1,4-Disubstituted imidazoles are potential antibacterial agents functioning as inhibitors of enoyl acyl carrier protein reductase (FabI)

1,4-Disubstituted imidazole inhibitors of Staphylococcus aureus and Escherichia coli enoyl acyl carrier protein reductase (FabI) have been identified. Crystal structure data shows the inhibitor 1 bound in the enzyme active site of E. coli FabI.     

Structure of acyl carrier protein bound to FabI, the FASII enoyl reductase from Escherichia coli

Acyl carrier proteins play a central role in metabolism by transporting substrates in a wide variety of pathways including the biosynthesis of fatty acids and polyketides. However, despite their importance, there is a paucity of direct structural information concerning the interaction of ACPs with enzymes in these pathways. Here we report the structure of an acyl-ACP substrate bound to the Escherichia coli fatty acid biosynthesis enoyl reductase enzyme (FabI), based on a combination of x-ray crystallography and molecular dynamics simulation. The structural data are in agreement with kinetic studies on wild-type and mutant FabIs, and reveal that the complex is primarily stabilized by interactions between acidic residues in the ACP helix alpha2 and a patch of basic residues adjacent to the FabI substrate-binding loop. Unexpectedly, the acyl-pantetheine thioester carbonyl is not hydrogen-bonded to Tyr(156), a conserved component of the short chain alcohol dehydrogenase/reductase superfamily active site triad. FabI is a proven target for drug discovery and the present structure provides insight into the molecular determinants that regulate the interaction of ACPs with target proteins.     

A pathogenic fungi diphenyl ether phytotoxin targets plant enoyl (acyl carrier protein) reductase

Cyperin is a natural diphenyl ether phytotoxin produced by several fungal plant pathogens. At high concentrations, this metabolite inhibits protoporphyrinogen oxidase, a key enzyme in porphyrin synthesis. However, unlike its herbicide structural analogs, the mode of action of cyperin is not light dependent, causing loss of membrane integrity in the dark. We report that this natural diphenyl ether inhibits Arabidopsis (Arabidopsis thaliana) enoyl (acyl carrier protein) reductase (ENR). This enzyme is also sensitive to triclosan, a synthetic antimicrobial diphenyl ether. Whereas cyperin was much less potent than triclosan on this target site, their ability to cause light-independent disruption of membrane integrity and inhibition of ENR is similar at their respective phytotoxic concentrations. The sequence of ENR is highly conserved within higher plants and a homology model of Arabidopsis ENR was derived from the crystal structure of the protein from Brassica napus. Cyperin mimicked the binding of triclosan in the binding pocket of ENR. Both molecules were stabilized by the pi-pi stacking interaction between one of their phenyl rings and the nicotinamide ring of the NAD(+). Furthermore, the side chain of tyrosine is involved in hydrogen bonding with a phenolic hydroxy group of cyperin. Therefore, cyperin may contribute to the virulence of the pathogens by inhibiting ENR and destabilizing the membrane integrity of the cells surrounding the point of infection.     

Structural elucidation of the specificity of the antibacterial agent triclosan for malarial enoyl acyl carrier protein reductase.

The human malaria parasite Plasmodium falciparum synthesizes fatty acids using a type II pathway that is absent in humans. The final step in fatty acid elongation is catalyzed by enoyl acyl carrier protein reductase, a validated antimicrobial drug target. Here, we report the cloning and expression of the P. falciparum enoyl acyl carrier protein reductase gene, which encodes a 50-kDa protein (PfENR) predicted to target to the unique parasite apicoplast. Purified PfENR was crystallized, and its structure resolved as a binary complex with NADH, a ternary complex with triclosan and NAD(+), and as ternary complexes bound to the triclosan analogs 1 and 2 with NADH. Novel structural features were identified in the PfENR binding loop region that most closely resembled bacterial homologs; elsewhere the protein was similar to ENR from the plant Brassica napus (root mean square for Calphas, 0.30 A). Triclosan and its analogs 1 and 2 killed multidrug-resistant strains of intra-erythrocytic P. falciparum parasites at sub to low micromolar concentrations in vitro. These data define the structural basis of triclosan binding to PfENR and will facilitate structure-based optimization of PfENR inhibitors.     

Kinetic and structural characteristics of the inhibition of enoyl (acyl carrier protein) reductase by triclosan.

Triclosan is used widely as an antibacterial agent in dermatological products, mouthwashes, and toothpastes. Recent studies imply that antibacterial activity results from binding to enoyl (acyl carrier protein) reductase (EACPR, EC 1.3.1.9). We first recognized the ability of triclosan to inhibit EACPR from Escherichia coli in a high throughput screen where the enzyme and test compound were preincubated with NAD(+), which is a product of the reaction. The concentration of triclosan required for 50% inhibition approximates to 50% of the enzyme concentration, indicating that the free compound is depleted by binding to EACPR. With no preincubation or added NAD(+), the degree of inhibition by 150 nM triclosan increases gradually over several minutes. The onset of inhibition is more rapid when NAD(+) is added. Gel filtration and mass spectrometry show that inhibition by triclosan is reversible. Steady-state assays were designed to avoid depletion of free inhibitor and changes in the degree of inhibition. The results suggest that triclosan binds to E-NAD(+) complex, with a dissociation constant around 20-40 pM. Triclosan follows competitive kinetics with respect to NADH, giving an inhibition constant of 38 pM at zero NADH and saturating NAD(+). Uncompetitive kinetics are observed when NAD(+) is varied, giving an inhibition constant of 22 pM at saturating NAD(+). By following regain of catalytic activity after dilution of EACPR that had been preincubated with triclosan and NAD(+), the rate constant for dissociation of the inhibitor (k(off)) is measured as 1.9 x 10(-4) s(-1). The association rate constant (k(on)) is estimated as 2.6 x 10(7) s(-1) M(-1) by monitoring the onset of inhibition during assays started by addition of EACPR. As expected, the ratio k(off)/k(on) = 7.1 pM is similar to the inhibition constants from the steady-state studies. The crystal structure of E. coli EACPR in a complex with coenzyme and triclosan has been determined at 1.9 A resolution, showing that this compound binds in a similar site to the diazaborine inhibitors. The high affinity of triclosan appears to be due to structural similarity to a tightly bound intermediate in catalysis.     

Synthesis of new 9-glycosyl-4,9-dihydropyrano [3,4-b]indole-1(3H)-ones as antibacterial agents

A series of new 9-glycosyl-4,9-dihydropyrano[3,4-b]indole-1(3H)-ones 3 was synthesized in moderate to low yields. 4,9-Dihydropyrano[3,4-b]indole-1(3H)-ones (1) were coupled with different acetobromoglycopyranoses 2 in refluxing toluene in the presence of silver oxide to afford one coupling product of the respective N-glycosides. α-L-Arabinopyranosides 3j and 3m were the most active glycosides among the tested compounds against certain Gram positive and Gram negative bacterial strains.     

Development of gallic acid formazans as novel enoyl acyl carrier protein reductase inhibitors for the treatment of tuberculosis

The enoyl acyl carrier protein reductase (InhA) of Mycobacterium tuberculosis (MTB) is an attractive target for developing novel antitubercular agents. A series of gallic acid formazans, were computationally designed and docked into the active site of InhA to understand their binding mode and potential to inhibit InhA. Nine compounds from the designed series were identified as potential InhA inhibitors, on the basis of good Glide score. These compounds were synthesized in the laboratory and evaluated for in vitro antitubercular activity against drug-sensitive and multi-drug resistant strains of MTB. Out of nine compounds, three compounds exhibited the most promising MIC of <2 μM against the sensitive strain of MTB, H37Rv. The compounds were evaluated against five resistant strains of MTB. Most of the compounds exhibited activity superior to the standard, linezolid, against all these resistant strains. The mechanism of action of these compounds was concluded to be InhA inhibition, through InhA enzyme inhibition study. Insignificant cytotoxicity of these compounds was observed on RAW 264.7 cell line. Inactivity of all these compounds against gram positive and gram negative bacteria indicated their specificity against MTB. The compounds were further analyzed for ADME properties and showed potential as good oral drug candidates. The results clearly identified some novel, selective and specific InhA inhibitors against sensitive and resistant strains of MTB.     

Synthesis and structure-activity relationships of 5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-based hydroxamic acids as HB-EGF shedding inhibitors

HB-EGF Shedding inhibitors have been expected to become effective medicines for skin diseases caused by the proliferation of keratinocytes. In order to discover novel HB-EGF shedding inhibitors and clarify their structure-activity relationships, 5,6,7,8-tetrahydronaphthylidine-based hydroxamic acid and 5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-based hydroxamic acids have been synthesized. Among the synthesized compounds, the ethoxyethoxy derivative 3o and the methoxypropoxy derivative 3p exhibited much more potent HB-EGF shedding inhibitory activity than CGS 27023A. The structural modification of 5,6,7,8-tetrahydropyrido[3,4-b]pyrazine-based hydroxamic acids enabled us to establish the following structure-activity relationships; the existence of the hydroxamic acid, the sulfonamide, and the phenyl moieties are crucial for a potent HB-EGF shedding inhibitory activity, and the stereochemistry of the alpha carbon of hydroxamic acid is also important. In addition, from the comparison of their HB-EGF shedding inhibitory activities with their MMPs inhibitory activities, we found that the S1' pocket of the responsible enzyme for HB-EGF shedding is deep unlike that of MMP-1.     

2,3,4,5-Tetrahydro-1H-pyrido[2,3-b and e][1,4]diazepines as inhibitors of the bacterial enoyl ACP reductase, FabI

In the search for new antibacterial agents, the enzyme FabI has been identified as an attractive target. Employing a structure guided approach, the previously reported ene-amide series of FabI inhibitors were expanded to include 2,3,4,5-tetrahydro-1H-pyrido[2,3-b and e][1,4]diazepines. These novel series incorporate additional H-bonding functions and can be more water soluble than their naphthyridinone progenitors; diazepine 16c is shown to be efficacious in a mouse infection model.     

Structure-activity relationship for a series of 2-substituted 1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indoles: potent subtype-selective inhibitors of N-methyl-D-aspartate (NMDA) receptors.

A series of 2-substituted 1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indoles was synthesized as potential antagonists for the NR1A/2B subtype of N-methyl-D-aspartate (NMDA) receptors. Assayed by electrical recording under steady-state conditions, 7-hydroxy-2-(4-phenylbutyl)- 1,2,3,4-tetrahydropyrido-[3,4-b]indole (30) was the most potent compound in the series having an IC50 value of 50 nM at the NR1A/2B receptors.     

X-ray structural analysis of Plasmodium falciparum enoyl acyl carrier protein reductase as a pathway toward the optimization of triclosan antimalarial efficacy

The x-ray crystal structures of five triclosan analogs, in addition to that of the isoniazid-NAD adduct, are described in relation to their integral role in the design of potent inhibitors of the malarial enzyme Plasmodium falciparum enoyl acyl carrier protein reductase (PfENR). Many of the novel 5-substituted analogs exhibit low micromolar potency against in vitro cultures of drug-resistant and drug-sensitive strains of the P. falciparum parasite and inhibit purified PfENR enzyme with IC50 values of <200 nM. This study has significantly expanded the knowledge base with regard to the structure-activity relationship of triclosan while affording gains against cultured parasites and purified PfENR enzyme. In contrast to a recent report in the literature, these results demonstrate the ability to improve the in vitro potency of triclosan significantly by replacing the suboptimal 5-chloro group with larger hydrophobic moieties. The biological and x-ray crystallographic data thus demonstrate the flexibility of the active site and point to future rounds of optimization to improve compound potency against purified enzyme and intracellular Plasmodium parasites.     

Inactivation mechanism of the beta-ketoacyl-[acyl carrier protein] reductase of bacterial type-II fatty acid synthase by epigallocatechin gallate.

Epigallocatechin gallate (EGCG), a major compound from green tea, reversibly inhibits beta-ketoacyl-[acyl carrier protein] reductase (FabG) from Escherichia coli. In this study, we found that EGCG exhibited an atypical time-dependent inhibition of FabG, which possibly resulted from the EGCG-induced aggregation of FabG. It was observed that FabG inactivation and aggregation occurred nearly simultaneously, with a lag time that decreased with increasing EGCG concentration. These results suggest that some chemical reactions, required for aggregation and inactivation, occurred during the lag time. Since EGC was detected by HPLC after the incubation of EGCG with FabG, EGCG probably covalently modified FabG. These further results showed that 1 tetramer of FabG must be modified by several, possibly 4, EGCG molecules before the formation of FabG aggregates. FabG aggregation was a first-order reaction independent of protein concentration. Due to an initial lag time, the first-order rate of aggregation gradually increased, reaching a maximal and constant value. The effect of increasing concentration of EGCG on the first-order rate constant for aggregation indicated that EGCG bound to FabG by affinity labeling. Based on the results, we propose a mechanism for the interaction of EGCG with FabG:EGCG first binds reversibly to each subunit of FabG, followed by covalent modification and then aggregation of the 4 EGCG-modified subunits.     

Synthesis and biological evaluation of novel isoxazolo[4,3-e]indoles as antibacterial agents

The synthesis of a new series of 8-bromo-6-alkyl-1-aryl-6H-isoxazolo[4,3-e]indole derivatives is described. All the newly synthesized compounds were screened for their antibacterial activity against Escherichia coli HB101, Staphylococcus aureus pathogens (methicillin resistant S. aureus and methicillin susceptible S. aureus), Pseudomonas aeruginosa, and Bacillus subtilis; also MIC values of these compounds were determined.     

Synthesis, molecular modeling and QSAR studies in chiral 2,3-disubstituted-1,2,3,4-tetrahydro-9H-pyrido(3,4-b)indoles as potential modulators of opioid antinociception.

In view of coexistence of opioid and cholecystokinin (CCK) in the brain areas concerned with pain processing, some semirigid racemic and chiral analogues of a potent CCK receptor antagonist (benzotript) have been synthesized and tested for their modulatory role on opioid antinociception, which may be mediated by CCK-B receptor. Some of these compounds, 3e, 3g, 3h, 4a, 4b and 4h, exhibited antinociceptive potentiation comparable to benzotript and proglumide. In order to identify the essential chemical structural features important for this potentiation, molecular modeling and quantitative structure activity relationship (QSAR) studies have been carried out in the S and R enantiomers of some of these semi-rigid compounds. The 3D-biophore models, common to all molecules of the training set have been derived. These models with superimposition (match value >0.25) depicted three biophoric sites one each for, pi/hydrophobic interactions, hydrogen bonding and ionic interactions among the phenyl/pyrrole ring, indole nitrogen, amidic oxygen, pyridyl nitrogen and lone pair of amidic oxygen. The total hydrophobicity and S absolute stereochemistry are found to positively contribute to potentiation of antinociception induced by morphine and the resulting quantitative pharmacophoric model with good correlation is found to well describe the observed activity.     

An efficient synthesis of highly functionalized novel chromeno[4,3-b]pyrroles and indolizino[6,7-b]indoles as potent antimicrobial and antioxidant agents

A facile and efficient synthesis of novel chromeno[4,3-b]pyrroles has been accomplished by intramolecular 1,3-dipolar cycloaddition which on subsequent Pictet-Spengler cyclisation in presence of p-toluenesulfonic acid yielded indolizino[6,7-b]indoles. The synthesized chromenopyrroles and indolizinoindoles were evaluated for their antimicrobial and antioxidant activities. Compounds 7b, 7e, 7a and 7d exhibited respectively, good antibacterial and antifungal activities against tested pathogens when compared to reference control.     

Synthesis and evaluation of pyrazolo[3,4-b]pyridine CDK1 inhibitors as anti-tumor agents

A series of 3,5-disubstituted pyrazolo[3,4-b]pyridine cyclin-dependent kinase (CDK) inhibitors was synthesized. These compounds showed potent and selective CDK inhibitory activities and inhibited in vitro cellular proliferation in cultured human tumor cells. Selected compounds were evaluated in an in vivo tumor xenograft model. The synthesis and biological evaluation of these pyrazolo[3,4-b]pyridines and related compounds are reported.     

Structure of beta-ketoacyl-[acyl carrier protein] reductase from Escherichia coli: negative cooperativity and its structural basis.

The structure of beta-ketoacyl-[acyl carrier protein] reductase (FabG) from Escherichia coli was determined via the multiwavelength anomalous diffraction technique using a selenomethionine-labeled crystal containing 88 selenium sites in the asymmetric unit. The comparison of the E. coli FabG structure with the homologous Brassica napus FabG.NADP(+) binary complex reveals that cofactor binding causes a substantial conformational change in the protein. This conformational change puts all three active-site residues (Ser 138, Tyr 151, and Lys 155) into their active configurations and provides a structural mechanism for allosteric communication between the active sites in the homotetramer. FabG exhibits negative cooperative binding of NADPH, and this effect is enhanced by the presence of acyl carrier protein (ACP). NADPH binding also increases the affinity and decreases the maximum binding of ACP to FabG. Thus, unlike other members of the short-chain dehydrogenase/reductase superfamily, FabG undergoes a substantial conformational change upon cofactor binding that organizes the active-site triad and alters the affinity of the other substrate-binding sites in the tetrameric enzyme.     

Synthesis and biological activity of diaryl ether inhibitors of malarial enoyl acyl carrier protein reductase. Part 2: 2'-substituted triclosan derivatives

2'-Substituted analogs of triclosan have been synthesized to target inhibition of the key malarial enzyme Plasmodium falciparum enoyl acyl carrier protein reductase (PfENR). Many of these compounds exhibit good potency (EC50<500 nM) against in vitro cultures of drug-resistant and drug-sensitive strains of the P. falciparum parasite and modest (IC50=1-20 microM) potency against purified PfENR enzyme. Compared to triclosan, this survey of 2'-substituted derivatives has afforded gains in excess of 20- and 30-fold versus the 3D7 and Dd2 strains of parasite, respectively.     

Synthesis and biological evaluation of novel alkyl amide functionalized trifluoromethyl substituted pyrazolo[3,4-b]pyridine derivatives as potential anticancer agents

A series of novel alkyl amide functionalized trifluoromethyl substituted pyrazolo[3,4-b]pyridine derivatives 5, 6 and 7 were prepared starting from 6-phenyl-4-(trifluoromethyl)-1H-pyrazolo[3,4-b]pyridin-3-amine 3 via selective N-alkylation, followed by reaction with different primary aliphatic amines, cyclic secondary amines or l-amino acids under different set of conditions. All the synthesized compounds 5, 6 and 7 were screened for anticancer activity against four cancer cell lines such as A549—Lung cancer (CCL-185), MCF7—Breast cancer (HTB-22), DU145—Prostate cancer (HTB-81) and HeLa—Cervical cancer (CCL-2). The compounds 5i and 6e are found to have promising bioactivity at micro molar concentration.