifaceted importance in living organisms. In plants, the Smirnoff-Wheeler pathway is the primary route for AsA biosynthesis, and understanding the mechanistic details behind its component enzymes has implications for plant biology, nutritional science, and biotechnology. As part of an initiative to determine the structures of all six core enzymes of the pathway, the present study focuses on three of them in the model species Myrciaria dubia (camu-camu): GDP-d-mannose 3',5'-epimerase (GME), l-galactose dehydrogenase (l-GalDH), and l-galactono-1,4-lactone dehydrogenase (l-GalLDH). We provide insights into substrate and cofactor binding and the conformational changes they induce. The MdGME structure reveals a distorted substrate in the active site, pertinent to the catalytic mechanism. Mdl-GalDH shows that the way in which NAD+ association affects loop structure over the active site is not conserved when compared with its homologue in spinach. Finally, the structure of Mdl-GalLDH is described for the first time. This allows for the rationalization of previously identified residues which play important roles in the active site or in the formation of the covalent bond with FAD. In conclusion, this study enhances our understanding of AsA biosynthesis in plants, and the information provided should prove useful for biotechnological applications.

Mechanistic implications of the ternary complex structural models for the photoenzyme protochlorophyllide oxidoreductase.

The photoenzyme protochlorophyllide oxidoreductase (POR) is an important enzyme for understanding biological H-transfer mechanisms. It uses light to catalyse the reduction of protochlorophyllide to chlorophyllide, a key step in chlorophyll biosynthesis. Although a wealth of spectroscopic data have provided crucial mechanistic insight, a structural rationale for POR photocatalysis has proved challenging and remains hotly debated. Recent structural models of the ternary enzyme-substrate complex, derived from crystal and electron microscopy data, show differences in the orientation of the protochlorophyllide substrate and the architecture of the POR active site, with significant implications for the catalytic mechanism. Here, we use a combination of computational and experimental approaches to investigate the compatibility of each structural model with the hypothesised reaction mechanisms and propose an alternative structural model for the cyanobacterial POR ternary complex. We show that a strictly conserved tyrosine, previously proposed to act as the proton donor in POR photocatalysis, is unlikely to be involved in this step of the reaction but is crucial for Pchlide binding. Instead, an active site cysteine is important for both hydride and proton transfer reactions in POR and is proposed to act as the proton donor, either directly or through a water-mediated network. Moreover, a conserved glutamine is important for Pchlide binding and ensuring efficient photochemistry by tuning its electronic properties, likely by interacting with the central Mg atom of the substrate. This optimal 'binding pose' for the POR ternary enzyme-substrate complex illustrates how light energy can be harnessed to facilitate enzyme catalysis by this unique enzyme.

An enzyme that selectively S-nitrosylates proteins to regulate insulin signaling.

Acyl-coenzyme A (acyl-CoA) species are cofactors for numerous enzymes that acylate thousands of proteins. Here, we describe an enzyme that uses S-nitroso-CoA (SNO-CoA) as its cofactor to S-nitrosylate multiple proteins (SNO-CoA-assisted nitrosylase, SCAN). Separate domains in SCAN mediate SNO-CoA and substrate binding, allowing SCAN to selectively catalyze SNO transfer from SNO-CoA to SCAN to multiple protein targets, including the insulin receptor (INSR) and insulin receptor substrate 1 (IRS1). Insulin-stimulated S-nitrosylation of INSR/IRS1 by SCAN reduces insulin signaling physiologically, whereas increased SCAN activity in obesity causes INSR/IRS1 hypernitrosylation and insulin resistance. SCAN-deficient mice are thus protected from diabetes. In human skeletal muscle and adipose tissue, SCAN expression increases with body mass index and correlates with INSR S-nitrosylation. S-nitrosylation by SCAN/SNO-CoA thus defines a new enzyme class, a unique mode of receptor tyrosine kinase regulation, and a revised paradigm for NO function in physiology and disease.

Recent advances on patents of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors as antimalarial agents.

INTRODUCTION: Pyrimidine nucleotides are essential for the parasite's growth and replication. Parasites have only a de novo pathway for the biosynthesis of pyrimidine nucleotides. Dihydroorotate dehydrogenase (DHODH) enzyme is involved in the rate-limiting step of the pyrimidine biosynthesis pathway. DHODH is a biochemical target for the discovery of new antimalarial agents. AREA COVERED: This review discussed the development of patented PfDHODH inhibitors published between 2007 and 2023 along with their chemical structures and activities. EXPERT OPINION: PfDHODH enzyme is involved in the rate-limiting fourth step of the pyrimidine biosynthesis pathway. Thus, inhibition of PfDHODH using species-selective inhibitors has drawn much attention for treating malaria because they inhibit parasite growth without affecting normal human functions. Looking at the current scenario of antimalarial drug resistance with most of the available antimalarial drugs, there is a huge need for targeted newer agents. Newer agents with unique mechanisms of action may be devoid of drug toxicity, adverse effects, and the ability of parasites to quickly gain resistance, and PfDHODH inhibitors can be those newer agents. Many PfDHODH inhibitors were patented in the past, and the dependency of Plasmodium on de novo pyrimidine provided a new approach for the development of novel antimalarial agents.

3ß-hydroxysteroid-Δ24 reductase dampens anti-viral innate immune responses by targeting K27 ubiquitination of MAVS and STING.

IMPORTANCE: The precise regulation of the innate immune response is essential for the maintenance of homeostasis. MAVS and STING play key roles in immune signaling pathways activated by RNA and DNA viruses, respectively. Here, we showed that DHCR24 impaired the antiviral response by targeting MAVS and STING. Notably, DHCR24 interacts with MAVS and STING and inhibits TRIM21-triggered K27-linked ubiquitination of MAVS and AMFR-triggered K27-linked ubiquitination of STING, restraining the activation of MAVS and STING, respectively. Together, this study elucidates how one cholesterol key enzyme orchestrates two antiviral signal transduction pathways.

A Novel mechanism of herbicide action through disruption of pyrimidine biosynthesis.

A lead aryl pyrrolidinone anilide identified using high-throughput in vivo screening was optimized for efficacy, crop safety, and weed spectrum, resulting in tetflupyrolimet. Known modes of action were ruled out through in vitro enzyme and in vivo plant-based assays. Genomic sequencing of aryl pyrrolidinone anilide-resistant Arabidopsis thaliana progeny combined with nutrient reversal experiments and metabolomic analyses confirmed that the molecular target of the chemistry was dihydroorotate dehydrogenase (DHODH), the enzyme that catalyzes the fourth step in the de novo pyrimidine biosynthesis pathway. In vitro enzymatic and biophysical assays and a cocrystal structure with purified recombinant plant DHODH further confirmed this enzyme as the target site of this class of chemistry. Like known inhibitors of other DHODH orthologs, these molecules occupy the membrane-adjacent binding site of the electron acceptor ubiquinone. Identification of a new herbicidal chemical scaffold paired with a novel mode of action, the first such finding in over three decades, represents an important leap in combatting weed resistance and feeding a growing worldwide population.

Deciphering Interactions between Potential Inhibitors and the Plasmodium falciparum DHODH Enzyme: A Computational Perspective.

Malaria is a parasitic disease that, in its most severe form, can even lead to death. Insect-resistant vectors, insufficiently effective vaccines, and drugs that cannot stop parasitic infestations are making the fight against the disease increasingly difficult. It is known that the enzyme dihydroorotate dehydrogenase (DHODH) is of paramount importance for the synthesis of pyrimidine from the Plasmodium precursor, that is, for its growth and reproduction. Therefore, its blockade can lead to disruption of the parasite's life cycle in the vertebrate host. In this scenario, PfDHODH inhibitors have been considered candidates for a new therapy to stop the parasitic energy source. Given what is known, in this work, we applied molecular fractionation with conjugated caps (MFCC) in the framework of the quantum formalism of density functional theory (DFT) to evaluate the energies of the interactions between the enzyme and the different triazolopyrimidines (DSM483, DMS557, and DSM1), including a complex carrying the mutation C276F. From these results, it was possible to identify the main features of each system, focusing on the wild-type and mutant PfDHODH and examining the major amino acid residues that are part of the four complexes. Our analysis provides new information that can be used to develop new drugs that could prove to be more effective alternatives to present antimalarial drugs.

Diverse evolutionary pathways challenge the use of collateral sensitivity as a strategy to suppress resistance.

Drug resistance remains a major obstacle to malaria control and eradication efforts, necessitating the development of novel therapeutic strategies to treat this disease. Drug combinations based on collateral sensitivity, wherein resistance to one drug causes increased sensitivity to the partner drug, have been proposed as an evolutionary strategy to suppress the emergence of resistance in pathogen populations. In this study, we explore collateral sensitivity between compounds targeting the Plasmodium dihydroorotate dehydrogenase (DHODH). We profiled the cross-resistance and collateral sensitivity phenotypes of several DHODH mutant lines to a diverse panel of DHODH inhibitors. We focus on one compound, TCMDC-125334, which was active against all mutant lines tested, including the DHODH C276Y line, which arose in selections with the clinical candidate DSM265. In six selections with TCMDC-125334, the most common mechanism of resistance to this compound was copy number variation of the dhodh locus, although we did identify one mutation, DHODH I263S, which conferred resistance to TCMDC-125334 but not DSM265. We found that selection of the DHODH C276Y mutant with TCMDC-125334 yielded additional genetic changes in the dhodh locus. These double mutant parasites exhibited decreased sensitivity to TCMDC-125334 and were highly resistant to DSM265. Finally, we tested whether collateral sensitivity could be exploited to suppress the emergence of resistance in the context of combination treatment by exposing wildtype parasites to both DSM265 and TCMDC-125334 simultaneously. This selected for parasites with a DHODH V532A mutation which were cross-resistant to both compounds and were as fit as the wildtype parent in vitro. The emergence of these cross-resistant, evolutionarily fit parasites highlights the mutational flexibility of the DHODH enzyme.

Malaria affects around 240 million people around the world every year. The microscopic parasite responsible for the disease are carried by certain mosquitoes and gets transmitted to humans through bites. These parasites are increasingly acquiring genetic mutations that make anti-malaria medication less effective, creating an urgent need for alternative treatment approaches. Several new malaria drugs being explored in preclinical research work by binding to an enzyme known as DHODH and preventing it from performing its usual role in the parasite. Previous work found that, in some cases, malaria parasites that evolved resistance to one type of DHODH inhibitor (by acquiring mutations in their DHODH enzyme) then became more vulnerable to another kind. It may be possible to leverage this 'collateral sensitivity' by designing treatments which combine two DHODH inhibitors and therefore make it harder for the parasites to evolve resistance. To investigate this possibility, Mandt et al. first tested several DHODH inhibitors to find the one that was most potent against drug-resistant parasites. In subsequent experiments, they combined TCMDC-125334, the best candidate that emerged from these tests, with a DHODH inhibitor that works well against vulnerable parasites. However, the parasites still rapidly evolved resistance. Further work identified a new DHODH mutation that allowed the parasites to evade both drugs simultaneously. Together, these findings suggest that the DHODH enzyme may not be the best target for new malaria drugs because many it can acquire many possible mutations that confer resistance. Such results may inform other studies that aim to harness collateral sensitivity to fight against a range of harmful agents.

Discovery of dual-target natural antimalarial agents against DHODH and PMT of Plasmodium falciparum: pharmacophore modelling, molecular docking, quantum mechanics, and molecular dynamics simulations.

Malaria is a lethal disease that claims thousands of lives worldwide annually. The objective of this study was to identify new natural compounds that can target two P. falciparum enzymes; P. falciparum Dihydroorotate dehydrogenase (PfDHODH) and P. falciparum phosphoethanolamine methyltransferase (PfPMT). To accomplish this, e-pharmacophore modelling and molecular docking were employed against PfDHODH. Following this, 1201 natural compounds with docking scores of ≤ -7 kcal/mol were docked into the active site of the second enzyme PMT. The top nine compounds were subjected to further investigation using MM-GBSA free binding energy calculations and ADME analysis. The results revealed favourable free binding energy values better than the references, as well as acceptable pharmacokinetic properties. Compounds ZINC000013377887, ZINC000015113777, and ZINC000085595753 were scrutinized to assess their interaction stability with the PfDHODH enzyme, and chemical stability reactivity using molecular dynamics (MD) simulation and density functional theory (DFT) calculations. These findings indicate that the three natural compounds are potential candidates for dual PfDHODH and PfPMT inhibitors for malaria treatment.