Synthesis and Biological Evaluation of 1,3-Dideazapurine-Like 7-Amino-5-Hydroxymethyl-Benzimidazole Ribonucleoside Analogues as Aminoacyl-tRNA Synthetase Inhibitors.

Aminoacyl-tRNA synthetases (aaRSs) have become viable targets for the development of antimicrobial agents due to their crucial role in protein translation. A series of six amino acids were coupled to the purine-like 7-amino-5-hydroxymethylbenzimidazole nucleoside analogue following an optimized synthetic pathway. These compounds were designed as aaRS inhibitors and can be considered as 1,3-dideazaadenine analogues carrying a 2-hydroxymethyl substituent. Despite our intentions to obtain N1-glycosylated 4-aminobenzimidazole congeners, resembling the natural purine nucleosides glycosylated at the N9-position, we obtained the N3-glycosylated benzimidazole derivatives as the major products, resembling the respective purine N7-glycosylated nucleosides. A series of X-ray crystal structures of class I and II aaRSs in complex with newly synthesized compounds revealed interesting interactions of these "base-flipped" analogues with their targets. While the exocyclic amine of the flipped base mimics the reciprocal interaction of the N3-purine atom of aminoacyl-sulfamoyl adenosine (aaSA) congeners, the hydroxymethyl substituent of the flipped base apparently loses part of the standard interactions of the adenine N1 and the N6-amine as seen with aaSA analogues. Upon the evaluation of the inhibitory potency of the newly obtained analogues, nanomolar inhibitory activities were noted for the leucine and isoleucine analogues targeting class I aaRS enzymes, while rather weak inhibitory activity against the corresponding class II aaRSs was observed. This class bias could be further explained by detailed structural analysis.

Synthesis and structural insights into the binding mode of the albomycin δ1 core and its analogues in complex with their target aminoacyl-tRNA synthetase.

Despite of proven efficacy and well tolerability, albomycin is not used clinically due to scarcity of material. Several attempts have been made to increase the production of albomycin by chemical or biochemical methods. In the current study, we have synthesized the active moiety of albomycin δ1 and investigated its binding mode to its molecular target seryl-trna synthetase (SerRS). In addition, isoleucyl and aspartyl congeners were prepared to investigate whether the albomycin scaffold can be extrapolated to target other aminoacyl-tRNA synthetases (aaRSs) from both class I and class II aaRSs, respectively. The synthesized analogues were evaluated for their ability to inhibit the corresponding aaRSs by an in vitro aminoacylation experiment using purified enzymes. It was observed that the diastereomer having the 5'S, 6'R-configuration (nucleoside numbering) as observed in the crystal structure, exhibits excellent inhibitory activity in contrast to poor activity of its companion 5'R,6'S-diasteromer obtained as byproduct during synthesis. Moreover, the albomycin core scaffold seems well tolerated for class II aaRSs inhibition compared with class I aaRSs. To understand this bias, we studied X-ray crystal structures of SerRS in complex with the albomycin δ1 core structure 14a, and AspRS in complex with compound 16a. Structural analysis clearly showed that diastereomer selectivity is attributed to the steric restraints of the active site of SerRS and AspRS.

Structural Insights into the Binding of Natural Pyrimidine-Based Inhibitors of Class II Aminoacyl-tRNA Synthetases.

The pyrimidine-containing Trojan horse antibiotics albomycin and a recently discovered cytidine-containing microcin C analog target the class II seryl- and aspartyl-tRNA synthetases (serRS and aspRS), respectively. The active components of these compounds are competitive inhibitors that mimic the aminoacyl-adenylate intermediate. How they effectively substitute for the interactions mediated by the canonical purine group is unknown. Employing nonhydrolyzable aminoacyl-sulfamoyl nucleosides substituting the base with cytosine, uracil, and N3-methyluracil the structure-activity relationship of the natural compounds was evaluated. In vitro using E. coli serRS and aspRS, the best compounds demonstrated IC50 values in the low nanomolar range, with a clear preference for cytosine or N3-methyluracil over uracil. X-ray crystallographic structures of K. pneumoniae serRS and T. thermophilus aspRS in complex with the compounds showed the contribution of structured waters and residues in the conserved motif-2 loop in defining base preference. Utilizing the N3-methyluracil bound serRS structure, MD simulations of the fully modified albomycin base were performed to identify the interacting network that drives stable association. This analysis pointed to key interactions with a methionine in the motif-2 loop. Interestingly, this residue is mutated to a glycine in a second serRS (serRS2) found in albomycin-producing actinobacteria possessing self-immunity to this antibiotic. A comparative study demonstrated that serRS2 is poorly inhibited by the pyrimidine-containing intermediate analogs, and an equivalent mutation in E. coli serRS significantly decreased the affinity of the cytosine congener. These findings highlight the crucial role of dynamics and solvation of the motif-2 loop in modulating the binding of the natural antibiotics.

Synthesis and Biological Evaluation of Lipophilic Nucleoside Analogues as Inhibitors of Aminoacyl-tRNA Synthetases.

Emerging antibiotic resistance in pathogenic bacteria and reduction of compounds in the existing antibiotics discovery pipeline is the most critical concern for healthcare professionals. A potential solution aims to explore new or existing targets/compounds. Inhibition of bacterial aminoacyl-tRNA synthetase (aaRSs) could be one such target for the development of antibiotics. The aaRSs are a group of enzymes that catalyze the transfer of an amino acid to their cognate tRNA and therefore play a pivotal role in translation. Thus, selective inhibition of these enzymes could be detrimental to microbes. The 5'-O-(N-(L-aminoacyl)) sulfamoyladenosines (aaSAs) are potent inhibitors of the respective aaRSs, however due to their polarity and charged nature they cannot cross the bacterial membranes. In this work, we increased the lipophilicity of these existing aaSAs in an effort to promote their penetration through the bacterial membrane. Two strategies were followed, either attaching a (permanent) alkyl moiety at the adenine ring via alkylation of the N6-position or introducing a lipophilic biodegradable prodrug moiety at the alpha-terminal amine, totaling eight new aaSA analogues. All synthesized compounds were evaluated in vitro using either a purified Escherichia coli aaRS enzyme or in presence of total cellular extract obtained from E. coli. The prodrugs showed comparable inhibitory activity to the parent aaSA analogues, indicating metabolic activation in cellular extracts, but had little effect on bacteria. During evaluation of the N6-alkylated compounds against different microbes, the N6-octyl containing congener 6b showed minimum inhibitory concentration (MIC) of 12.5 µM against Sarcina lutea while the dodecyl analogue 6c displayed MIC of 6.25 µM against Candida albicans.

Comparative analysis of pyrimidine substituted aminoacyl-sulfamoyl nucleosides as potential inhibitors targeting class I aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases (aaRSs) catalyse the ATP-dependent coupling of an amino acid to its cognate tRNA. Being vital for protein translation aaRSs are considered a promising target for the development of novel antimicrobial agents. 5'-O-(N-aminoacyl)-sulfamoyl adenosine (aaSA) is a non-hydrolysable analogue of the aaRS reaction intermediate that has been shown to be a potent inhibitor of this enzyme family but is prone to chemical instability and enzymatic modification. In an attempt to improve the molecular properties of this scaffold we synthesized a series of base substituted aaSA analogues comprising cytosine, uracil and N3-methyluracil targeting leucyl-, tyrosyl- and isoleucyl-tRNA synthetases. In in vitro assays seven out of the nine inhibitors demonstrated Kiapp values in the low nanomolar range. To complement the biochemical studies, X-ray crystallographic structures of Neisseria gonorrhoeae leucyl-tRNA synthetase and Escherichia coli tyrosyl-tRNA synthetase in complex with the newly synthesized compounds were determined. These highlighted a subtle interplay between the base moiety and the target enzyme in defining relative inhibitory activity. Encouraged by this data we investigated if the pyrimidine congeners could escape a natural resistance mechanism, involving acetylation of the amine of the aminoacyl group by the bacterial N-acetyltransferases RimL and YhhY. With RimL the pyrimidine congeners were less susceptible to inactivation compared to the equivalent aaSA, whereas with YhhY the converse was true. Combined the various insights resulting from this study will pave the way for the further rational design of aaRS inhibitors.

Family-wide analysis of aminoacyl-sulfamoyl-3-deazaadenosine analogues as inhibitors of aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases (aaRSs) are enzymes that precisely attach an amino acid to its cognate tRNA. This process, which is essential for protein translation, is considered a viable target for the development of novel antimicrobial agents, provided species selective inhibitors can be identified. Aminoacyl-sulfamoyl adenosines (aaSAs) are potent orthologue specific aaRS inhibitors that demonstrate nanomolar affinities in vitro but have limited uptake. Following up on our previous work on substitution of the base moiety, we evaluated the effect of the N3-position of the adenine by synthesizing the corresponding 3-deazaadenosine analogues (aaS3DAs). A typical organism has 20 different aaRS, which can be split into two distinct structural classes. We therefore coupled six different amino acids, equally targeting the two enzyme classes, via the sulfamate bridge to 3-deazaadenosine. Upon evaluation of the inhibitory potency of the obtained analogues, a clear class bias was noticed, with loss of activity for the aaS3DA analogues targeting class II enzymes when compared to the equivalent aaSA. Evaluation of the available crystallographic structures point to the presence of a conserved water molecule which could have importance for base recognition within class II enzymes, a property that can be explored in future drug design efforts.

Synthetic strategy and antiviral evaluation of diamide containing heterocycles targeting dengue and yellow fever virus.

High-throughput screening of a subset of the CD3 chemical library (Centre for Drug Design and Discovery; KU Leuven) provided us with a lead compound 1, displaying low micromolar potency against dengue virus and yellow fever virus. Within a project aimed at discovering new inhibitors of flaviviruses, substitution of its central imidazole ring led to synthesis of variably substituted pyrazine dicarboxylamides and phthalic diamides, which were evaluated in cell-based assays for cytotoxicity and antiviral activity against the dengue virus (DENV) and yellow fever virus (YFV). Fourteen compounds inhibited DENV replication (EC50 ranging between 0.5 and 3.4 µM), with compounds 6b and 6d being the most potent inhibitors (EC50 0.5 µM) with selectivity indices (SI) > 235. Compound 7a likewise exhibited anti-DENV activity with an EC50 of 0.5 µM and an SI of >235. In addition, good antiviral activity of seven compounds in the series was also noted against the YFV with EC50 values ranging between 0.4 and 3.3 µM, with compound 6n being the most potent for this series with an EC50 0.4 µM and a selectivity index of >34. Finally, reversal of one of the central amide bonds as in series 13 proved deleterious to the inhibitory activity.

Renaissance in Antibiotic Discovery: Some Novel Approaches for Finding Drugs to Treat Bad Bugs.

With the alarming resistance to currently used antibiotics, there is a serious worldwide threat to public health. Therefore, there is an urgent need to search for new antibiotics or new cellular targets which are essential for survival of the pathogens. However, during the past 50 years, only two new classes of antibiotics (oxazolidinone and lipopeptides) have reached the clinic. This suggests that the success rate in discovering new/novel antibiotics using conventional approaches is limited and that we must reconsider our antibiotic discovery approaches. While many new strategies are being pursued lately, this review primarily focuses only on a few of these novel/new approaches for antibiotic discovery. These include structure-based drug design (SBDD), the genomic approach, anti-virulence strategy, targeting nonmultiplying bacteria and the use of bacteriophages. In general, recent advancements in nuclear magnetic resonance, Xcrystallography, and genomic evolution have significant impact on antibacterial drug research. This review therefore aims to discuss recent strategies in searching new antibacterial agents making use of these technical novelties, their advantages, disadvantages and limitations.

N-acylated sulfonamide congeners of fosmidomycin lack any inhibitory activity against DXR.

The antibiotic fosmidomycin (3a) is an inhibitor of the non-mevalonate pathway for isoprenoid biosynthesis. Four analogues in which an acylated sulfonamide group is substituting for its phosphonate moiety have been synthesized in a fruitless effort to preserve one negative charge in order to increase the accompanying affinity for 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), the fosmidomycin target enzyme.

5'-(N-aminoacyl)-sulfonamido-5'-deoxyadenosine: attempts for a stable alternative for aminoacyl-sulfamoyl adenosines as aaRS inhibitors.

Synthesis of aminoacyl-sulfamoyl adenosines (aaSAs) and their peptidyl conjugates as aminoacyl tRNA synthetase (aaRS) inhibitors remains problematic due to the low yield of the aminoacylation and the subsequent conjugation reaction causing concomitant formation of a cyclic adenosine derivative. In an effort to reduce this undesirable side reaction, we aimed to prepare the corresponding aminoacyl sulfonamide (aaSoA) analogues as more stable alternatives for aaSA derivatives. Deletion of the 5'-oxygen in aaSA analogues should render the C-5' less electrophilic and therefore improve the stability of the aminoacyl sulfamate analogues. We therefore synthesized six sulfonamides and compared their activity against the respective aaSA analogues. However, except for the aspartyl derivative, the new compounds are not able to inhibit the corresponding aaRS. Possible reasons for this loss of activity are discussed by modeling and comparison of the newly synthesized aaSoA derivatives with their parent aaSA analogues.

Base substituted 5'-O-(N-isoleucyl)sulfamoyl nucleoside analogues as potential antibacterial agents.

Aminoacyl-sulfamoyl adenosines are well-known nanomolar inhibitors of the corresponding prokaryotic and eukaryotic tRNA synthetases in vitro. Inspired by the aryl-tetrazole containing compounds of Cubist Pharmaceuticals and the modified base as found in the natural antibiotic albomycin, the selectivity issue of the sulfamoylated adenosines prompted us to investigate the pharmacophoric importance of the adenine base. We therefore synthesized and evaluated several isoleucyl-sulfamoyl nucleoside analogues with either uracil, cytosine, hypoxanthine, guanine, 1,3-dideaza-adenine (benzimidazole) or 4-nitro-benzimidazole as the heterocyclic base. Based on the structure and antibacterial activity of microcin C, we also prepared their hexapeptidyl conjugates in an effort to improve their uptake potential. We further compared their antibacterial activity with the parent isoleucyl-sulfamoyl adenosine (Ile-SA), both in in vitro and in cellular assays. Surprisingly, the strongest in vitro inhibition was found for the uracil containing analogue 16f. Unfortunately, only very weak growth inhibitory properties were found as of low uptake. The results are discussed in the light of previous literature findings.

N-alkylated aminoacyl sulfamoyladenosines as potential inhibitors of aminoacylation reactions and microcin C analogues containing D-amino acids.

Microcin C analogues were recently envisaged as important compounds for the development of novel antibiotics. Two issues that may pose problems to these potential antibiotics are possible acquisition of resistance through acetylation and in vivo instability of the peptide chain. N-methylated aminoacyl sulfamoyladenosines were synthesized to investigate their potential as aminoacyl tRNA synthetase inhibitors and to establish whether these N-alkylated analogues would escape the natural inactivation mechanism via acetylation of the alpha amine. It was shown however, that these compounds are not able to effectively inhibit their respective aminoacyl tRNA synthetase. In addition, we showed that (D)-aspartyl-sulfamoyladenosine (i.e. with a (D)-configuration for the aspartyl moiety), is a potent inhibitor of aspartyl tRNA synthetase. However, we also showed that the inhibitory effect of (D)- aspartyl-sulfamoyladenosine is relatively short-lasting. Microcin C analogues with (D)-amino acids throughout from positions two to six proved inactive. They were shown to be resistant against metabolism by the different peptidases and therefore not able to release the active moiety. This observation could not be reversed by incorporation of (L)-amino acids at position six, showing that none of the available peptidases exhibit endopeptidase activity.

Aminoacyl-tRNA synthetase inhibitors as antimicrobial agents: a patent review from 2006 till present.

INTRODUCTION: Aminoacyl-tRNA synthetases (aaRSs) are one of the leading targets for development of antimicrobial agents. Although these enzymes are well conserved among prokaryotes, significant divergence has occurred between prokaryotic and eukaryotic aaRSs, which can be exploited in the discovery of broad-spectrum antibacterial agents. Although several aaRS inhibitors have been reported before, they failed as a result of poor selectivity and limited cell penetration. AREAS COVERED: This review covers January 2006 to April 2012 wherein several new analogues were claimed as aaRS inhibitors. Anacor Pharmaceuticals patented several boron-containing derivatives inhibiting the function of the editing domain of aaRSs. Two patents describe the combination of aaRS inhibitors with other antibacterial agents. Patents disclosing aaRS inhibitors for indications other than antimicrobial agents are not considered for review here. EXPERT OPINION: Several recently disclosed leads may form the foundation for development of potent and selective bacterial aaRS inhibitors. In comparison with, for example, terbinafine and itraconazole, compound C10 (AN2690) is a very promising candidate for treatment of ungual and periungual infections with improved nail penetration and low keratin binding. In addition, Raplidyne, Inc. reported bicyclic heteroaromatic compounds as potent and selective inhibitors of bacterial MetRS. These have proven to be particularly effective for treatment of Clostridium difficile-associated diarrhea. Finally, combination of aaRS inhibitors to attenuate resistance looks as a viable strategy to expand the lifespan of existing antibiotics.

Microcin C and albomycin analogues with aryl-tetrazole substituents as nucleobase isosters are selective inhibitors of bacterial aminoacyl tRNA synthetases but lack efficient uptake.

In 1998, Cubist Pharmaceuticals patented a series of aminoacyl tRNA synthetase (aaRS) inhibitors based on aminoacyl sulfamoyladenosines (aaSAs), in which the adenine was substituted by aryl-tetrazole moieties linked to the ribose fragment by a two-carbon spacer. Although potent and specific inhibitors of bacterial IleRS, these compounds did not prove successful in vivo due to low cell permeability and strong binding to serum albumin. In this work, we attempted to improve these compounds by combining them with microcin C (McC) or albomycin (i.e., siderophore-drug conjugate (SDC)) transport modules. We found that aryl-tetrazole variants of McC and albomycin still lacked antibacterial activity. However, these compounds were readily processed by E. coli aminopeptidases with the release of toxic aaRS inhibitors. Hence, the lack of activity in whole-cell assays was due to an inability of the new compounds to be taken up by the cells, thus indicating that the nucleotide moieties of McC and albomycin strongly contribute to facilitated transport of these compounds inside the cell.