Small molecule detectives
Robert Flaumenhaft, a research investigator at Beth Israel Deaconess Medical Center, wants to understand a major cause of disease and death. His research lab studies platelets – circulating cells that play a key role in arterial thrombosis, in which a blood clot blocks the flow of blood through an artery potentially leading to a heart attack or other complications. When platelets are activated (turned on), they begin releasing small particles called granules and binding to one another until a clot forms. Rob and his team have been searching for a chemical compound that can prevent platelet activation and potentially be developed into a drug to treat patients by preventing clots. With the help of the Broad Institute Probe Development Center, his group was able to narrow in on three promising compounds. You can see just how effective one of these is in the video below.
Part of a network of centers known as MLPCN (Molecular Libraries Probe Production Centers Network), the Broad’s probe development center has access to more than 300,000 chemical compounds, called probes because they can be used to explore and perturb cells’ biology. This library of compounds includes drugs, synthesized compounds, and molecules found in nature. Broad researchers can work with outside collaborators to set up a screen, which is a large-scale test in which cells are exposed to each of these molecules and their responses are observed, allowing the scientists to follow up on the most promising candidates and develop the best probes into tools for lab experiments or potentially even drugs.
Rob had initially worked with Institute of Chemistry and Cell Biology at Harvard Medical School to run a screen of about 16,000 compounds. Although the experiment turned up interesting results, Rob had difficulties following up on the most promising compounds. “With the success of that preliminary assay, we were looking for a way to expand to do some follow up work as well,” says Rob. “MLPCN was the perfect answer to what we were looking for.”
In order to carry out an experiment at this scale, however, the MLPCN team at the Broad needs a lot of starting material. Platelets are a very valuable resource – blood banks collect them and sell them to hospitals, which use them for transfusion purposes. In Rob’s lab, only small quantities of platelets are needed. But in the probe development center, the researchers needed enough platelets to test 300,000 compounds twice (the screens are performed in duplicate to confirm results) and needed millions of cells per compound.
Rob worked with Lynn VerPlank, a research scientist in the Broad’s Chemical Biology Platform, to contact blood banks across the country to request expired platelets. After five days on the shelf, platelets become too great an infection risk for the blood banks to sell them to hospitals, but they are perfectly suitable for biological research.
Lynn coordinated shipments from several blood banks, including one in California. “We were very dependent on when the blood banks had something,” she recalls. “It was very variable as to whether we were going to get a shipment from day to day.” She estimates that in total, the lab received about 20 liters of platelets over the course of the experiment (a normal, not concentrated microliter of blood contains 150,000-350,000 platelets).
Lynn had to tweak the protocol for handling cells to accommodate the unique needs of delicate platelets. She set up racks to gently rock the cells until it was time to put them to use. She then placed thousands of these cells into miniscule “wells” on a microplate, and each well was exposed to a different chemical compound. She then added a substance to activate the platelets and a “read reagent,” which would glow if the cells released granules. The researchers then measured light – if the well of cells glowed, the compound had not had an effect. This helped the team narrow down the list of candidate compounds from more than 300,000 to around 1,600 compounds.
Lynn and Rob then worked with medicinal chemists to take a closer look at the chemical structure of the compounds that seemed promising. They eliminated any compounds whose effects were too general and retested their hits. Eventually, they whittled the list down to 30 compounds.
“From there, Rob was key,” says Lynn. Rob took this manageable list of compounds back to his lab at Beth Israel to perform a palette of follow-up studies. Using a mouse model, Rob could look at clot formation in a living organism and see the effects of the different compounds in real time.
Rob hopes to continue developing the most promising small molecules from the MLPCN screen. The National Heart, Lung, and Blood Institute (NHLBI) at the NIH has set up a program designed to help researchers like Rob complete the next steps on the path to drug development.
“It’s exciting that there are now these resources available,” says Rob, referring to MLPCN and the NHLBI program. “Individual investigators can take a promising idea and develop probes that are interesting for their biology, and if the probes have potential medicinal use, they can take that forward.”
Learn more about the Broad’s MLPCN efforts here (http://www.broadinstitute.org/mlpcn).