Research Activities
(A list of current OTRADI projects can be found here)
OTRADI has completed the first two steps in the compound commercialization process, including primary screening of Oregon Collection compound library and dose-response assays for initial hits with anti-infectious disease activity. Results were sent to investigators for review. In ongoing experiments, OTRADI will produce data in support of step three-- assessing cytotoxicity for hits with sufficient potency from dose-response assays. Results produced by OTRADI are now included as supporting data on 14 grants to be submitted by 9 Oregon researchers for private and federal funding by mid-2009. Ongoing and future OTRADI activities include: supporting Phase I screen investigators with additional testing; providing supporting data for grants and scientific papers; expanding the Oregon Collection library; and soliciting proposals for additional novel screens.
OTRADI's compound/assay pipeline generally follows these steps. We first perform a cut-off screen in which we test two high concentrations (1uM and 10uM) of each compound for activity. For compounds that inhibit microorganism growth by at least 75% in the initial cut-off assays, we next perform dose-response assays to determine IC50 values. Next, we assay the compounds for cytotoxicity.
The Oregon Collection
The Oregon collection is made up of a number novel, locally-sourced, natural and synthetic chemical compounds contributed by chemists and scientists from the four major Oregon research universities: Oregon State University, Portland State University, Portland Veterans Affairs Medical Center/Oregon Health and Science University, and University of Oregon. Oregon university-based researchers have provided their compounds to OTRADI's research and testing team so that they may be assayed for their ability to kill microorganisms that cause infectious diseases.
Guidelines governing compounds submitted to OTRADI to be part of the Oregon Collection, are as follows: OTRADI receives a minimum of 10mg to 50mg of each compound. A permanent stock solution of each compound is made by dissolving in DMSO at an initial concentration of 10mM. OTRADI receives the compounds in a blinded fashion. Investigators submitting compounds keep a master list of each compound's identity and label compounds they send to OTRADI using the format of their initials and an identifying number that corresponds to their master list (i.e., JM1, JM2, JM3, JM4, etc.). Upon their deposition into the Oregon Collection, OTRADI gives each compound a unique identifier, which allows OTRADI to track the compound's progress through the experimental pipeline. Investigators are responsible for alerting OTRADI if their compounds are sensitive to freezing/thawing, exposure to light, etc.
Anti-Infectious Disease Assays
Anti-Malarial Assay
Human blood infected with Plasmodium falciparum is added to all wells of each 96-well plate. Each chemical is analyzed for anti-P. falciparum effects by treating triplicate wells with the chemical at two concentrations (1uM and 10uM). Negative control wells are left untreated with chemicals, and positive control wells are treated with the known anti-P. falciparum drug artemisinin. At 72-hours post-chemical treatment, cells are lysed and the fluorescence of each plate is measured using a 96-well plate reader. Parasite growth is determined using SYBR Green I, a dye that fluoresces when in contact with P. falciparum DNA. Wells treated with chemicals that are effective as anti-P. falciparum agents will show markedly lower fluorescence, as will the positive control wells. Wells treated with chemicals that are not effective anti-P. falciparum agents will show essentially no difference in fluorescence from the negative (chemical-free) control wells.
Anti-Bacterial Assay
Compounds are tested for their ability to inhibit the growth of either or both Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). E. coli is a gram-negative bacterium commonly implicated in food poisoning. S. aureus is a gram-positive bacterium that is responsible for the majority of post-surgical wound infections. To assay whether the chemicals tested were able to inhibit bacterial growth, bacteria was cultured until log-phase growth was observed. The culture was then diluted in media and added to each well of multiple 96-well plates. Chemicals to be analyzed for anti-bacterial growth inhibitory effects were then added to triplicate wells with each chemical tested at two concentrations (10uM and 50uM). Negative control wells were left untreated with chemicals, and positive control wells were treated with a panel of known anti-bacterial drugs (ampicillin, penicillin and gentamicin). At 20-hours post-chemical treatment, a spectrophotometer/plate reader was used to quantitate bacterial growth, measured using absorbance at 600nm (OD600). Growth of bacteria is directly related to absorbance, such that increased growth results in higher OD600 values at time of measurement. Wells treated with test chemicals that are effective as anti-bacterial agents will show markedly lower OD600 values, as will the positive control wells. Wells treated with chemicals that are not effective anti-bacterial agents will show essentially no difference in OD600 values from the negative (chemical-free) control wells.
Anti-Fungal Assay
Aspergillus flavus (A. flavus) is a filamentous fungi that is responsible for a range of human infections (ear, skin lesions) and ulcers. Growth of A. flavus is efficiently assayed using a 96-well plate reader. In short, a known concentration of A. flavus is added to all wells of each 96-well plate. Each chemical is analyzed for anti-fungal activity by treating triplicate wells with the chemical at two concentrations (1uM and 10uM). Negative control wells are left untreated with chemicals, and positive control wells are treated with the known anti-fungal drug amphotericin B. At 48-hours post-chemical treatment, the absorbance (OD630) of each plate is measured using a 96-well plate reader. Growth of A. flavus is directly related to absorbance, such that increased growth results in higher OD630 values at time of measurement. Wells treated with chemicals that are effective anti-fungal agents will show markedly lower absorbance, as will the positive control wells. Wells treated with chemicals that are not effective anti-fungal agents will show essentially no difference in absorbance from the negative (chemical-free) control wells.
Cytotoxicity Assay
The cytotoxicity assays used at OTRADI are based on the standard mammalian cytotoxicity regiment used by the National Cancer Institute to evaluate possible toxicity of novel compounds. The protocol and references can be found here. OTRADI currently has frozen stocks of the following mammalian cell lines available for cytotoxicity studies: HepG2 (human liver), HEK293 (human embryonic kidney), NIH/3T3 (mouse fibroblast), HeLa (human cervical adenocarcinoma), H9c2(2-1) (rat embryonic myocardium), L6 (rat myoblast), MRC-5 (human lung fibroblast), and Vero (African green monkey kidney). We can revive and propagate any of these cell lines to test the cytotoxicity of your compounds.
Future Research Activities
Other Assay Development
OTRADI's future plans include the custom design of assays to screen compounds for anti-cancer activity. Various assay protocols are currently in consideration for use which are modeled after the National Cancer Institute in vitro anticancer drug discovery protocol (which can be found here). Appropriate target cancer cell lines will be determined and added after discussion with participating investigators.