Nitrofurazone repurposing towards design and synthesis of novel apoptotic-dependent anticancer and antimicrobial agents: Biological evaluation, kinetic studies and molecular modeling.

Drug repurposing has gained much attention as a cost-effective strategy that plays an exquisite role in identifying undescribed biological activities in clinical drugs. In the present work, we report the repurposing of the antibacterial drug nitrofurazone (NFZ) as a potential anticancer agent against CaCo-2, MDA-MB 231 and HepG-2 cancer cell lines. Novel series of nitrofurazone analogs were then designed considering the important pharmacologic features present in NFZ. Synthesis and biological evaluation of the target compounds revealed their promising anticancer activities endowed with antimicrobial potential and possessing better lipophilicity than NFZ. Compound 7, exclusively, inhibited the growth of all tested cancer cells more potently than NFZ with the least cytotoxicity against normal cells, displaying anti Gram-positive bacterial activities and antifungal potential. Analysis of the stereo-electronic properties of compound 7 via investigating the energies of HOMO, LUMO, HOMO-LUMO energy gap and MEP maps demonstrated its high reactivity and the expected molecular mechanism of action through reduction of the 5-nitrofuryl moiety. Data of the bioactivity studies indicated that the potent anticancer activity of 7 is mainly through increasing intracellular ROS levels and induction of apoptosis via significantly down-regulating the expression of Bcl-2 while up-regulating BAX, p53 and caspase 3 expression levels. Compound 7 potently inhibited the cellular expression levels of antioxidant enzymes GPx1 and GR compared to NFZ. Antioxidant enzymes kinetic studies and blind molecular docking simulations disclosed the mechanistic and structural aspects of the interaction between 7 and both GR and GPx1. Thus, the successful discovery of 7 as a potential dual anticancer-antimicrobial nitrofurazone analog might validate the applicability of drug repurposing strategy in unravelling the unrecognized bioactivity of the present conventional drugs, besides furnishing the way towards more optimization and development studies.

Gly188Arg substitution eliminates substrate inhibition in arachidonate 11R-lipoxygenase.

Lipoxygenases (LOXs) are dioxygenases that catalyze the oxygenation of polyunsaturated fatty acids to hydroperoxyl derivates. These products are precursors for different lipid mediators which are associated with pathogenesis of various diseases such as asthma, atherosclerosis and cancer. Several LOXs suffer from substrate inhibition, a potential regulatory mechanism, yet it is unclear what is the cause of this phenomenon. One such enzyme is the coral 11R-LOX which displays a significant decrease in turnover rate at arachidonic acid concentrations above 30 µM. In this report, site-directed mutagenesis and inhibition assays were employed to shed light on the mechanism of substrate inhibition in 11R-LOX. We found that introduction of a positive charge to the active site entrance with Gly188Arg substitution completely eliminates the slow-down at higher substrate concentrations. Inhibition of 11R-LOX by its catalysis product, 11(R)-hydroperoxyeicosatetraenoic acid, suggests an uncompetitive mechanism. We reason that substrate inhibition in 11R-LOX is due to additional fatty acid binding by the enzyme:substrate complex at an allosteric site situated in the very vicinity of the active site entrance.

Identification of novel small-molecule inhibitors of α-methylacyl-CoA racemase (AMACR; P504S) and structure-activity relationships.

α-Methylacyl-CoA racemase (AMACR; P504S; EC 5.1.99.4) catalyses epimerization of 2-methylacyl-CoAs and is important for the degradation of branched-chain fatty acids and the pharmacological activation of ibuprofen and related drugs. It is also a novel drug target for prostate and other cancers. However, development of AMACR as a drug target has been hampered by the difficulties in assaying enzyme activity. Consequently, reported inhibitors have been rationally designed acyl-CoA esters, which are delivered as their carboxylate prodrugs. The novel colorimetric assay for AMACR based on the elimination of 2,4-dinitrophenolate was developed for high-throughput screening and 20,387 'drug-like compounds' were screened, with a throughput of 768 compounds assayed per day. Pyrazoloquinolines and pyrazolopyrimidines were identified as novel scaffolds and investigated as AMACR inhibitors. The most potent inhibitors have IC50 values of ~2 µM. The pyrazoloquinoline inhibitor 10a displayed uncompetitive inhibition, whilst 10j displayed mixed competitive inhibition. The pyrazolopyrimidine inhibitor 11k displayed uncompetitive inhibition. This is the first report of the identification of specific drug-like small-molecule AMACR inhibitors by high-throughput screening. Pyrazoloquinolines and pyrazolopyrimidines may also be useful as inhibitors of other CoA-utilizing enzymes.

A New Benzophenone C-Glucoside and Other Constituents of Pseuduvaria fragrans and Their α-Glucosidase Inhibitory Activity.

Phytochemical investigations of the leaves and stems of Pseuduvaria fragrans led to the isolation of a new benzophenone C-glucoside named pseuduvarioside (1), together with six known compounds including (-)-guaiol (2), (+)-isocorydine (3), cyathocaline (4), isoursoline (5), N-trans-coumaroyltyramine (6), and N-trans-feruloyltyramine (7). Their structures were characterized by NMR spectroscopy and mass spectrometry. All of the isolates were evaluated for inhibitory activity against the enzyme α-glucosidase. N-trans-coumaroyltyramine and N-trans-feruloyltyramine showed higher activity than the drug acarbose. Kinetic studies revealed that both tyramine-derived amides were uncompetitive inhibitors of the enzyme.

Kinetic characterization of the inhibition of protein tyrosine phosphatase-1B by Vanadyl (VO2+) chelates.

Protein tyrosine phosphatases (PTPases) are a prominent focus of drug design studies because of their roles in homeostasis and disorders of metabolism. These studies have met with little success because (1) virtually all inhibitors hitherto exhibit only competitive behavior and (2) a consensus sequence H/V-C-X5-R-S/T characterizes the active sites of PTPases, leading to low specificity of active site directed inhibitors. With protein tyrosine phosphatase-1B (PTP1B) identifed as the target enzyme of the vanadyl (VO2+) chelate bis(acetylacetonato)oxidovanadium(IV) [VO(acac)2] in 3T3-L1 adipocytes [Ou et al. J Biol Inorg Chem 10: 874-886, 2005], we compared the inhibition of PTP1B by VO(acac)2 with other VO2+-chelates, namely, bis(2-ethyl-maltolato)oxidovanadium(IV) [VO(Et-malto)2] and bis(3-hydroxy-2-methyl-4(1H)pyridinonato)oxidovanadium(IV) [VO(mpp)2] under steady-state conditions, using the soluble portion of the recombinant human enzyme (residues 1-321). Our results differed from those of previous investigations because we compared inhibition in the presence of the nonspecific substrate p-nitrophenylphosphate and the phosphotyrosine-containing undecapeptide DADEpYLIPQQG mimicking residues 988-998 of the epidermal growth factor receptor, a relevant, natural substrate. While VO(Et-malto)2 acts only as a noncompetitive inhibitor in the presence of either subtrate, VO(acac)2 exhibits classical uncompetitive inhibition in the presence of DADEpYLIPQQG but only apparent competitive inhibition with p-nitrophenylphosphate as substrate. Because uncompetitive inhibitors are more potent pharmacologically than competitive inhibitors, structural characterization of the site of uncompetitive binding of VO(acac)2 may provide a new direction for design of inhibitors for therapeutic purposes. Our results suggest also that the true behavior of other inhibitors may have been masked when assayed with only p-nitrophenylphosphate as substrate.

Inhibitory effects promoted by 5'-nucleotides on the ecto-3'-nucleotidase activity of Leishmania amazonensis.

The protozoan parasite Leishmania amazonensis is the etiological agent of cutaneous leishmaniasis. During its life cycle, the flagellated metacyclic promastigote forms are transmitted to vertebrate hosts by sandfly bites, and they develop into amastigotes inside macrophages, where they multiply. L. amazonensis possesses a bifunctional enzyme, called 3'-nucleotidase/nuclease (3'NT/NU), which is able to hydrolyze extracellular 3'-monophosphorylated nucleosides and nucleic acids. 3'NT/NU plays an important role in the generation of extracellular adenosine and has been described as a key enzyme in the acquisition of purines by trypanosomatids. Furthermore, it has been observed that 3'NT/NU also plays a valuable role in the establishment of parasitic infection. In this context, this study aimed to investigate the modulation of the 3'-nucleotidase (3'NT) activity of L. amazonensis by several nucleotides. It was observed that 3'NT activity is inhibited by micromolar concentrations of guanosine and guanine nucleotides. The inhibition promoted by 5'-GMP on the 3'NT activity of L. amazonensis is reversible and uncompetitive because the addition of the inhibitor decreased the kinetic parameters Km and Vmax. Finally, we found that the addition of 5'-GMP is able to reverse the stimulation promoted by 3'-AMP in a macrophage-parasite interaction assay. The determination of compounds that can inhibit the 3'NT activity of Leishmania is very important because this enzyme does not occur in mammals, making it a potential therapeutic target.

Exploring the inhibitory activity of Withaferin-A against Pteridine reductase-1 of L. donovani.

Withaferin A is an abundant withanolide present in Withania somnifera leaves and to some extent in roots. It has been known for its profound anti-cancer properties, but its role in counteracting the Leishmania donovani infection has to be explored. Pteridine reductase 1 (PTR1) is involved in pteridine salvage and an important enzyme for the parasite growth, which could be targeted for the development of an efficient antileishmanial drug. We employed molecular docking studies to identify the binding mode of withaferin A with PTR1 in silico. We further cloned, expressed, and purified PTR1 of L. donovani and performed the enzyme kinetics using the Michaelis-Menten equation and enzyme inhibition studies with withaferin A by plotting the Lineweaver-Burk graph, which followed an uncompetitive mode of inhibition. We also showed the inhibition of the enzyme in the crude lysate of treated parasites. Thus, our study contributes towards understanding the mode of action of withaferin A against L. donovani parasite.

Thiol oxidation is crucial in the desensitization of the mitochondrial F1FO-ATPase to oligomycin and other macrolide antibiotics.

BACKGROUND: The macrolide antibiotics oligomycin, venturicidin and bafilomycin, sharing the polyketide ring and differing in the deoxysugar moiety, are known to block the transmembrane ion channel of ion-pumping ATPases; oligomycins are selective inhibitors of mitochondrial ATP synthases. METHODS: The inhibition mechanism of macrolides was explored on swine heart mitochondrial F1FO-ATPase by kinetic analyses. The amphiphilic membrane toxicant tributyltin (TBT) and the thiol reducing agent dithioerythritol (DTE) were used to elucidate the nature of the macrolide-enzyme interaction. RESULTS: When individually tested, the macrolide antibiotics acted as uncompetitive inhibitors of the ATPase activity. Binary mixtures of macrolide inhibitors I1 and I2 pointed out a non-exclusive mechanism, indicating that each macrolide binds to its binding site on the enzyme. When co-present, the two macrolides acted synergistically in the formed quaternary complex (ESI1I2), thus mutually strengthening the enzyme inhibition. The enzyme inhibition by macrolides displaying a shared mechanism was dose-dependently reduced by TBT≥1µM. The TBT-driven enzyme desensitization was reversed by DTE. CONCLUSIONS: The macrolides tested share uncompetitive inhibition mechanism by binding to a specific site in a common macrolide-binding region of FO. The oxidation of highly conserved thiols in the ATP synthase c-ring of FO weakens the interaction between the enzyme and the macrolides. The native macrolide-inhibited enzyme conformation can be restored by reducing crucial thiols oxidized by TBT. GENERAL SIGNIFICANCE: The findings, by elucidating the macrolide inhibitory mechanism on FO, indirectly cast light on the F1FO torque generation involving crucial amino acid residues and may address drug design and antimicrobial therapy.

Kinetics studies with fruit bromelain (Ananas comosus) in the presence of cysteine and divalent ions.

The kinetics of cysteine and divalent ion modulation viz. Ca(2+), Cu(2+), Hg(2+) of fruit bromelain (EC 3.4.22.33) have been investigated in the present study. Kinetic studies revealed that at pH 4.5, cysteine induced V-type activation of bromelain catalyzed gelatin hydrolysis. At pH 3.5, Ca(2+) inhibited the enzyme noncompetitively, whereas, both K-and V-type activations of bromelain were observed in the presence of 0.5 mM Ca(2+) at pH 4.5 and 7.5. Bromelain was inhibited competitively at 0.6 mM Cu(2+) ions at pH 3.5, which changed to an uncompetitive inhibition at pH 4.5 and 7.5. An un-competitive inhibition of bromelain catalyzed gelatin hydrolysis was observed in the presence of 0.6 mM Hg(2+) at pH 3.5 and 4.5. These findings suggest that divalent ions modulation of fruit bromelain is pH dependent.

Trypsin is inhibited by phytocompounds liquiritin and terpinen-4-ol from the herb Glycyrrhiza glabra: in vitro and in silico studies.

Serine proteases are a class of hydrolytic enzymes involved in various physiological functions like digestion, coagulation, fibrinolysis and immunity. The present study evaluates the serine protease inhibitory potential of phytochemicals liquiritin and terpinen-4-ol present in the herb Glycyrrhiza glabra L. using trypsin as the model enzyme. In silico studies showed that both the compounds have a significant binding affinity towards trypsin with a binding energy of -26.66 kcal/mol and -19.79 kcal/mol for liquiritin and terpinen-4-ol, respectively. Their binding affinity was confirmed through in vitro enzyme inhibition assays. The mode of inhibition was found to be uncompetitive. In order to explain the mode of inhibition, docking of the ligands to the enzyme-substrate complex was also done and binding energy was calculated after MD simulation. The energy values showed that the binding affinities of these compounds towards the enzyme substrate complex are more than that towards the enzyme alone. This explains the uncompetitive mode of inhibition.Communicated by Ramaswamy H. Sarma.

Biohydrogen production from lignocellulosic hydrolysate: Unveiling the synergistic impact of substrate concentration and furfural inhibition.

Lignocellulosic biomass offers substantial potential as an ideal feedstock for dark fermentative hydrogen production due to its sustainability and cost-effectiveness. The current study examined the influence of furfural on fermentative hydrogen production using lignocellulosic hydrolysate in the presence of furfural. Synthetic lignocellulosic hydrolysate, consisting primarily of 76% xylose, 10% glucose, 9% arabinose, and a mixture of other sugars such as galactose and mannose (85% pentose sugars and 15% hexose sugars), was employed as the substrate. Various substrate concentrations ranging from 2 to 32 g/L were tested, along with furfural concentrations of 0, 1, and 2 g/L. The investigation aimed to assess the effects of initial substrate concentration, initial furfural concentration, furfural-to-biomass ratio (F/B), and furfural-to-substrate ratio (F/S) on biohydrogen production yields. The maximum specific substrate utilization rates at different substrate concentrations were effectively characterized using Haldane's substrate inhibition model. Among the tested concentrations, the 16 g/L emerged as the optimal substrate concentration. The initial furfural concentration was identified as the most significant parameter impacting biohydrogen production, with complete inhibition observed at a furfural concentration of 2 g/L. Higher F/S ratios at substrate concentrations ranging from 2 to 16 g/L resulted in reduced maximum specific hydrogen production rates (MSHPR) and hydrogen yields. Substrate inhibition was observed at 24 g/L and 32 g/L. Lactate was the predominant metabolite in all batches containing 2-g/L furfural, as well as in batches with 1-g/L furfural and substrate concentrations of 24 and 32 g/L. Furfural at a concentration of 1 g/L was not inhibitory in any of the batches. Overall, the mixed cultures in this study could efficiently produce hydrogen from lignocellulosic hydrolysates and degrade furfural, providing new insights into fermentative hydrogen-producing bacteria with furfural tolerance.

Allosteric lariat peptide inhibitors of Abl kinase.

Going against tradition: although most kinase inhibitors are ATP competitive, lariat peptides inhibit Abl kinase activity in an ATP-uncompetitive manner. Further, lariat peptides discriminated Src family kinases, and recognize the allosteric region that lies adjacent to the ATP binding pocket in the Abl kinase catalytic cleft.

Discovery of E2730, a novel selective uncompetitive GAT1 inhibitor, as a candidate for anti-seizure medication.

OBJECTIVE: As of 2022, 36 anti-seizure medications (ASMs) have been licensed for the treatment of epilepsy, however, adverse effects (AEs) are commonly reported. Therefore, ASMs with a wide margin between therapeutic effects and AEs are preferred over ASMs that are associated with a narrow margin between efficacy and risk of AEs. E2730 was discovered using in vivo phenotypic screening and characterized as an uncompetitive, yet selective, inhibitor of γ-aminobutyric acid (GABA) transporter 1 (GAT1). Here, we describe the preclinical characteristics of E2730. METHODS: Anti-seizure effects of E2730 were evaluated in several animal models of epilepsy: corneal kindling, 6 Hz-44 mA psychomotor seizure, amygdala kindling, Fragile X syndrome, and Dravet syndrome models. Effects of E2730 on motor coordination were assessed in accelerating rotarod tests. The mechanism of action of E2730 was explored by [3 H]E2730 binding assay. The GAT1-selectivity over other GABA transporters was examined by GABA uptake assay of GAT1, GAT2, GAT3, or betaine/GABA transporter 1 (BGT-1) stably expressing HEK293 cells. To further investigate the mechanism for E2730-mediated inhibition of GAT1, in vivo microdialysis and in vitro GABA uptake assays were conducted under conditions of different GABA concentrations. RESULTS: E2730 showed anti-seizure effects in the assessed animal models with an approximately >20-|fold margin between efficacy and motor incoordination. [3 H]E2730 binding on brain synaptosomal membrane was abolished in GAT1-deficient mice, and E2730 selectively inhibited GAT1-mediated GABA uptake over other GABA transporters. In addition, results of GABA uptake assays showed that E2730-mediated inhibition of GAT1 positively correlated to the level of ambient GABA in vitro. E2730 also increased extracellular GABA concentration in hyperactivated conditions but not under basal levels in vivo. SIGNIFICANCE: E2730 is a novel, selective, uncompetitive GAT1 inhibitor, which acts selectively under the condition of increasing synaptic activity, contributing to a wide margin between therapeutic effect and motor incoordination.

α-Glucosidase inhibition of sesquiterpenoids from the heartwood of Mansonia gagei.

Nine undescribed sesquiterpenoids, along with ten known compounds, were isolated from the ethyl acetate extract of Mansonia gagei heartwood. Their structures were determined by spectroscopic data analysis (FTIR, 1D, 2D NMR, and HRESIMS), and their absolute configurations were established by ECD calculation. The isolated compounds were evaluated for their inhibitory effect against α-glucosidase from yeast. The results showed that mansonone U, mansonialactam, heliclactone and mansonone S exhibited exceptionally potent activities when compared to the positive control, acarbose, with IC50 values of 12.38 ± 0.71, 0.20 ± 0.05, 13.12 ± 2.85, and 12.05 ± 1.91 µM, respectively. Among them, mansonialactam possessed the most potent inhibitory activity against yeast α-glucosidase, and it showed an uncompetitive inhibition mode.

A mechanism of uncompetitive inhibition of the serotonin transporter.

The serotonin transporter (SERT/SLC6A4) is arguably the most extensively studied solute carrier (SLC). During its eponymous action - that is, the retrieval of serotonin from the extracellular space - SERT undergoes a conformational cycle. Typical inhibitors (antidepressant drugs and cocaine), partial and full substrates (amphetamines and their derivatives), and atypical inhibitors (ibogaine analogues) bind preferentially to different states in this cycle. This results in competitive or non-competitive transport inhibition. Here, we explored the action of N-formyl-1,3-bis (3,4-methylenedioxyphenyl)-prop-2-yl-amine (ECSI#6) on SERT: inhibition of serotonin uptake by ECSI#6 was enhanced with increasing serotonin concentration. Conversely, the KM for serotonin was lowered by augmenting ECSI#6. ECSI#6 bound with low affinity to the outward-facing state of SERT but with increased affinity to a potassium-bound state. Electrophysiological recordings showed that ECSI#6 preferentially interacted with the inward-facing state. Kinetic modeling recapitulated the experimental data and verified that uncompetitive inhibition arose from preferential binding of ECSI#6 to the K+-bound, inward-facing conformation of SERT. This binding mode predicted a pharmacochaperoning action of ECSI#6, which was confirmed by examining its effect on the folding-deficient mutant SERT-PG601,602AA: preincubation of HEK293 cells with ECSI#6 restored export of SERT-PG601,602AA from the endoplasmic reticulum and substrate transport. Similarly, in transgenic flies, the administration of ECSI#6 promoted the delivery of SERT-PG601,602AA to the presynaptic specialization of serotonergic neurons. To the best of our knowledge, ECSI#6 is the first example of an uncompetitive SLC inhibitor. Pharmacochaperones endowed with the binding mode of ECSI#6 are attractive, because they can rescue misfolded transporters at concentrations, which cause modest transport inhibition.

Calotropin and corotoxigenin 3-O-glucopyranoside from the desert milkweed Asclepias subulata inhibit the Na+/K+-ATPase activity.

Na+/K+-ATPase is an essential transmembrane enzyme found in all mammalian cells with critical functions for cell ion homeostasis. The inhibition of this enzyme by several cardiotonic steroids (CTS) has been associated with the cytotoxic effect on cancer cell lines of phytochemicals such as ouabain and digitoxin. This study evaluated the inhibitory capacity of cardenolides calotropin and corotoxigenin 3-O-glucopyranoside (C3OG) from Asclepias subulata over the Na+/K+-ATPase activity in vitro and silico. The inhibitory assays showed that calotropin and C3OG decreased the Na+/K+-ATPase activity with IC50 values of 0.27 and 0.87 µM, respectively. Furthermore, the molecules presented an uncompetitive inhibition on Na+/K+-ATPase activity, with Ki values of 0.2 µM to calotropin and 0.5 µM to C3OG. Furthermore, the molecular modeling indicated that calotropin and C3OG might interact with the Thr797 and Gln111 residues, considered essential to the interaction with the Na+/K+-ATPase. Besides, these cardenolides can interact with amino acid residues such as Phe783, Leu125, and Ala323, to establish hydrophobic interactions on the binding site. Considering the results, these provide novel evidence about the mechanism of action of cardenolides from A. subulata, proposing that C3OG is a novel cardenolide that deserves further consideration for in vitro cellular antiproliferative assays and in vivo studies as an anticancer molecule.

Uncompetitive, adduct-forming SARM1 inhibitors are neuroprotective in preclinical models of nerve injury and disease.

Axon degeneration is an early pathological event in many neurological diseases. The identification of the nicotinamide adenine dinucleotide (NAD) hydrolase SARM1 as a central metabolic sensor and axon executioner presents an exciting opportunity to develop novel neuroprotective therapies that can prevent or halt the degenerative process, yet limited progress has been made on advancing efficacious inhibitors. We describe a class of NAD-dependent active-site SARM1 inhibitors that function by intercepting NAD hydrolysis and undergoing covalent conjugation with the reaction product adenosine diphosphate ribose (ADPR). The resulting small-molecule ADPR adducts are highly potent and confer compelling neuroprotection in preclinical models of neurological injury and disease, validating this mode of inhibition as a viable therapeutic strategy. Additionally, we show that the most potent inhibitor of CD38, a related NAD hydrolase, also functions by the same mechanism, further underscoring the broader applicability of this mechanism in developing therapies against this class of enzymes.

An insight on the nature of biochemical interactions between glycyrrhizin, myricetin and CYP3A4 isoform.

Drug interaction studies are imperative to gain insights into the beneficial or harmful effects of therapeutic and dietary agents. This study investigated the mechanism of modulatory roles of glycyrrhizin (GLH) and myricetin (MYC) on the human CYP3A4 isoform using in silico and in vitro methods. While MYC had concentration-dependent inhibitory effect on CYP3A4 (IC50 : 10.5 ± 0.55 µM) with characteristic Km and Vmax values of 1.13 µM and 1.54 nM/min, respectively, GLH exhibited no inhibitory effect on CYP3A4 activity in vitro. These observations are consistent with the results of in silico evaluations where the effect of MYC compared well with that of ketoconazole (a known CYP3A4 inhibitor) against CYP3A4. Overall, the established interactions between the study compounds and CYP3A4 could potentiate clinically vital drug-drug interactions and has lent credence to the mechanism of modulatory effect of MYC and GLH on CYP3A4 that could guide their safe use as therapeutic agents. PRACTICAL IMPLICATIONS: Myricetin (MYR) and glycyrrhizin (GLH) occur freely in commonly ingested foods and their supplements are recommended for the treatment of several debilitating diseases such as diabetes, cancer, and cardiovascular complications. This study provided an insight on the possible interactions that could be established between these compounds (MYR and GLH) and CYP3A4 when ingested and metabolized by the liver. The results suggested possibilities of potential clinical drug-drug interactions and advocates for their cautious use within the therapeutic dose in food supplements or medications to avoid probable liver damage.

Metabolic targeting of cancer by a ubiquinone uncompetitive inhibitor of mitochondrial complex I.

SMIP004-7 is a small molecule inhibitor of mitochondrial respiration with selective in vivo anti-cancer activity through an as-yet unknown molecular target. We demonstrate here that SMIP004-7 targets drug-resistant cancer cells with stem-like features by inhibiting mitochondrial respiration complex I (NADH:ubiquinone oxidoreductase, complex I [CI]). Instead of affecting the quinone-binding site targeted by most CI inhibitors, SMIP004-7 and its cytochrome P450-dependent activated metabolite(s) have an uncompetitive mechanism of inhibition involving a distinct N-terminal region of catalytic subunit NDUFS2 that leads to rapid disassembly of CI. SMIP004-7 and an improved chemical analog selectively engage NDUFS2 in vivo to inhibit the growth of triple-negative breast cancer transplants, a response mediated at least in part by boosting CD4+ and CD8+ T cell-mediated immune surveillance. Thus, SMIP004-7 defines an emerging class of ubiquinone uncompetitive CI inhibitors for cell autonomous and microenvironmental metabolic targeting of mitochondrial respiration in cancer.

Reversible Mechanisms of Enzyme Inhibition and Resulting Clinical Significance.

Inhibition of a drug-metabolizing enzyme by the reversible interaction of a drug with the enzyme, thus decreasing the metabolism of another drug, is a major cause of clinically significant drug-drug interactions. This chapter defines the four reversible mechanisms of inhibition exhibited by drugs: competitive, noncompetitive, uncompetitive, and mixed competitive/noncompetitive. An in vitro procedure to determine the potential of a drug to be a reversible inhibitor is also provided. Finally, a number of examples of clinically significant drug-drug interactions resulting from reversible inhibition are described.