2-(3-Indolyl)acetamides and their oxazoline analogues: Anticancer SAR study.

We previously studied 2-aryl-2-(3-indolyl)acetohydroxamates as potential agents against melanoma. These compounds were ineffective in a mouse melanoma xenograft model, most likely due to unfavorable metabolic properties, specifically due to glucuronidation of the N-hydroxyl of the hydoxamic moiety. In the present work, we prepared a series of analogues, 2-aryl-2-(3-indolyl)acetamides and their oxazoline derivatives, which do not contain the N-hydroxyl group. We investigated the structure-activity relationship in both series of compounds and found that the 2-naphthyl is a preferred group at C-2 of the indole in the amide series, whereas the tetralin moiety is favorable in the same location in the oxazoline series. Overall, three compounds in the amide series have GI50 values as low as 0.2-0.3 µM and the results clearly indicate that the N-hydroxyl group is not necessary for high potency in vitro.

An antibiotic preorganized for ribosomal binding overcomes antimicrobial resistance.

We report the design conception, chemical synthesis, and microbiological evaluation of the bridged macrobicyclic antibiotic cresomycin (CRM), which overcomes evolutionarily diverse forms of antimicrobial resistance that render modern antibiotics ineffective. CRM exhibits in vitro and in vivo efficacy against both Gram-positive and Gram-negative bacteria, including multidrug-resistant strains of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. We show that CRM is highly preorganized for ribosomal binding by determining its density functional theory-calculated, solution-state, solid-state, and (wild-type) ribosome-bound structures, which all align identically within the macrobicyclic subunits. Lastly, we report two additional x-ray crystal structures of CRM in complex with bacterial ribosomes separately modified by the ribosomal RNA methylases, chloramphenicol-florfenicol resistance (Cfr) and erythromycin-resistance ribosomal RNA methylase (Erm), revealing concessive adjustments by the target and antibiotic that permit CRM to maintain binding where other antibiotics fail.

Structural basis of Cfr-mediated antimicrobial resistance and mechanisms to evade it.

The bacterial ribosome is an essential drug target as many clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent resistance mechanisms to PTC-acting drugs in Gram-positive bacteria is C8-methylation of the universally conserved A2503 nucleobase by Cfr methylase in 23S ribosomal RNA. Despite its clinical importance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. Here, we report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-transfer RNAs. These structures reveal an allosteric rearrangement of nucleotide A2062 upon Cfr-mediated methylation of A2503 that likely contributes to the reduced potency of some PTC inhibitors. Additionally, we provide the structural bases behind two distinct mechanisms of engaging the Cfr-methylated ribosome by the antibiotics iboxamycin and tylosin.

Structural basis of Cfr-mediated antimicrobial resistance and mechanisms for its evasion.

The ribosome is an essential drug target as many classes of clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent mechanisms of resistance to PTC-acting drugs is C8-methylation of the universally conserved adenine residue 2503 (A2503) of the 23S rRNA by the methyltransferase Cfr. Despite its clinical significance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. In this work, we developed a method to express a functionally-active Cfr-methyltransferase in the thermophilic bacterium Thermus thermophilus and report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-tRNAs. Our structures reveal that an allosteric rearrangement of nucleotide A2062 upon Cfr-methylation of A2503 is likely responsible for the inability of some PTC inhibitors to bind to the ribosome, providing additional insights into the Cfr resistance mechanism. Lastly, by determining the structures of the Cfr-methylated ribosome in complex with the antibiotics iboxamycin and tylosin, we provide the structural bases behind two distinct mechanisms of evading Cfr-mediated resistance.

Synthesis of ß-Carbolines with Electrocyclic Cyclization of 3-Nitrovinylindoles.

The ß-carboline motif is common in drug discovery and among numerous biologically active natural products. However, its synthetic preparation relies on multistep sequences and heavily depends on the type of substitution required in the core of the desired ß-carboline target. Herein, we demonstrate that this structural motif can be accessed with the microwave-assisted electrocyclic cyclization of heterotrienic aci (alkylideneazinic acid) forms of 3-nitrovinylindoles. The reaction can start with 3-nitrovinylindoles themselves under two sets of conditions. The first one involves microwave irradiation of butanolic solutions of 3-nitrovinylindoles, whereas the second one consists of prior Boc protection of indolic nitrogen, where the protecting group cleanly comes off during the course of the reaction. Alternatively, the reaction can start with 3-nitrovinylindoles prepared in situ using various processes. Finally, the reaction may utilize indoles with ß-nitrostyrenes, likely involving the intermediacy of spirocyclic oxazolines, which rearrange to similar heterotrienic systems undergoing cyclization to ß-carbolines. As part of this study, several natural products, namely, alkaloids norharmane, harmane, and eudistomin N, were synthesized.

Structure-Activity Relationships of the Antimicrobial Peptide Natural Product Apidaecin.

With the growing crisis of antimicrobial resistance, it is critical to continue to seek out new sources of novel antibiotics. This need has led to renewed interest in natural product antimicrobials, specifically antimicrobial peptides. Nonlytic antimicrobial peptides are highly promising due to their unique mechanisms of action. One such peptide is apidaecin (Api), which inhibits translation termination through stabilization of the quaternary complex of the ribosome-apidaecin-tRNA-release factor. Synthetic derivatives of apidaecin have been developed, but structure-guided modifications have yet to be considered. In this work, we have focused on modifying key residues in the Api sequence that are responsible for the interactions that stabilize the quaternary complex. We present one of the first examples of a highly modified Api peptide that maintains its antimicrobial activity and interaction with the translation complex. These findings establish a starting point for further structure-guided optimization of Api peptides.

One-Pot Synthesis of Polynuclear Indole Derivatives by Friedel-Crafts Alkylation of γ-Hydroxybutyrolactams.

The Friedel-Crafts reaction of novel 3,5-diarylsubstituted 5-hydroxy-1,5-dihydro-2H-pyrrol-2-ones was used for low cost, one-pot preparation of polycyclic indole derivatives structurally similar to Ergot alkaloids.

Insights into the ribosome function from the structures of non-arrested ribosome-nascent chain complexes.

During protein synthesis, the growing polypeptide threads through the ribosomal exit tunnel and modulates ribosomal activity by itself or by sensing various small molecules, such as metabolites or antibiotics, appearing in the tunnel. While arrested ribosome-nascent chain complexes (RNCCs) have been extensively studied structurally, the lack of a simple procedure for the large-scale preparation of peptidyl-tRNAs, intermediates in polypeptide synthesis that carry the growing chain, means that little attention has been given to RNCCs representing functionally active states of the ribosome. Here we report the facile synthesis of stably linked peptidyl-tRNAs through a chemoenzymatic approach based on native chemical ligation and use them to determine several structures of RNCCs in the functional pre-attack state of the peptidyl transferase centre. These structures reveal that C-terminal parts of the growing peptides adopt the same uniform ß-strand conformation stabilized by an intricate network of hydrogen bonds with the universally conserved 23S rRNA nucleotides, and explain how the ribosome synthesizes growing peptides containing various sequences with comparable efficiencies.

Nitrovinylindoles as Heterotrienes: Electrocyclic Cyclization En Route to ß-Carbolines: Total Synthesis of Alkaloids Norharmane, Harmane, and Eudistomin N.

Unusual cascade transformation was developed involving microwave assisted electrocyclic cyclization of aci (alkylideneazinic acid) forms of nitrovinylindoles acting as heterotrienes. Subsequent one-pot reduction allowed for efficient access to ß-carbolines, including several natural products, alkaloids norharmane, harmane and eudistomin N.

Structural basis for the inability of chloramphenicol to inhibit peptide bond formation in the presence of A-site glycine.

Ribosome serves as a universal molecular machine capable of synthesis of all the proteins in a cell. Small-molecule inhibitors, such as ribosome-targeting antibiotics, can compromise the catalytic versatility of the ribosome in a context-dependent fashion, preventing transpeptidation only between particular combinations of substrates. Classic peptidyl transferase center inhibitor chloramphenicol (CHL) fails to inhibit transpeptidation reaction when the incoming A site acceptor substrate is glycine, and the molecular basis for this phenomenon is unknown. Here, we present a set of high-resolution X-ray crystal structures that explain why CHL is unable to inhibit peptide bond formation between the incoming glycyl-tRNA and a nascent peptide that otherwise is conducive to the drug action. Our structures reveal that fully accommodated glycine residue can co-exist in the A site with the ribosome-bound CHL. Moreover, binding of CHL to a ribosome complex carrying glycyl-tRNA does not affect the positions of the reacting substrates, leaving the peptide bond formation reaction unperturbed. These data exemplify how small-molecule inhibitors can reshape the A-site amino acid binding pocket rendering it permissive only for specific amino acid residues and rejective for the other substrates extending our detailed understanding of the modes of action of ribosomal antibiotics.

Synthetic Studies toward 1,2,3,3a,4,8b-Hexahydropyrrolo[3,2-b]indole Core. Unusual Fragmentation with 1,2-Aryl Shift.

Base-assisted transformations of 2-(3-oxoindolin-2-yl)acetonitriles were investigated. Unexpectedly, attempted reactions of substrates possessing nonprotected nitrogen atoms were accompanied by unusual extrusions of 2-arylacetonitriles, followed by a 1,2-aryl shift to afford 3-hydroxyindolin-2-ones. On the other hand, the reactions for N-alkyl derivatives of oxoindolines took the expected route by only providing 1,2,3,3a,4,8b-hexahydropyrrolo[3,2-b]indoles.

Direct Conversion of 3-(2-Nitroethyl)-1H-Indoles into 2-(1H-Indol-2-yl)Acetonitriles.

The recently discovered [4+1]-spirocyclization of nitroalkenes to indoles provided a convenient new approach to 2-(1H-indol-2-yl)acetonitriles. However, this reaction was complicated by the formation of inert 3-(2-nitroethyl)-1H-indole byproducts. Herein, we offer a workaround this problem that allows for effective transformation of the unwanted byproducts into acetonitrile target molecules.

The Role of Release Factors in the Hydrolysis of Ester Bond in Peptidyl-tRNA.

Class I release factors (RFs) recognize stop codons in the sequences of mRNAs and are required for the hydrolysis of peptidyl-tRNA in the ribosomal P site during the final step of protein synthesis in bacteria, resulting in the release of a complete polypeptide chain from the ribosome. A key role in this process belongs to the highly conserved GGQ motif in RFs. Mutations in this motif can reduce the hydrolysis rate or even completely inhibit the reaction. Previously, it was hypothesized that the amino acid residues of GGQ (especially glutamine) are essential for the proper coordination of the water molecule for subsequent hydrolysis of the ester bond. However, available structures of the 70S ribosome termination complex do not allow unambiguous identification of the exact orientation of the carbonyl group in peptidyl-tRNA relative to the GGQ, as well as of the position of the catalytic water molecule in the peptidyl transferase center (PTC). This mini-review summarizes key facts and hypotheses on the role of GGQ in the catalysis of peptide release, as well as suggests and discusses future experiments aimed to produce high-quality structural data for deciphering the precise mechanism of RF-mediated catalysis.

Structure of Erm-modified 70S ribosome reveals the mechanism of macrolide resistance.

Many antibiotics inhibit bacterial growth by binding to the ribosome and interfering with protein biosynthesis. Macrolides represent one of the most successful classes of ribosome-targeting antibiotics. The main clinically relevant mechanism of resistance to macrolides is dimethylation of the 23S rRNA nucleotide A2058, located in the drug-binding site, a reaction catalyzed by Erm-type rRNA methyltransferases. Here, we present the crystal structure of the Erm-dimethylated 70S ribosome at 2.4 Å resolution, together with the structures of unmethylated 70S ribosome functional complexes alone or in combination with macrolides. Altogether, our structural data do not support previous models and, instead, suggest a principally new explanation of how A2058 dimethylation confers resistance to macrolides. Moreover, high-resolution structures of two macrolide antibiotics bound to the unmodified ribosome reveal a previously unknown role of the desosamine moiety in drug binding, laying a foundation for the rational knowledge-based design of macrolides that can overcome Erm-mediated resistance.

Preparation of spiro[indole-3,5'-isoxazoles] via Grignard conjugate addition/spirocyclization sequence.

A highly efficient one-pot procedure combining conjugate addition of Grignard reagents to (2-nitroalkenyl)indoles and sub-sequent Brønsted acid-assisted spirocyclization allowed for preparation of 4'H-spiro[indole-3,5'-isoxazoles] in a diastereomerically selective fashion. Utilization of alkyl Grignard reagents provided an easy access to 4'-alkylsubstituted derivatives hardly available by other means.

Preparation of Stereodefined 2-(3-Oxoindolin-2-yl)-2-Arylacetonitriles via One-Pot Reaction of Indoles with Nitroalkenes.

Recently discovered reactivity of nitrostyrenes in phosphorous acid to facilitate the diastereoselective [4 + 1]-cycloaddition of indoles in combination with unusual oxazoline ring cleavage and subsequent 1,2-alkyl shift afforded stereochemically defined 2-(3-oxoindolin-2-yl)-2-arylacetonitriles as sole products.

Synthesis of Spiro[indole-3,5'-isoxazoles] with Anticancer Activity via a Formal [4 + 1]-Spirocyclization of Nitroalkenes to Indoles.

An acid-assisted [4 + 1]-cycloaddition of indoles with nitrostyrenes affords 4' H-spiro[indole-3,5'-isoxazoles] in a diastereomerically pure form. Several of these spirocyclic molecules exhibit promising anticancer activity by reducing viability and inducing differentiation of neuroblastoma cells.

Outside Mainstream Electronic Databases: Review of Studies Conducted in the USSR and Post-Soviet Countries on Electric Current-Assisted Consolidation of Powder Materials.

This paper reviews research articles published in the former USSR and post-soviet countries on the consolidation of powder materials using electric current that passes through the powder sample and/or a conductive die-punch set-up. Having been published in Russian, many of the reviewed papers are not included in the mainstream electronic databases of the scientific articles and thus are not known to the scientific community. The present review is aimed at filling this information gap. In the paper, the electric current-assisted sintering techniques based on high- and low-voltage approaches are presented. The main results of the theoretical modeling of the processes of electromagnetic field-assisted consolidation of powder materials are discussed. Sintering experiments and related equipment are described and the major experimental results are analyzed. Sintering conditions required to achieve the desired properties of the sintered materials are provided for selected material systems. Tooling materials used in the electric current-assisted consolidation set-ups are also described.