Duke University | Chemical Biology Program

Case Studies in Chemical Biology

Glaucoma

Glaucoma is a major disease of aging, affecting nearly 1% of the population of the United States. Although the term glaucoma represents a family of diseases, the most common manifestations involve an increase in intraocular pressure, arising from a failure of drainage of fluid through the trabecular meshwork, a tiny organ in the front of the eye. The primary treatment for glaucoma is pharmacological, utilizing prostaglandins that upregulate the alternate uveoscleral outflow pathway.

Duke researchers are developing second-generation prostaglandins for the treatment of glaucoma. Although the lead compound showed extraordinary potency against the F-prostanoid receptor and unparalleled specificity, leading to a greatly enhanced safety profile, the drug was extraordinarily insoluble and could not be formulated at the concentrations required for pharmacological effect. Staff in the small molecule synthesis facility developed a novel esterification strategy that produced a novel form of the drug that was both remarkably soluble and showed exceptional corneal penetration. Further enhancements to the strategy used a symmetric ester and resulted in a crystalline material that was prepared as GMP material on a kg scale. The approach was patented, and may serve as a platform technology for the development of novel ophthamological therapeutics.

The isopropyl ester of this novel prostaglandin (left) is too insoluble for formulation as an effective ophthamological drug. The hydrophilic ester shown at right is both highly soluble and shows excellent corneal penetration. The strategy was patented by Duke researchers and provides a proprietary platform strategy for the topical delivery of ophthamological drugs.

Prostaglandins

LpxC-inhibitors

The development and wide application of antibiotics have saved millions of lives and enabled complicated surgical procedures and new cancer treatments to be performed without the dire consequences of bacterial infections. Unfortunately, medicine's triumph over bacterial pathogens is increasingly challenged by the emergence of multidrug-resistant bacteria. Gram-negative bacteria are responsible for approximately half of the reported multidrug-resistant infections, and some of these pathogens, such as certain Pseudomonas and Acinetobacter strains, are resistant to all commercially available antibiotic treatments.1-3 To circumvent established resistance mechanisms, Duke researchers are working on previously unexploited molecular targets, such as the lipid A biosynthetic pathway that produces the major lipid component of the outermost monolayer of Gram-negative bacteria.

The zinc-dependent deacetylase LpxC is an essential enzyme in lipid A biosynthesis and an attractive antibiotic target. CHIR-090, the most potent LpxC-inhibitor discovered to date, effectively kills Pseudomonas aerugiona—the predominant cause of morbidity and mortality in cystic fibrosis patients—in disc diffusion tests.4 In order to facilitate the development of broad-spectrum LpxC-targeting antibiotics, Duke researchers undertook structural studies of various LpxC/inhibitor complexes to elucidate the molecular details of LpxC inhibitors and their interactions with LpxC. CHIR-090 was resynthesized in the Duke Small Molecule Synthesis Facility to allow site-specific incorporation of 13C and 15N nuclei and to support structural studies by NMR. The use of the isotopically enriched CHIR-090 has made it feasible to probe the protein-inhibitor interface using NMR experiments with unparalleled sensitivity and significantly reduced artifacts, both of which are crucial for the successful structural determination of the LpxC/CHIR-090 complex.5

The molecular details revealed in the LpxC/CHIR-090 complex not only explain the specificity and high-affinity binding of CHIR-090, but also provide insights to guide the further optimization of existing LpxC inhibitors as novel antibiotics for treating Gram-negative infections.

Reference:
  1. Greenberg et al. PLoS Pathog 2006; 2: e28.
  2. Talbot et al. Clin Infect Dis 2006; 42: 657-68.
  3. Livermore and Woodford. Trends Microbiol 2006; 14: 413-20.
  4. McClerren et al. Biochemistry 2005; 44: 16574-83.
  5. Barb et al. Proc Natl Acad Sci USA 2007; 104: 18433-18438.
AaLpxC CHIR 090

Icmt-inhibitors

Many key regulatory proteins, including members of the Ras family of GTPases, are modified at their C-terminus by a process termed prenylation. This processing is initiated by the addition of an isoprenoid lipid, and the proteins are further modified by a proteolytic event and methylation of the C-terminal prenylcysteine. Although the biological consequences of isoprenoid addition have been characterized extensively, the contributions of prenylcysteine methylation to the functions of the modified proteins are not well understood. This reaction is catalyzed by the enzyme isoprenylcysteine carboxylmethyltransferase (Icmt). Genetic disruption studies in mouse cells provided the first evidence that blocking Icmt activity has profound consequences on oncogenic transformation. Through screening of a diverse chemical library obtained by the Duke Center for Chemical Biology, a novel and selective small-molecule inhibitor of Icmt, cysmethynil, was discovered.1,2 Cysmethynil was authenticated as an Icmt inhibitor through resynthesis in the Duke Small Molecule Synthesis Facility; the synthesis facility has also provided gram quantities of cysmethynil to support animal studies. Treatment of cancer cells with cysmethynil resulted in mislocalization of Ras and impaired epidermal growth factor signaling. In a human colon cancer cell line, cysmethynil treatment resulted in reduction in Ras-dependent oncogenic transformation in a mechanism-based manner. In a recent study at the Duke-NUS Graduate Medical School Singapore, cysmethynil treatment was found to markedly reduced tumor burden in xenograft mice bearing multiple human tumor cell lines. During the course of these studies, it was discovered that cysmethynil treatment induced autophagy in cancer cells and that the resultant over-exuberant autophagy resulted in autophagic cell death.3 These findings have provided a compelling rationale for development of Icmt inhibitors as a new approach to anticancer drug development.

  1. Winter-Vann et al. Proc. Natl. Acad. Sci. USA, 2005, 102, 4336.
  2. Baron et al. Biochemistry, 2007 46:544.
  3. Wang et al. J. Biol. Chem. 2008, 283:18678.
autophagy

Inhibition of Icmt induces autophagy in human prostate cancer cells. PC3 cell were treated with the small molecule inhibitor of Icmt, cysmethynil, for 48 h and then stained by acridine orange. The acidic autophagolysosomes induced by Icmt inhibition stain orange-red. From Wang et al (2008) A small molecule inhibitor of isoprenylcysteine carboxymethyltransferase induces autophagic cell death in PC3 prostate cancer cells. J Biol Chem. 283:18678-18684.