The Fridman research group is engaged in the use of organic chemistry, especially carbohydrate chemistry, to solve key biological and medicinal problems. Our interdisciplinary studies involve the use of organic chemistry, microbiology, cell biology, molecular biology, and microscopy to study and/or alter the mode of action of biologically active molecules with the focus on novel approaches for the development of antimicrobial and antitumor agents.

Our current research covers three major topics: I) rational design of membrane targeting antibiotics, II) development of chemical approaches to circumvent the action of resistance mechanisms that compromise ribosome targeting antibiotics, and III) structure-function studies and rational design of novel antitumor agents.

 

To date, disruption of the bacterial membrane bi-layer has been poorly exploited as a strategy for the development of antibiotics. Bacterial membrane-disrupting antibiotics offer several advantages over antimicrobial agents that target intracellular bacterial targets. First, membrane disruption is not dependent on the bacterial cell cycle state and is therefore a promising strategy for the eradication of dormant bacteria and treatment of persistent infections. Second, antimicrobial agents that act outside the bacterial cell evade intracellular resistance mechanisms and are expected to have prolonged clinical efficacy. Finally, cell permeability, which is often a significant challenge for drug designers, is not necessary for the design of membrane-targeting antibiotics. A major challenge, however, lies in avoiding cytotoxicity to eukaryotic cells through non-selective membrane disruption. In contrast to most eukaryotic cell membranes, both Gram-positive and Gram-negative bacterial membranes are highly negatively charged due to a high content of anionic lipids such as cardiolipin and phosphatidylglycerol. The outer leaflet of the outer membrane of Gram-negative bacteria contains the negatively charged core of the lipopolysaccharide (LPS), whereas negatively charged techoic acids are major constituents of Gram-positive bacteria cell walls. Hence, both Gram-positive and Gram-negative bacterial membranes attract positively charged organic compounds through ionic interactions.

We have been particularly interested in using the scaffolds of positively charged aminoglycosides (AGs) for the development of membrane-targeting cationic amphiphilic antimicrobial agents by the attachment of hydrophobic residues to one or more positions on the AG. We have focused on the pseudo trisaccharide tobramycin (TOB), which is penta-positively charged under physiological conditions. So far we synthesized several families of amphiphilic aminoglycoside analogues by attaching a hydrophobic residue by modifying one or more of thier alcohols. Through microbiological tests as well as confocal microscopy, we found evidence that the most potent analogues target bacterial membranes; the parent drug targets the ribosome. To test the selectivity of these compounds for bacterial cells, we conducted hemolysis tests and demostrated that undesired hemolytic effects depended on aliphatic chain length and the type of chemical linkage between the hydrophilic and hydrophobic parts of the molecules.

i 1

A large percentage of the currently available repertoire of clinically used antibiotics acts by targeting the prokaryotic ribosome and inhibition of protein synthyesis. In this research program, we utilize the information obtained from the solved structure of the bacterial ribosome and recent year's progress in the understanding of how ribosome targeting antibiotics bind to their ribosomal target site for the development of novel families of antimicrobial agents. In designing the proposed novel ribosome-targeting antibiotics, we focus on strategies to evade several bacterial resistance mechanisms and offer new directions for structure based development of broad spectrum antibiotics.

The design of the novel antimicrobial agents is based on the incorporation of binding motifs from P-site and A-site targeting antibiotics and form novel molecules that will interact with the bacterial ribosome using novel sets of interactions. We study the protein synthesis inhibition potency of the novel antimicrobials by in-vitro and in-vivo translation assays. We also study the ability of these antimicrobials to evade the action of a broad variety of antibiotics deactivating enzymes which are currently one of the leading causes for the loss of clinical efficacy of clinically used ribosome-targeting antibiotics. The antimicrobial activity of the novel antibiotics is studied by several microbiological tests on a broad spectrum of pathogenic and drug resistant bacteria.

i 2

Anthraquinone-derived antitumor agents, such as doxorubicin and mitoxantrone, are widely used chemotherapeutic agents and are effective against a broad spectrum of both solid tumors and leukemias. The clinical importance of these drugs as antitumor agents is greatly overshadowed, however, by their dose limiting cardiotoxicity and the rapid evolution of tumor resistance to these chemotherapeutic agents.

Small chemical and structural differences between both natural and semi-synthetic members of the anthracycline family of antitumor agents greatly affect their pharmacokinetics, spectrum of efficacy, and limiting cardiotoxicity dose. The mechanism of action of these antitumor agents is likely through formation of a ternary complex between double-stranded DNA, the enzyme topoisomerase II, and the drug, which leads to the induction of apoptosis. However, these drugs have additional activities that hamper several essential cellular processes and result in fatal damage. The marked differences in the activity of anthracycline analogues with high structural similarities and the multiple cellular processes that are affected by these important chemotherapeutic agents suggest that it should be possible to determine the dominant cellular targets and modes of action through biological analysis of pre-designed chemical structures.


In this research program we design anthraquinone structures based on those of clinically used anthracyclines and study their mode of action in a set of molecular biology and cell biology assays. Incubation of tumor cells with some of the synthetic compounds induces cell death in less than one cell cycle indicating that these compounds do not directly target the cell division mechanism. We utilize the natural fluorescence of anthracyclines and their synthetic analogues for confocal microscopy studies and determine the effect of structural motifs on the sub-cellular target localization of these molecules and hence identify their possible cellular targets.

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