UC Irvine

PART I

The amidohydrolase urease is critical for the survival and pathogenesis of Helicobacter pylori. Urease catalyzes the hydrolysis of one molecule of urea to two molecules of ammonia. It is through the generation and excretion of basic ammonia that H. pylori can survive the acidic gut. It has been well established that inhibition of the enzyme renders the pathogen defenseless against the harsh human stomach. Therefore it is not surprising that urease has become an ideal target for H. pylori eradication. However, despite considerable research efforts, few practical urease inhibitors exist. Additionally, increasing antibiotic resistance has generated a need for effective therapies against H. pylori. Urease’s unique ability to generate a basic product simplifies activity detection using pH indicators such as phenol red. The first phenol red-based (pH-based) high-throughput screening (HTS) assay for small molecule inhibitors of H. pylori urease has been developed and optimized. The phenol-red based assay, which allows for rapid screening at room temperature, has exhibited high quality and reproducibility, with an OD Z’- factor = 0.6109 ± 0.2123. The assay has been utilized to screen upwards of 200,000 compounds against recombinant H. pylori urease, with 255 primary hits (0.13%), 61 reconfirmed hits, and 26 valid inhibitors. Additionally several novel compounds that exhibit low and submicromolar inhibition against H. pylori urease have been identified. Further characterization of these inhibitors is ongoing, including co-crystal structures of the inhibitors with H. pylori urease. Overall, the phenol red assay unlocks opportunities to discover and develop highly effective, direct treatment against H. pylori urease that will eradicate the chronic bacterial infection in humans.

PART II

Cytochrome P450 represents a superfamily of heme-containing oxidase enzymes responsible for transmuting xenobiotics into more water-soluble compounds. Amongst the cytochrome P450 isoforms, CYP3A4 is the most profuse, and one of the most clinically relevant xenobiotic metabolizing enzymes in humans. Moreover, CYP3A4 is the most chemically versatile P450, with the ability to oxidize a variety of molecules ranging in size and chemical structure. In addition to drugs, CYP3A4 is involved in the transformation of toxins, environmental pollutants, and carcinogens. Variations in expression and activity of CYP3A4 can greatly affect drug efficacy or toxicity. Additionally, various synthetic or naturally derived agents such as furanocoumarin derivatives can either enhance or inhibit CYP3A4 activity. Although inhibition of CYP3A4 can result in undesirable drug toxicity and/or drug-drug interactions, carefully controlled inhibition can be exploited to increase the bioavailability of drugs that would otherwise be rapidly metabolized. Controlled inhibition is already being exploited in HIV and HCV combination treatment with ritonavir. Initially designed as an HIV protease inhibitor, ritonavir was later found to be an effective CYP3A4 inhibitor. However, neither ritonavir, nor its derivatives were developed using pharmacophore models, rational design, or CYP3A4 crystal structures. Therefore, little is understood on the mechanism of inhibition. Using the ritonavir-CYP3A4 crystal structure as a guide, a preliminary pharmacophore model of CYP3A4 inhibition was proposed. Through scaffold modification and rational design, the requirements for successful CYP3A4 inhibition are now more clearly understood. To date, several modest, yet highly potent CYP3A4 inhibitors have been developed. Ultimately, these rationally designed pharmacoenhancers may assist in more effective combination treatment and lower drug costs.