Targeting FtsZ Activity Using Novel Antibacterials: Screening Assays, Small Molecule Hits, and Synthetic Pathways to Potent Natural Products

Summary The emergence of bacterial strains resistant to current drugs has prompted a renewed effort to discover new methods for fighting infectious disease. A promising new target is FtsZ, a key mediator of bacterial cell division that is highly conserved among bacteria. Because FtsZ, much like its eukaryotic analog tubulin, consumes GTP during Z-ring assembly in mitosis, it is susceptible to both antagonists and agonists that interfere with its GTPase activity. Discoveries from two multidisciplinary groups at Harvard are presented as synergistic approaches to targeting FtsZ activity and developing first-in-class therapeutics against bacterial infection.

The Inventions

Primary and Secondary Screening Assays Yielding Small Molecule Hits Against FtsZ Activity

Professor Marc Kirschner (Department of Systems Biology) has developed a series of primary and secondary screening assays that have identified 16 small molecules targeting FtsZ activity. This number includes small molecules that exhibit bactericidal activity at levels as low as 2 µg/ml, and compounds which target protein-protein interactions between FtsZ and other divisional components in the septal ring. Primary and secondary assays include a variety of high throughput GTPase assays as well as inventive in-vivo assays leveraging enhanced sensitivity to compounds that may either inhibit or activate FtsZ activity. The assays, which include proprietary screening materials, may lead to additional hit discovery as well as highly specific screens for compound validation and development.

Novel Synthetic Pathway to Natural Product FtsZ Inhibitors and their Analogs

Complementing these broad screening capabilities, are discoveries led by Dr. Jared Shaw at the Broad Institute, relevant to natural compounds and their analogs which target FtsZ GTPase activity. The Harvard team developed a novel, high yield synthetic route to produce hydroxybenzylated flavanone natural products and analogs. Previously, it was demonstrated that such natural products, including dichamanetin and 2''-hydroxy-5''-benzyliso-uvarinol-B, displayed clinically relevant MIC values against a wide spectrum of microbes. However, despite excellent antimicrobial activities, a common limitation to their use in the clinic has been a lack of bulk quantities.

To overcome this obstacle, Shaw and colleagues have synthesized dichamanetin and 2'-hydroxy-5'-benzylisouvarinol-B utilizing a common core structure with a new zinc chloride-mediated benzylic coupling reaction. In vivo antimicrobial data demonstrate potent inhibition of E. coli FtsZ GTPase activity using the synthesized dichamanetin (IC50 = 12.5±0.5) and 2'-hydroxy-5'-benzylisouvarinol-B (IC50 = 8.3±0.5). Data also indicate that these compounds display excellent antimicrobial activity against antibiotic resistant E. coli bacteria. To our knowledge, this represents the first report of efficient synthesis of compounds related to dichamanetin and 2'-hydroxy-5'-benzylisouvarinol-B.

Applications Commercial Applications and Markets Compounds targeting FtsZ may be developed as broad spectrum antibacterials, relevant to both gram positive and gram negative bacteria. Increasing numbers of drug-resistant infections, including MRSA and a shortage of pipeline development by pharmaceuticals have created significant opportunities for novel antibacterials. The CDC estimates that 2 million individuals will acquire a hospital bacterial infection annually, with 90,000 of these cases resulting in death. Overall, the total market for antibacterials is valued at approximately $25B, with the hospital anti-bacterial market representing $8B.

Advantages In combination, the inventions represent a powerful approach to discovering and developing new anti-bacterial candidates targeting FtsZ activity. Using the proposed screens, it may be possible to yield highly specific and potent antibacterials with a low level of anticipated resistance. Crucial to this rationale is the screening focus on protein-protein interactions of the FtsZ protein, as opposed to the FtsZ protein itself. In addition, FtsZ orthologs have a high degree of sequence conservation across various bacteria, especially in domains involved in GTP binding and hydrolysis, in subunit interactions required for polymerization, and in interaction with other cell division proteins such as ftsA and ZipA.

With respect to synthesizing dichamanetin and 2'-hydroxy-5'-benzylisouvarinol-B, previous attempts at synthesis have resulted in a far less selective reaction (produced a mixture of 3 products) and extremely low yields (<5%). The conditions demonstrated by Shaw et al. for the first time have led to high yield synthesis of the reported natural products as well as several unnatural derivatives and analogs.

Publications

1. Sameer Urgaonkar et al. Synthesis of Antimicrobial Natural Products Targeting FtsZ:(±)-Dichamanetin and (±)-2''-Hydroxy-5''-benzylisouvarinol-B. Org. Lett., Vol. 7, No. 25, 2005. 2. Margalit et al. Targeting cell division: small-molecule inhibitors of FtsZ GTPase perturb cytokinetic ring assembly and induce bacterial lethality. Proc Natl Acad Sci, Vol. 101, No.32, 11821-6, 2004.

Patent Status US Number: 7,011,946 and patent applications covering the relevant inventions have been filed and are pending. For Further Information Please Contact the Director of Business Development Laura Brass Email: laura_brass@harvard.edu Telephone: (617) 495-3067

Inventor(s): Shaw, Jared T.

Type of Offer: Licensing



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