BOSTON — Researchers at Panacos Pharmaceuticals have developed a high-throughput cell-based assay that can identify non-peptide, small molecule HIV fusion inhibitors.
The technique could enable the company to develop an orally delivered HIV fusion inhibitor to compete against the only inhibitor currently on the market — the subcutaneously injected Fuzeon, sold by Trimeris and Roche.
Karl Salzwedel, director of drug discovery for Watertown, Mass-based Panacos, presented his team’s findings at the Assay Development and High Throughput Screening conference, held here last week.
According to Salzwedel, the high-throughput cell-based assay uses antibodies specific for the fusion-active conformation of the HIV Env protein to detect conformational changes in viral Env. He said that Panacos scientists used this approach to screen approximately 385,000 compounds.
Salzwedel reported that hit-to-lead optimization reduced the IC50 of one of the series to approximately 0.5 nM in the screening assay. He said that activity was specific for HIV-1, with no activity against HIV-2. The compounds were non-cytotoxic at concentrations up to 100 µM, yielding a therapeutic index of greater than 10,000.
Panacos’ lead compounds, which the company is currently optimizing, are small, drug-like molecules. Salzwedel said that Panacos scientists have also demonstrated oral bioavailability of up to 30 percent in rodent models, so the compounds could possibly be administered in tablet form.
Fuzeon, co-marketed by Trimeris and Roche, is currently the only fusion inhibitor available worldwide. But with a 36-amino acid peptide, it is a large molecule and is therefore difficult to manufacture, said Salzwedel. It must also be administered by subcutaneous injection twice daily.
“I believe the reason that we were so successful in our approach is because we used cell-expressed viral Env protein as our screening target,” said Salzwedel. “As a result, we were able to identify compounds that inhibit fusion by a different mechanism of action from that of Fuzeon.”
The Panacos compounds appear to bind the Env protein both before and after CD4 engagement, and trigger a conformational change. In doing so, they prevent the subsequent conformational change that is required for the two gp41 peptide domains to form a six-helix bundle and infect CD4 cells.
It is more of an allosteric effect, Salzwedel said, as opposed to trying to directly compete with the interaction of the gp41 peptide domains and prevent 6HB formation.
Using the assay, researchers were able to identify seven structurally distinct and tractable hit series, Salzwedel said. Of these, three series were found to share a mechanism of action and resistance profile different from those of Fuzeon, so they were active against most Fuzeon-resistant virus isolates.
“I believe the reason that we were so successful in our approach is because we used cell-expressed viral Env protein as our screening target. As a result, we were able to identify compounds that inhibit fusion by a different mechanism of action from that of Fuzeon.”
The company’s compounds are in the hit-to-lead or lead optimization stage, depending on the chemical series, Salzwedel told CBA News in a follow-up interview this week. The company has one series that is currently in the lead-optimization stage and hopes to identify a candidate to enter preclinical testing by the end of this year or early next year.
Panacos’ other antiretroviral drug-development program is focused on the maturation of the HIV Gag protein, which is required for the virus to become infectious, Salzwedel said.
He said that Panacos’ maturation inhibitor, bevirimat (PA-457), is currently in Phase 2B clinical trials. Bevirimat blocks the cleavage of the Gag protein at the capsid and SP1 juncture, which is the rate-limiting step in Gag processing.
Salzwedel feels that those in the field of HIV drug discovery will continue looking for HIV fusion inhibitors and maturation inhibitors. Traditionally, HIV infection has been treated with reverse transcriptase and protease inhibitors.
“The problem is that the targets for transcriptase and protease inhibitors are becoming exhausted,” said Salzwedel. Few opportunities are left to overcome the resistant forms of the virus because they are resistant to multiple classes of currently available drugs, he said.
The real drive in the field over the last several years has been to identify drugs that work by novel mechanisms and target other viral proteins, or, in some cases, cellular proteins, Salzwedel said. Panacos is focusing on fusion or maturation.