Mode of interaction of .beta.-hydroxy-.beta.-methylglutaryl coenzyme A reductase with strong binding inhibitors: compactin and related compounds

Nakamura, Charles E.; Abeles, Robert H.

doi:10.1021/bi00327a014

Cited by 109 publications

(42 citation statements)

References 30 publications

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“…It was earlier reported that the inhibition of HMGR by mevastatin is competitive with respect to HMG-CoA in case of rat liver enzyme (Endo et al 1976). Mevastatin-mediated inhibition of yeast HMGR is reported to be competitive with respect to HMG-CoA (Nakamura and Abeles 1985). However, when the effect of mevastatin was tested on the native parasite HMGR enzyme, it caused 70 % inhibition at 20 μM of mevastatin concentration.…”

Section: Discussionmentioning

confidence: 97%

Antileishmanial effect of mevastatin is due to interference with sterol metabolism

2015

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Visceral leishmaniasis (VL) is one of the most severe forms of leishmaniasis which is fatal if left untreated. Sterol biosynthetic pathway in Leishmania is currently being explored for its therapeutic potential. In the present study, we have evaluated the antileishmanial efficacy of mevastatin, a known inhibitor of 3-hydroxy-3-methyl glutaryl-CoA reductase (HMGR) enzyme. Mevastatin inhibited Leishmania donovani promastigotes and intracellular amastigotes with an 50% inhibitory concentration (IC50) value of 23.8 ± 4.2 and 7.5 ± 1.1 μM, respectively, without exhibiting toxicity towards host cell line. Mevastatin also inhibited recombinant L. donovani HMGR (LdHMGR) enzyme activity with an IC50 value of 42.2 ± 3.0 μM. Kinetic analysis revealed that the inhibition of recombinant LdHMGR activity by mevastatin was competitive with HMG-CoA. Mevastatin-treated parasites exhibited 66% reduction in ergosterol levels with respect to untreated parasites. Incubation of mevastatin-treated L. donovani promastigotes with ergosterol resulted in revival of cell growth, whereas cholesterol supplementation failed to cause reversal in cell death. To further prove the specificity of mevastatin for HMGR enzyme, HMGR-overexpressing parasites were used which showed almost threefold resistance to mevastatin. It also induced morphological changes in the parasite accompanied by lipid body accumulation. Hence, antileishmanial effect of mevastatin was due to the inhibition of HMGR, which eventually leads to reduction in ergosterol levels and hence parasite death. The present study may have implications in the treatment of visceral form of leishmaniasis.

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Section: Discussionmentioning

confidence: 97%

Antileishmanial effect of mevastatin is due to interference with sterol metabolism

2015

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“…As shown experimentally, the K, values of these sulfones are much smaller than the K , of the substrate, but their affinity is not as high as that of the sulfoxides. vrobablv because the sulfur-oxvgen bond methylglutaryl-CoA reductase and the fungal metabolite compactin [18], the high affinity of this strong competitive inhibitor has been shown to arise from the presence, in one molecule, of a substrate part and a hydrophobic region permitting the binding to the active site as well as the anchoring to a hydrophobic pocket of the enzyme. The sulfoxides VIII and X have an affinity towards 3-hydroxy-3-methylglutaryl-CoA similar to that of compactin but are purely hydrophilic.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of 3‐Hydroxy‐3‐Methylglutaryl‐CoA Reductase and Citrate Synthase by Sulfoxides and Sulfones of Substrate‐analogue CoA‐thioether Derivatives

Karl¹,

Hupperich²,

Thomer³

et al. 1995

European Journal of Biochemistry

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Sulfoxides and sulfones were prepared by specific oxidation of 3-hydroxy-3-methylglutaryl-CoAanalogue CoA-thioether derivatives and their kinetic properties were determined with 3-hydroxy-3-methylglutaryl-CoA reductase. The oxidized CoA-thioether derivatives with a hydroxyl group at C3 were powerful competitive inhibitors, their K, values being much smaller than the K,,, for 3-hydroxy-3-methylSulfoxides and sulfones of substrate analogues of citrate synthase were also prepared. When tested in the appropriate reaction with citrate synthase, the sulfoxide and sulfone derivatives were competitive inhibitors, but their K, values were greater than the K, values of the corresponding unmodified substrates.Keywords. 3-Hydroxy-3-methylglutaryl-CoA reductase ; citrate synthase ; substrate-analogue inhibitors.glutqrl-CoA.3-Hydroxy-3-m1~thylglutaryl-CoA reductase represents the key regulatory enzyme in cholesterol biosynthesis. This enzyme catalyzes the two-step reduction of 3-hydroxy-3-methylglutarylCoA by two equivalents of NADPH to eventually yield mevalonate. In search of substrate-based inhibitors of 3-hydroxy-3-methylglutaryl-CoA, reductase, we previously reported the influence of substitutions in the acyl moiety of 3-hydroxy-3-methylglutaryl-CoA on substrate binding [l]. A further target for modification was the thioester group of 3-hydroxy-3-methylglutarylCoA. The replacement of the thioester oxygen by hydrogen yielded non-reducible substrate analogues without significantly affecting the affinity towards 3-hydroxy-3-methylglutaryl-CoA reductase [ 11. With some substrate-analogue CoA-thioethers, a drastic increase in affinity towards the reductase was found on introduction of oxygen at the thioether sulfur to yield a sulfoxide [2]. In this study, we investigate the requirements for this increased affinity with structurally related CoA-sulfoxide derivatives. Attempts are also described to enhance the inhibitory power of these compounds by introduction of a second oxygen at the sulfur to yield a CoA-sulfone derivative. To probe the oxidation of CoA-tihioethers to CoA-sulfones we used the simplest CoA-thioether, S-ethyl-CoA, and the more complicated S-(3-carboxy-3-hydroxypropyl)-CoA, substrate analogues of acetyl-CoA and malyl-CoA. Acetyl-CoA is a substrate of citrate synthase and reacts, with oxaloacetate to yield citrate via transiently formed citryl-CoA. Malyl-CoA is an analogue of this intermediate and is hydrolyzed in the presence of citrate synthase but not cleaved to acetyl-CoA and oxoacid [3]. The mechanism of hydrolysis of citryl-CoA is as yet unknown. A highaffinity analogue of malyl-CoA, interacting specifically with the Correspondence ttq H. Eggerer,

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“…In a third set of experiments to support ClFDP binding to the C site, a series of pairwise preincubation-dilution assays was undertaken. These experiments were modeled after those described by Nakamura and Abeles to determine the site specificity of slow-onset and slow-release reversible inhibitors such as compactin for HMGCoA reductase, when high substrate concentrations render traditional substrate protection assays ineffective due to substrate inhibition (29). For hRNR the SA of α at 0.5 mM CDP, 15 mM Mg 2þ , and 3 mM ATP is reduced by half when [CDP] is increased to 10 mM, and increasing [Mg 2þ ] does not prevent the observed substrate inhibition.…”

Section: Which Imposes No Restrictions About [I] Relative To [E] (mentioning

confidence: 99%

Clofarabine 5′-di and -triphosphates inhibit human ribonucleotide reductase by altering the quaternary structure of its large subunit

Aye

Stubbe²

2011

Proc. Natl. Acad. Sci. U.S.A.

172

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Human ribonucleotide reductases (hRNRs) catalyze the conversion of nucleotides to deoxynucleotides and are composed of α-and β-subunits that form active α n β m (n, m ¼ 2 or 6) complexes. α binds NDP substrates (CDP, UDP, ADP, and GDP, C site) as well as ATP and dNTPs (dATP, dGTP, TTP) allosteric effectors that control enzyme activity (A site) and substrate specificity (S site). Clofarabine (ClF), an adenosine analog, is used in the treatment of refractory leukemias. Its mode of cytotoxicity is thought to be associated in part with the triphosphate functioning as an allosteric inhibitor of hRNR. Studies on the mechanism of inhibition of hRNR by ClF di-and triphosphates (ClFDP and ClFTP) are presented. ClFTP is a reversible inhibitor (K i ¼ 40 nM) that rapidly inactivates hRNR. However, with time, 50% of the activity is recovered. D57N-α, a mutant with an altered A site, prevents inhibition by ClFTP, suggesting its A site binding. ClFDP is a slow-binding, reversible inhibitor (K Ã i ¼ 17 nM; t 1∕2 ¼ 23 min). CDP protects α from its inhibition. The altered off-rate of ClFDP from E • ClFDP Ã by ClFTP (A site) or dGTP (S site) and its inhibition of D57N-α together implicate its C site binding. Size exclusion chromatography of hRNR or α alone with ClFDP or ClFTP, AEATP or dGTP, reveals in each case that α forms a kinetically stable hexameric state. This is the first example of hexamerization of α induced by an NDP analog that reversibly binds at the active site.allosteric regulation | oligomerization | slow-binding inhibitor

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Mode of interaction of .beta.-hydroxy-.beta.-methylglutaryl coenzyme A reductase with strong binding inhibitors: compactin and related compounds

Cited by 109 publications

References 30 publications

Antileishmanial effect of mevastatin is due to interference with sterol metabolism

Antileishmanial effect of mevastatin is due to interference with sterol metabolism

Inhibition of 3‐Hydroxy‐3‐Methylglutaryl‐CoA Reductase and Citrate Synthase by Sulfoxides and Sulfones of Substrate‐analogue CoA‐thioether Derivatives

Clofarabine 5′-di and -triphosphates inhibit human ribonucleotide reductase by altering the quaternary structure of its large subunit

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