Micha Fridman

Exploring the Substrate Promiscuity of Drug-Modifying Enzymes for the Chemoenzymatic Generation of N-Acylated Aminoglycosides

Aminoglycosides are broad‐spectrum antibiotics commonly used for the treatment of serious bacterial infections. Decades of clinical use have led to the widespread emergence of bacterial resistance to this family of drugs limiting their efficacy in the clinic. Here, we report the development of a methodology that utilizes aminoglycoside acetyltransferases (AACs) and unnatural acyl coenzyme A analogues for the chemoenzymatic generation of N‐acylated aminoglycoside analogues. Generation of N‐acylated aminoglycosides is followed by a simple qualitative test to assess their potency as potential antibacterials. The studied AACs (AAC(6′)‐APH(2′′) and AAC(3)‐IV) show diverse substrate promiscuity towards a variety of aminoglycosides as well as acyl coenzyme A derivatives. The enzymes were also used for the sequential generation of homo‐ and hetero‐di‐N‐acylated aminoglycosides. Following the clinical success of the N‐acylated amikacin and arbekacin, our chemoenzymatic approach offers access to regioselectively N‐acylated aminoglycosides in quantities that allow testing of the antibacterial potential of the synthetic analogues making it possible to decide which molecules will be worth synthesizing on a larger scale.

6′′-Thioether Tobramycin Analogues: Towards Selective Targeting of Bacterial Membranes†

Decades of widespread clinical use of the bacterial ribosome A-site targeting aminoglycosides (AGs) enhanced the evolution of resistance to these antibiotics and reduced their clinical efficacy.[1] Three modes of action lead to bacterial resistance to AGs: reduction in the intracellular concentration of the antibiotics by efflux pump proteins or through reduced membrane permeability; structural modifications of the 16S ribosomal RNA leading to reduced target affinity; and deactivation by AG-modifying enzymes (AMEs).[1c, 2] AMEs are divided into three families: AG nucleotidyltransferases (ANTs), AG phosphotransferases (APHs), and AG acetyltransferases (AACs).[1b, 3]

Cationic Pillararenes Potently Inhibit Biofilm Formation without Affecting Bacterial Growth and Viability

It is estimated that up to 80% of bacterial infections are accompanied by biofilm formation. Since bacteria in biofilms are less susceptible to antibiotics than are bacteria in the planktonic state, biofilm-associated infections pose a major health threat, and there is a pressing need for antibiofilm agents. Here we report that water-soluble cationic pillararenes differing in the quaternary ammonium groups efficiently inhibited the formation of biofilms by clinically important Gram-positive pathogens. Biofilm inhibition did not result from antimicrobial activity; thus, the compounds should not inhibit growth of natural bacterial flora. Moreover, none of the cationic pillararenes caused detectable membrane damage to red blood cells or toxicity to human cells in culture. The results indicate that cationic pillararenes have potential for use in medical applications in which biofilm formation is a problem.

Design of membrane targeting tobramycin-based cationic amphiphiles with reduced hemolytic activity

Tobramycin-based cationic amphiphiles differing in the chemical bond linking their hydrophobic and hydrophilic parts were synthesized and biologically evaluated. Several compounds demonstrated potent antimicrobial activities compared to the parent drug. One analogue exhibited a significant reduction in red blood cells hemolysis, demonstrating that it is possible to maintain the antimicrobial potency of these molecules while reducing their undesired hemolytic effect through chemical modifications.

Tobramycin and Nebramine as Pseudo‐oligosaccharide Scaffolds for the Development of Antimicrobial Cationic Amphiphiles

Antimicrobial cationic amphiphiles derived from aminoglycoside pseudo-oligosaccharide antibiotics interfere with the structure and function of bacterial membranes and offer a promising direction for the development of novel antibiotics. Herein, we report the design and synthesis of cationic amphiphiles derived from the pseudo-trisaccharide aminoglycoside tobramycin and its pseudo-disaccharide segment nebramine. Antimicrobial activity, membrane selectivity, mode of action, and structure-activity relationships were studied. Several cationic amphiphiles showed marked antimicrobial activity, and one amphiphilic nebramine derivative proved effective against all of the tested strains of bacteria; furthermore, against several of the tested strains, this compound was well over an order of magnitude more potent than the parent antibiotic tobramycin, the membrane-targeting antimicrobial peptide mixture gramicidin D, and the cationic lipopeptide polymyxin B, which are in clinical use.

Synthesis and evaluation of hetero- and homodimers of ribosome-targeting antibiotics: antimicrobial activity, in vitro inhibition of translation, and drug resistance.

In this study, we describe the synthesis of a full set of homo- and heterodimers of three intact structures of different ribosome-targeting antibiotics: tobramycin, clindamycin, and chloramphenicol. Several aspects of the biological activity of the dimeric structures were evaluated including antimicrobial activity, inhibition of in vitro bacterial protein translation, and the effect of dimerization on the action of several bacterial resistance mechanisms that deactivate tobramycin and chloramphenicol. This study demonstrates that covalently linking two identical or different ribosome-targeting antibiotics may lead to (i) a broader spectrum of antimicrobial activity, (ii) improved inhibition of bacterial translation properties compared to that of the parent antibiotics, and (iii) reduction in the efficacy of some drug-modifying enzymes that confer high levels of resistance to the parent antibiotics from which the dimers were derived.

Di-N-Methylation of Anti-Gram-Positive Aminoglycoside-Derived Membrane Disruptors Improves Antimicrobial Potency and Broadens Spectrum to Gram-Negative Bacteria.

The effect of di-N-methylation of bacterial membrane disruptors derived from aminoglycosides (AGs) on antimicrobial activity is reported. Di-N-methylation of cationic amphiphiles derived from several diversely structured AGs resulted in a significant increase in hydrophobicity compared to the parent compounds that improved their interactions with membrane lipids. The modification led to an enhancement in antibacterial activity and a broader antimicrobial spectrum. While the parent compounds were either modestly active or inactive against Gram-negative pathogens, the corresponding di-N-methylated compounds were potent against the tested Gram-negative as well as Gram-positive bacterial strains. The reported modification offers a robust strategy for the development of broad-spectrum membrane-disrupting antibiotics for topical use.

Site-Selective Displacement of Tobramycin Hydroxyls for Preparation of Antimicrobial Cationic Amphiphiles

A short site-selective strategy for the activation and derivatization of alcohols of the clinically important aminoglycoside tobramycin is reported. The choice of amine protecting group affected the site-selective conversion of secondary alcohols of tobramycin into leaving groups. Temperature-dependent, chemoselective sequential nucleophilic displacements resulted in hetero- and homodithioether tobramycin-based cationic amphiphiles that demonstrated marked antimicrobial activity and impressive membrane selectivity.

Using biological performance similarity to inform disaccharide library design

Designing better small-molecule discovery libraries requires having methods to assess the consequences of different synthesis decisions on the biological performance of resulting library members. Since we are particularly interested in how stereochemistry affects performance in biological assays, we prepared a disaccharide library containing systematic stereochemical variations, assayed the library for different biological effects, and developed methods to assess the similarity of performance between members across multiple assays. These methods allow us to ask which subsets of stereochemical features best predict similarity in patterns of biological performance between individual members and which features produce the greatest variation of outcomes. We anticipate that the data-analysis approach presented here can be generalized to other sets of biological assays and other chemical descriptors. Methods to assess which structural features of library members produce the greatest similarity in performance for a given set of biological assays should help prioritize synthesis decisions in second-generation library development targeting the underlying cell-biological processes. Methods to assess which structural features of library members produce the greatest variation in performance should help guide decisions about what synthetic methods need to be developed to make optimal small-molecule screening collections.

Inhibition of glioma progression by a newly discovered CD38 inhibitor.

Glioma, the most common cancer of the central nervous system, has very poor prognosis and no effective treatment. It has been shown that activated microglia/macrophages in the glioma tumor microenvironment support progression. Hence, inhibition of the supporting effect of these cells may constitute a useful therapeutic approach. Recently, using a syngeneic mouse glioma progression model, we showed that the ectoenzyme CD38 regulated microglia activation and, in addition, that the loss of CD38 from the tumor microenvironment attenuated glioma progression and prolonged the life span of the tumor‐bearing mice. These studies, which employed wild‐type (WT) and Cd38−/− C57BL/6J mice, suggest that inhibition of CD38 in glioma microenvironment may be used as a new therapeutic approach to treat glioma. Our study tested this hypothesis. Initially, we found that the natural anthranoid, 4,5‐dihydroxyanthraquinone‐2‐carboxylic acid (rhein), and its highly water‐soluble tri‐potassium salt form (K‐rhein) are inhibitors of CD38 enzymatic (nicotinamide adenine dinucleotide glycohydrolase) activity (IC50 = 1.24 and 0.84 μM, respectively, for recombinant mouse CD38). Treatment of WT, but not Cd38−/− microglia with rhein and K‐rhein inhibited microglia activation features known to be regulated by CD38 (lipopolysaccharide/IFN‐γ‐induced activation, induced cell death and NO production). Furthermore, nasal administration of K‐rhein into WT, but not Cd38−/− C57BL/6J, mice intracranially injected with GL261 cells substantially and significantly inhibited glioma progression. Hence, these results serve as a proof of concept, demonstrating that targeting CD38 at the tumor microenvironment by small‐molecule inhibitors of CD38, for example, K‐rhein, may serve as a useful therapeutic approach to treat glioma.