Retinol binding protein 4 antagonists and protein synthesis inhibitors: Potential for therapeutic development.

Retinol-binding protein 4 (RBP4) is a serum protein that transports Vitamin A. RBP4 is correlated with numerous diseases and metabolic syndromes, including insulin resistance in type 2 diabetes, cardiovascular diseases, obesity, and macular degeneration. Recently, RBP4 antagonists and protein synthesis inhibitors are under development to regulate the effect of RBP4. Several RBP4 antagonists, especially BPN-14136, have demonstrated promising safety profiles and potential therapeutic benefits in animal studies. Two RBP4 antagonists, specifically tinlarebant (Belite Bio) and STG-001 (Stargazer) are currently undergoing clinical trials. Some antidiabetic drugs and nutraceuticals have been reported to reduce RBP4 expression, but more clinical data is needed to evaluate their therapeutical benefits. As regulating RBP4 levels or its activities would benefit a wide range of patients, further research is highly recommended to develop clinically useful RBP4 antagonists or protein synthesis inhibitors.

Identifying the Cellular Target of Cordyheptapeptide A and Synthetic Derivatives.

Cordyheptapeptide A is a lipophilic cyclic peptide from the prized Cordyceps fungal genus that shows potent cytotoxicity in multiple cancer cell lines. To better understand the bioactivity and physicochemical properties of cordyheptapeptide A with the ultimate goal of identifying its cellular target, we developed a solid-phase synthesis of this multiply N-methylated cyclic heptapeptide which enabled rapid access to both side chain- and backbone-modified derivatives. Removal of one of the backbone amide N-methyl (N-Me) groups maintained bioactivity, while membrane permeability was also preserved due to the formation of a new intramolecular hydrogen bond in a low dielectric solvent. Based on its cytotoxicity profile in the NCI-60 cell line panel, as well as its phenotype in a microscopy-based cytological assay, we hypothesized that cordyheptapeptide was acting on cells as a protein synthesis inhibitor. Further studies revealed the molecular target of cordyheptapeptide A to be the eukaryotic translation elongation factor 1A (eEF1A), a target shared by other lipophilic cyclic peptide natural products. This work offers a strategy to study and improve cyclic peptide natural products while highlighting the ability of these lipophilic compounds to effectively inhibit intracellular disease targets.

Search for Shorter Portions of the Proline-Rich Antimicrobial Peptide Fragment Bac5(1-25) That Retain Antimicrobial Activity by Blocking Protein Synthesis.

The spread of antibiotic-resistant pathogens has boosted the search for new antimicrobial drugs. Proline-rich antimicrobial peptides are promising lead compounds for the development of next-generation antibiotics, given their very low cytotoxicity and their good antimicrobial activity targeting the bacterial ribosome. Bac5(1-25) is an N-terminal fragment of the bovine proline-rich antimicrobial peptide Bac5, whose mode of action has been recently described. In this work we tested a number of Bac5(1-25) fragments, and we characterized their antimicrobial activity against Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus, Salmonella enterica, and Pseudomonas aeruginosa. We evaluated their cytotoxicity toward human cells and their efficacy in inhibiting bacterial protein synthesis. This allowed us to identify some shorter fragments of Bac5(1-25) with a good balance between antibacterial efficacy, protein synthesis inhibition, and ease/cost-effectiveness of synthesis, suitable as lead compounds to develop new antibacterials.

Large-scale preparation of key building blocks for the manufacture of fully synthetic macrolide antibiotics.

Key building blocks for the production of fully synthetic macrolides have been scaled-up in first time pilot plant and kilo-lab campaigns. These building blocks have supported the discovery of new macrolide antibiotics as well as ongoing preclinical studies.

Dual effect of chloramphenicol peptides on ribosome inhibition.

Chloramphenicol peptides were recently established as useful tools for probing nascent polypeptide chain interaction with the ribosome, either biochemically, or structurally. Here, we present a new 10mer chloramphenicol peptide, which exerts a dual inhibition effect on the ribosome function affecting two distinct areas of the ribosome, namely the peptidyl transferase center and the polypeptide exit tunnel. According to our data, the chloramphenicol peptide bound on the chloramphenicol binding site inhibits the formation of both acetyl-phenylalanine-puromycin and acetyl-lysine-puromycin, showing, however, a decreased peptidyl transferase inhibition compared to chloramphenicol-mediated inhibition per se. Additionally, we found that the same compound is a strong inhibitor of green fluorescent protein synthesis in a coupled in vitro transcription-translation assay as well as a potent inhibitor of lysine polymerization in a poly(A)-programmed ribosome, showing that an additional inhibitory effect may exist. Since chemical protection data supported the interaction of the antibiotic with bases A2058 and A2059 near the entrance of the tunnel, we concluded that the extra inhibition effect on the synthesis of longer peptides is coming from interactions of the peptide moiety of the drug with residues comprising the ribosomal tunnel, and by filling up the tunnel and blocking nascent chain progression through the restricted tunnel. Therefore, the dual interaction of the chloramphenicol peptide with the ribosome increases its inhibitory effect and opens a new window for improving the antimicrobial potency of classical antibiotics or designing new ones.

Inhibition of protein synthesis and malaria parasite development by drug targeting of methionyl-tRNA synthetases.

Aminoacyl-tRNA synthetases (aaRSs) are housekeeping enzymes that couple cognate tRNAs with amino acids to transmit genomic information for protein translation. The Plasmodium falciparum nuclear genome encodes two P. falciparum methionyl-tRNA synthetases (PfMRS), termed PfMRS(cyt) and PfMRS(api). Phylogenetic analyses revealed that the two proteins are of primitive origin and are related to heterokonts (PfMRS(cyt)) or proteobacteria/primitive bacteria (PfMRS(api)). We show that PfMRS(cyt) localizes in parasite cytoplasm, while PfMRS(api) localizes to apicoplasts in asexual stages of malaria parasites. Two known bacterial MRS inhibitors, REP3123 and REP8839, hampered Plasmodium growth very effectively in the early and late stages of parasite development. Small-molecule drug-like libraries were screened against modeled PfMRS structures, and several "hit" compounds showed significant effects on parasite growth. We then tested the effects of the hit compounds on protein translation by labeling nascent proteins with (35)S-labeled cysteine and methionine. Three of the tested compounds reduced protein synthesis and also blocked parasite growth progression from the ring stage to the trophozoite stage. Drug docking studies suggested distinct modes of binding for the three compounds, compared with the enzyme product methionyl adenylate. Therefore, this study provides new targets (PfMRSs) and hit compounds that can be explored for development as antimalarial drugs.

Importance of the 6'-hydroxy group and its configuration for apramycin activity.

A series of apramycin derivatives was prepared and investigated for antibacterial activity and the ability to inhibit protein synthesis in cell-free translation assays. The effect of various modifications at the 6'- and N7'-positions on antiribosomal activity is discussed in terms of their influence on drug binding to specific residues in the decoding A-site. These studies contribute to the development of a structure-activity relationship for the antibacterial activity of the apramycin class of aminoglycosides and to the future design and development of more active and less toxic antibiotics.

Functionalization of tetracycline and evaluation of its antibacterial activity including against resistant bacteria.

Ever growing resistance of pathogenic bacteria against the existing antibiotics has forced researchers to look for new methods and techniques to design effective antimicrobial agents. In the present study a new tetracycline-based antimicrobial polymer (AMP) was synthesized from tetracycline and methacrylic acid (MAAc) using lipase as catalyst. The AMP, thus obtained, was transformed into nanoparticles via an emulsion method. The AMP and its nano-form were characterized by FTIR, NMR, XRD, SEM and EDAX. The antibacterial activity of the AMP was studied against both resistant (-) [P. aeruginosa] and susceptible (+) [S. aureus] bacteria. The synthesized AMP, including its nanoform, was observed to be more potent and efficient antimicrobial agent than the precursor tetracycline.

Antibacterial activity of new oxazolidin-2-one analogues in methicillin-resistant Staphylococcus aureus strains.

Staphylococcus aureus is one of the most common causes of nosocomial infections. The purpose of this study was the synthesis and in vitro evaluation of antimicrobial activity of 10 new 3-oxazolidin-2-one analogues on 12 methicillin resistant S. aureus (MRSA) clinical isolates. S. aureus confirmation was achieved via catalase and coagulase test. Molecular characterization of MRSA was performed by amplification of the mecA gene. Antimicrobial susceptibility was evaluated via the Kirby-Bauer disc diffusion susceptibility test protocol, using commonly applied antibiotics and the oxazolidinone analogues. Only (R)-5-((S)-1-dibenzylaminoethyl)-1,3-oxazolidin-2-one (7a) exhibited antibacterial activity at 6.6 µg. These results, allow us to infer that molecules such as 7a can be potentially used to treat infections caused by MRSA strains.

A novel ketolide, RBx 14255, with activity against multidrug-resistant Streptococcus pneumoniae.

We present here the novel ketolide RBx 14255, a semisynthetic macrolide derivative obtained by the derivatization of clarithromycin, for its in vitro and in vivo activities against sensitive and macrolide-resistant Streptococcus pneumoniae. RBx 14255 showed excellent in vitro activity against macrolide-resistant S. pneumoniae, including an in-house-generated telithromycin-resistant strain (S. pneumoniae 3390 NDDR). RBx 14255 also showed potent protein synthesis inhibition against telithromycin-resistant S. pneumoniae 3390 NDDR. The binding affinity of RBx 14255 toward ribosomes was found to be more than that for other tested drugs. The in vivo efficacy of RBx 14255 was determined in murine pulmonary infection induced by intranasal inoculation of S. pneumoniae ATCC 6303 and systemic infection with S. pneumoniae 3390 NDDR strains. The 50% effective dose (ED50) of RBx 14255 against S. pneumoniae ATCC 6303 in a murine pulmonary infection model was 3.12 mg/kg of body weight. In addition, RBx 14255 resulted in 100% survival of mice with systemic infection caused by macrolide-resistant S. pneumoniae 3390 NDDR at 100 mg/kg four times daily (QID) and at 50 mg/kg QID. RBx 14255 showed favorable pharmacokinetic properties that were comparable to those of telithromycin.

Asymmetric synthesis of the aminocyclitol pactamycin, a universal translocation inhibitor.

An asymmetric total synthesis of the aminocyclopentitol pactamycin is described. The title compound is delivered in 15 steps from 2,4-pentanedione. Critical to this approach was the exploitation of a complex symmetry-breaking reduction strategy to assemble the C1, C2, and C7 relative stereochemistry within the first four steps of the synthesis. Multiple iterations of this reduction strategy are described, and a thorough analysis of stereochemical outcomes is detailed. In the final case, an asymmetric Mannich reaction was developed to install a protected amine directly at the C2 position. Symmetry-breaking reduction of this material gave way to a remarkable series of stereochemical outcomes leading to the title compound without recourse to nonstrategic downstream manipulations. This synthesis is immediately accommodating to the preparation of structural analogs.

Synthesis of novel isoquinolinone and 1,2-dihydroisoquinoline scaffolds via Ugi reaction and ring opening reaction of furans.

Combining the Ugi reaction with ring opening reaction of furans for the synthesis of novel isoquinolinone and 1,2-dihydroisoquinoline scaffolds has been developed. The isoquinolinone and 1,2-dihydroisoquinoline derivatives with unsaturated carbonyl moiety may open up many opportunities for further functionalizations.

Synthetic silvestrol analogues as potent and selective protein synthesis inhibitors.

Misregulation of protein translation plays a critical role in human cancer pathogenesis at many levels. Silvestrol, a cyclopenta[b]benzofuran natural product, blocks translation at the initiation step by interfering with assembly of the eIF4F translation complex. Silvestrol has a complex chemical structure whose functional group requirements have not been systematically investigated. Moreover, silvestrol has limited development potential due to poor druglike properties. Herein, we sought to develop a practical synthesis of key intermediates of silvestrol and explore structure-activity relationships around the C6 position. The ability of silvestrol and analogues to selectively inhibit the translation of proteins with high requirement on the translation-initiation machinery (i.e., complex 5'-untranslated region UTR) relative to simple 5'UTR was determined by a cellular reporter assay. Simplified analogues of silvestrol such as compounds 74 and 76 were shown to have similar cytotoxic potency and better ADME characteristics relative to those of silvestrol.

Synthesis of N²-modified 7-methylguanosine 5'-monophosphates as nematode translation inhibitors.

Preparative scale synthesis of 14 new N(2)-modified mononucleotide 5' mRNA cap analogues was achieved. The key step involved use of an S(N)Ar reaction with protected 2-fluoro inosine and various primary and secondary amines. The derivatives were tested in a parasitic nematode, Ascaris suum, cell-free system as translation inhibitors. The most effective compound with IC(50) ∼0.9µM was a N(2)-p-metoxybenzyl-7-methylguanosine-5'-monophosphate 35.

Antibiotic optimization and chemical structure stabilization of thiomuracin A.

Synthetic studies of the antimicrobial secondary metabolite thiomuracin A (1) were initiated to improve chemical stability and physicochemical properties. Functional group modifications of 1 included removing the C2-C7 side chain, derivatizing the C84 epoxide region, and altering the C44 hydroxyphenylalanine motif. The resulting derivatives simplified and stabilized the chemical structure and were evaluated for antibacterial activity relative to 1. The simplified structure and improved organic solubility of the derivatives facilitated isolation yields from fermentation broths and simplified the procedures involved for the process. These advancements increased material supply for continued medicinal chemistry optimization and culminated in the identification of 2, a structurally simplified and chemically stable analogue of 1 which retained potent antibiotic activity.

Synthesis of rocaglamide hydroxamates and related compounds as eukaryotic translation inhibitors: synthetic and biological studies.

The rocaglates/rocaglamides are a class of natural products known to display potent anticancer activity. One such derivative, silvestrol, has shown activity comparable to taxol in certain settings. Here, we report the synthesis of various rocaglamide analogues and identification of a hydroxamate derivative (-)-9 having activity similar to silvestrol in vitro and ex vivo for inhibition of protein synthesis. We also show that (-)-9 synergizes with doxorubicin in vivo to reduce Eµ-Myc driven lymphomas.

Design, synthesis, and evaluation of novel fluoroquinolone-aminoglycoside hybrid antibiotics.

A series of new hybrid structures containing fluoroquinolone (ciprofloxacin) and aminoglycoside (neomycin) antibiotics linked via 1,2,3-triazole moiety were designed and synthesized, and their antibacterial activities were determined against both Gram-negative and Gram-positive bacteria, including resistant strains. The nature of spacers in both the ciprofloxacin and neomycin parts greatly influenced the antibacterial activity. The majority of hybrids was significantly more potent than the parent neomycin and overcame most prevalent types of resistance associated with aminoglycosides. Selected hybrids inhibited bacterial protein synthesis with the potencies similar to or better than that of neomycin and were up to 32-fold more potent inhibitors than ciprofloxacin for the fluoroquinolone targets, DNA gyrase and toposiomerase IV, indicating a balanced dual mode of action. Significant delay of resistance formation was observed in both E. coli and B. subtilis to the treatment with ciprofloxacin-neomycin hybrid in comparison to that of each drug separately or their 1:1 mixture.

Phosphorothioate analogs of m7GTP are enzymatically stable inhibitors of cap-dependent translation.

We report synthesis and properties of a pair of new potent inhibitors of translation, namely two diastereomers of 7-methylguanosine 5'-(1-thiotriphosphate). These new analogs of mRNA 5'cap (referred to as m(7)GTPalphaS (D1) and (D2)) are recognized by translational factor eIF4E with high affinity and are not susceptible to hydrolysis by Decapping Scavenger pyrophosphatase (DcpS). The more potent of diastereomers, m(7)GTPalphaS (D1), inhibited cap-dependent translation in rabbit reticulocyte lysate approximately 8-fold and approximately 15-fold more efficiently than m(7)GTP and m(7)GpppG, respectively. Both analogs were also significantly more stable in RRL than unmodified ones.

Synthetic studies toward sordarin: building blocks for the terpenoid core and for analogues thereof.

Avenues to bi- and tricyclic building blocks for the elaboration of sordaricin and its analogues are described. The target molecules were obtained through inter- and intramolecular Diels-Alder reactions of a number of previously unknown cyclopentadienes. Unusual properties of 3-cyanoenones and 1-cyanocyclopentadienes have been unveiled and circumvented.

FR290581, a novel sordarin derivative: synthesis and antifungal activity.

Sordarin is a unique natural product antifungal agent that is an inhibitor of elongation factor 2. To improve biological activity, we synthesized various compounds by novel modification of the aglycone, sordaricin. As a result, we have discovered the novel sordarin derivative FR290581. This compound exhibited superior activity and a good pharmacokinetic profile, and also displayed good in vivo activity against Candida albicans.