Development of a natural product optimization strategy for inhibitors against MraY, a promising antibacterial target.

MraY (phospho-N-acetylmuramoyl-pentapeptide-transferase) inhibitory natural products are attractive molecules as candidates for a new class of antibacterial agents to combat antimicrobial-resistant bacteria. Structural optimization of these natural products is required to improve their drug-like properties for therapeutic use. However, chemical modifications of these natural products are painstaking tasks due to complex synthetic processes, which is a bottleneck in advancing natural products to the clinic. Here, we develop a strategy for a comprehensive in situ evaluation of the build-up library, which enables us to streamline the preparation of the analogue library and directly assess its biological activities. We apply this approach to a series of MraY inhibitory natural products. Through construction and evaluation of the 686-compound library, we identify promising analogues that exhibit potent and broad-spectrum antibacterial activity against highly drug-resistant strains in vitro as well as in vivo in an acute thigh infection model. Structures of the MraY-analogue complexes reveal distinct interaction patterns, suggesting that these analogues represent MraY inhibitors with unique binding modes. We further demonstrate the generality of our strategy by applying it to tubulin-binding natural products to modulate their tubulin polymerization activities.

Discovery of a Novel Fluoroquinolone Antibiotic Candidate WFQ-228 with Potent Antimicrobial Activity and the Potential to Overcome Major Drug Resistance.

WFQ-101 with a unique N-1 substituent, 5-amino-4-fluoro-2-(hydroxymethyl)phenyl group, was selected as a lead compound through combination screening based on antimicrobial activity and the efflux index against quinolone-resistant (QR) Pseudomonas aeruginosa (P. aeruginosa). Through structural optimization, we identified WFQ-228 as a novel fluoroquinolone antibiotic candidate. WFQ-228 had potent and superior activity in comparison to levofloxacin (LVX) and ciprofloxacin (CIP) against clinical isolates of P. aeruginosa, Escherichia coli and Acinetobacter baumannii, including QR strains. Furthermore, WFQ-228 demonstrated the potential to overcome major mechanisms of drug resistance; its antimicrobial activity was less affected by both pump-mediated efflux and mutations of the quinolone resistance-determining region in P. aeruginosa compared with LVX and CIP. These results suggest that WFQ-228 is a promising candidate for further evaluation in the treatment of infections caused by QR Gram-negative pathogens.

Evaluation of the Effects of S-Allyl-L-cysteine, S-Methyl-L-cysteine, trans-S-1-Propenyl-L-cysteine, and Their N-Acetylated and S-Oxidized Metabolites on Human CYP Activities.

Three major organosulfur compounds of aged garlic extract, S-allyl-L-cysteine (SAC), S-methyl-L-cysteine (SMC), and trans-S-1-propenyl-L-cysteine (S1PC), were examined for their effects on the activities of five major isoforms of human CYP enzymes: CYP1A2, 2C9, 2C19, 2D6, and 3A4. The metabolite formation from probe substrates for the CYP isoforms was examined in human liver microsomes in the presence of organosulfur compounds at 0.01-1 mM by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Allicin, a major component of garlic, inhibited CYP1A2 and CYP3A4 activity by 21-45% at 0.03 mM. In contrast, a CYP2C9-catalyzed reaction was enhanced by up to 1.9 times in the presence of allicin at 0.003-0.3 mM. SAC, SMC, and S1PC had no effect on the activities of the five isoforms, except that S1PC inhibited CYP3A4-catalyzed midazolam 1'-hydroxylation by 31% at 1 mM. The N-acetylated metabolites of the three compounds inhibited the activities of several isoforms to a varying degree at 1 mM. N-Acetyl-S-allyl-L-cysteine and N-acetyl-S-methyl-L-cysteine inhibited the reactions catalyzed by CYP2D6 and CYP1A2, by 19 and 26%, respectively, whereas trans-N-acetyl-S-1-propenyl-L-cysteine showed weak to moderate inhibition (19-49%) of CYP1A2, 2C19, 2D6, and 3A4 activities. On the other hand, both the N-acetylated and S-oxidized metabolites of SAC, SMC, and S1PC had little effect on the reactions catalyzed by the five isoforms. These results indicated that SAC, SMC, and S1PC have little potential to cause drug-drug interaction due to CYP inhibition or activation in vivo, as judged by their minimal effects (IC50>1 mM) on the activities of five major isoforms of human CYP in vitro.

Pharmacokinetics and N-acetylation metabolism of S-methyl-l-cysteine and trans-S-1-propenyl-l-cysteine in rats and dogs.

1. Pharmacokinetics and N-acetylation metabolism of S-methyl-L-cysteine (SMC) and trans-S-1-propenyl-L-cysteine (S1PC) were examined in rats and dogs. SMC and S1PC (2-5 mg/kg) were well absorbed in both species with high bioavailability (88-100%). 2. SMC and S1PC were excreted only to a small extent in the urine of rats and dogs. The small renal clearance values (<0.03 l/h/kg) indicated the extensive renal reabsorption of SMC and S1PC, which potentially contributed to their long elimination half-lives (>5 h) in dogs. 3. S1PC, but not SMC, underwent N-acetylation extensively in vivo, which can be explained by the relative activities of N-acetylation of S1PC/SMC and deacetylation of their N-acetylated forms, N-acetyl-S1PC/N-acetyl-SMC, in the liver and kidney in vitro. The activities for S1PC N-acetylation were similar to or higher than those for N-acetyl-S1PC deacetylation in liver S9 fractions of rat and dog, whereas liver and kidney S9 fractions of rat and dog had little activity for SMC N-acetylation or considerably higher activities for N-acetyl-SMC deacetylation. 4. Our study demonstrated that the pharmacokinetics of SMC and S1PC in rats and dogs was characterized by high bioavailability and extensive renal reabsorption; however, the extent of undergoing the N-acetylation metabolism was extremely different between SMC and S1PC.

Pharmacokinetics of S-Allyl-l-cysteine in Rats Is Characterized by High Oral Absorption and Extensive Renal Reabsorption.

BACKGROUND: S-Allylcysteine (SAC) is a key component of aged garlic extract, one of many garlic products. However, information on its pharmacokinetics has been scant except for data from a few animal studies. OBJECTIVE: We designed this study to determine the overall pharmacokinetics of SAC in rats. METHODS: After oral or intravenous administration of SAC to rats at a dose of 5 mg/kg, the plasma concentration-time profile of SAC and its metabolites, as well as the amounts excreted in bile and urine, were analyzed by using liquid chromatography tandem mass spectrometry. RESULTS: After oral administration, SAC was well absorbed with a bioavailability of 98%. Two major metabolites of SAC, N-acetyl-S-allylcysteine (NAc-SAC) and N-acetyl-S-allylcysteine sulfoxide (NAc-SACS), were detected in plasma, but their concentrations were markedly lower than those of SAC. SAC was metabolized to a limited extent, but most of the orally absorbed SAC was excreted into urine in the form of its N-acetylated metabolites. The amounts of SAC, NAc-SAC, and NAc-SACS excreted in urine over 24 h were 2.9%, 80%, and 11% of the orally administered SAC, respectively. The very low renal clearance (0.016 L ⋅ h(-1) ⋅ kg(-1)) of SAC indicated that it undergoes extensive renal reabsorption. These results collectively suggested that SAC was ultimately metabolized to NAc-SAC and NAc-SACS through the cycles of urinary excretion, renal reabsorption, and systemic recirculation. CONCLUSION: The pharmacokinetics of SAC in rats were characterized by high oral absorption, limited metabolism, and extensive renal reabsorption, all of which potentially contribute to its high and relatively long-lasting plasma concentrations.

Discovery of WQ-3810: Design, synthesis, and evaluation of 7-(3-alkylaminoazetidin-1-yl)fluoro-quinolones as orally active antibacterial agents.

Novel 7-(3-alkylaminoazetidin-1-yl)fluoroquinolones were designed, synthesized, and evaluated for their antibacterial activities and oral absorption rates. Against Gram-negative bacteria, 10a-e, which have various alkyl groups containing different numbers of carbon atoms (C0-C3) at the C-7 alkylaminoazetidine position, showed potent and similar antibacterial activities, whereas the activity of 10f (C4, t-Bu) was significantly lower than those of 10a-e. Conversely, the oral absorption rates of 10a-e in rats increased depending on the number of carbon atoms in the alkyl groups; 10d (C3, n-Pr) and 10e (C3, i-Pr) had high oral absorption rates (>90% at 10 mg/kg). These results demonstrated that the introduction of alkyl groups onto C-7 aminoazetidine is useful for the improvement of the oral absorption rates of these drugs while maintaining their antibacterial activities. As a conclusion, from this series of fluoroquinolones, WQ-3810 (10e), having 3-isopropylaminoazetidine as the C-7 substituent, was identified as an orally active antibacterial agent with a potent in vitro activity.

Metabolism, excretion, and pharmacokinetics of S-allyl-L-cysteine in rats and dogs.

The metabolism, excretion, and pharmacokinetics of S-allyl-l-cysteine (SAC), an active key component of garlic supplements, were examined in rats and dogs. A single dose of SAC was administered orally or i.v. to rats (5 mg/kg) and dogs (2 mg/kg). SAC was well absorbed (bioavailability >90%) and its four metabolites-N-acetyl-S-allyl-l-cysteine (NAc-SAC), N-acetyl-S-allyl-l-cysteine sulfoxide (NAc-SACS), S-allyl-l-cysteine sulfoxide (SACS), and l-γ-glutamyl-S-allyl-l-cysteine-were identified in the plasma and/or urine. Renal clearance values (<0.01 l/h/kg) of SAC indicated its extensive renal reabsorption, which contributed to the long elimination half-life of SAC, especially in dogs (12 hours). The metabolism of SAC to NAc-SAC, principal metabolite of SAC, was studied in vitro and in vivo. Liver and kidney S9 fractions of rats and dogs catalyzed both N-acetylation of SAC and deacetylation of NAc-SAC. After i.v. administration of NAc-SAC, SAC appeared in the plasma and its concentration declined in parallel with that of NAc-SAC. These results suggest that the rate and extent of the formation of NAc-SAC are determined by the N-acetylation and deacetylation activities of liver and kidney. Also, NAc-SACS was detected in the plasma after i.v. administration of either NAc-SAC or SACS, suggesting that NAc-SACS could be formed via both N-acetylation of SACS and S-oxidation of NAc-SAC. In conclusion, this study demonstrated that the pharmacokinetics of SAC in rats and dogs is characterized by its high oral bioavailability, N-acetylation and S-oxidation metabolism, and extensive renal reabsorption, indicating the critical roles of liver and kidney in the elimination of SAC.

In vitro activity of WQ-3810, a novel fluoroquinolone, against multidrug-resistant and fluoroquinolone-resistant pathogens.

The aim of this study was to examine the in vitro antibacterial activity of WQ-3810, a new fluoroquinolone, against clinically relevant pathogens such as Acinetobacter baumannii, Escherichia coli and Streptococcus pneumoniae, including multidrug-resistant (MDR) and fluoroquinolone-resistant (FQR) isolates, compared with those of ciprofloxacin, levofloxacin, moxifloxacin and gemifloxacin. WQ-3810 demonstrated the most potent activity against the antimicrobial-resistant pathogens tested. Against A. baumannii, including MDR isolates, the potency of WQ-3810 [minimum inhibitory concentration for 90% of the organisms (MIC(90))=1 mg/L] was more than eight-fold higher than that of ciprofloxacin (64 mg/L) and levofloxacin (8 mg/L). Against E. coli and S. pneumoniae, including FQR isolates, WQ-3810 (MIC(90)=4 mg/L and 0.06 mg/L, respectively) was also more active than ciprofloxacin (64 mg/L and 2 mg/L) and levofloxacin (32 mg/L and 2 mg/L). Furthermore, WQ-3810 was the most potent among the fluoroquinolones tested against meticillin-resistant Staphylococcus aureus (MRSA) and Neisseria gonorrhoeae, including FQR isolates. In particular, WQ-3810 demonstrated highly potent activity against FQR isolates of A. baumannii, E. coli and S. pneumoniae with amino acid mutation(s) in the quinolone resistance-determining region of DNA gyrase and/or topoisomerase IV, which are the target enzymes of fluoroquinolones. An enzyme inhibition study performed using FQR E. coli DNA gyrase suggested that the potent antibacterial activity of WQ-3810 against drug-resistant isolates partly results from the strong inhibition of the target enzymes. In conclusion, this study demonstrated that WQ-3810 exhibits extremely potent antibacterial activity over the existing fluoroquinolones, particularly against MDR and FQR pathogens.

The mechanism causing the difference in kinetic properties between rat CYP2D4 and human CYP2D6 in the oxidation of dextromethorphan and bufuralol.

The capacity to oxidize bufuralol (BF) and dextromethorphan (DEX) was compared kinetically between human CYP2D6 and four rat CYP2D (CYP2D1, -2D2, -2D3 and -2D4) isoenzymes in a yeast cell expression system. In BF 1''-hydroxylation and DEX O-demethylation, only CYP2D4 showed hook-shaped Eadie-Hofstee plots, the other four CYP2D enzymes exhibiting linear plots. In DEX N-demethylation, rat CYP2D2 did not show any detectable activity under the conditions used, whereas the other four enzymes yielded linear Eadie-Hofstee plots. To elucidate the mechanisms causing the nonlinear kinetics, four CYP2D4 mutants, CYP2D4-F109I, -V123F, -L216F and -A486F, were prepared. CYP2D4-V123F, -L216F and -A486F yielded linear or linear-like Eadie-Hofstee plots for BF 1''-hydroxylation, whereas only CYP2D4-A486F exhibited linear plots for DEX O-demethylation. The substitution of Phe-109 by isoleucine did not have any effect on the oxidative capacity of CYP2D4 for either BF or DEX. These results suggest that the introduction of phenylalanine in the active-site cavity of CYP2D4 simplifies complicated interactions between the substrates and the amino acid residues, but the mechanisms causing the simplification differ between BF and DEX.

The roles of amino acid residues at positions 216 and 219 in the structural stability and metabolic functions of rat cytochrome P450 2D1 and 2D2.

We examined the effects of the mutual substitution of amino acid residues at positions 216 and 219 between rat CYP2D1 and CYP2D2 on their microsomal contents and enzymatic functions using a yeast cell expression system and 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) as a substrate. CYP2D1 has amino acid residues, leucine and valine, at positions of 216 and 219, respectively, whereas CYP2D2 has phenylalanine and aspartic acid at the same positions. In reduced carbon monoxide-difference spectroscopic analysis, the substitution of Asp-219 of CYP2D2 by valine markedly increased a peak at 450 nm and concomitantly decreased a peak at 420 nm, while the replacement of Phe-216 of CYP2D2 with leucine gave no observable change. The double substitution of Phe-216 and Asp-219 by leucine and valine, respectively, yielded a typical CYP spectrum. The substitution of Val-219 of CYP2D1 by aspartic acid decreased the CYP content to one-half, whereas the replacement of Leu-216 with phenylalanine did not have any effect. The double substitution of Leu-216 and Val-219 of CYP2D1 by phenylalanine and aspartic acid, respectively, diminished the CYP content by 90%. CYP2D1 catalyzed both 5-MeO-DIPT N-deisopropylation and O-demethylation at relatively low levels, while CYP2D2 catalyzed 5-MeO-DIPT O-demethylation efficiently. The substitution of the amino acid at position 216 substantially increased 5-MeO-DIPT oxidation activities of the two CYP2D enzymes. The replacement of the amino acid at position 219 increased the 5-MeO-DIPT O- and N-dealkylation activities of CYP2D1, whereas it decreased the 5-MeO-DIPT O-demethylation activity of CYP2D2. These results indicate that amino acid residues at positions 216 and 219 have important roles in the enzymatic functions of rat CYP2D1 and CYP2D2.