May 15, 2007 (Vol. 27, No. 10)

Gail Dutton

Advantages for Protein-based Therapies Are Lower Toxicity and Better Efficacy

Emerging work in peptide drug development will begin paying off in the next few years as new compounds march toward clinical trials. Benefits, so far, include lower toxicity and greater efficacy than existing drugs and in some cases, new classes of compounds that are otherwise unavailable.

Aileron Therapeutics ( is hot on the trail of a novel drug class that, in animal research, mimics the natural apoptosis process. These stapled alpha-helical peptides actually penetrate cells, allowing them to block intra-cellular protein-protein interactions that can’t be addressed by small molecules or biologics, according to Huw M. Nash, Ph.D., vp, corporate development, in his presentation at Cambridge Healthtech’s “Protein-Protein Interactions as Drug Targets,” held this week in La Jolla, CA.

Alpha-helical peptides recognize linear, extended binding sites on a target protein. However, as Dr. Nash explains, they lose their structure when they are synthesized as isolated peptides. Aileron’s solution, developed by researchers at Harvard University and Dana Farber Cancer Research Institute, “installs a hydrocarbon bridge along the nonbinding face of the peptides, allowing them to stabilize the biologically active alpha-helical conformation and obtain good drug-like properties, including resistance to proteolytic degradation and active transport across cell membranes. Fluid-phase endocytosis through the cell membrane appears to be involved. This applies to a diversity of stapled peptide sequences that target cytosolic or nuclear proteins.”

Aileron’s first drug discovery programs are focused on stapled peptides that can mimic the apoptosis-inducing BID protein by directly activating BAX/BAK. Dr. Nash says that he is particularly excited about the immediate possibilities for treating cancers that have become resistant to current cancer therapies, “especially those with lower levels of the BID or the related BIM proteins,” which are obvious candidates for functional replacement by their stapled-peptide mimics.

Targeting Oncology

In initial studies of mice injected with luciferase-expressing leukemia cells, median survival for those injected for seven days with 10mg/kg of Aileron’s first lead stapled peptide was 11 days, up from five days for untreated controls. A significant decrease in tumor growth was also observed. “The half-life for the lead stapled peptide after intravenous injection was observed to be 1.6 hours, unusually long for a peptide. We’re very happy with that,” Dr. Nash says. He adds that clinical trials are planned for the end of 2008 or early 2009.

Proteolix (, on the other hand, is already in the clinic. It took its lead compound from discovery to Phase I trials within a year, according to a presentation to be made at Cambridge Healthtech’s “TIDES” conference next week in Las Vegas. Carfilzomib, a peptide-based proteasome inhibitor, is being developed to treat hematological cancers.

Chris Molineaux, Ph.D., vp, development, compares the compound to bortezomib (Millennium Pharmaceuticals’ Velcad®), noting some important differences. While both are peptides, carfilzomib has four amino acids with an epoxyketone warhead, versus Velcade’s two amino acids and boronate warhead. “Carfilzomib may bind differently to the proteasome,” Dr. Molineaux says. “With carfilzomib, the peptide, not the epoxyketone warhead, drives the binding to the target enzyme. For bortezomib, the warhead itself is critical.”

In preclinical studies, Proteolix’ compound appears to be a more specific proteasome inhibitor. In looking at a panel of enzymes that may reflect off-target activity, “bortezomib has been shown to inhibit several serine proteases with micromolar or submicromolar activity, while carfilzomib, at a higher concentration, inhibits only a few.” Early preclinical studies, he says, indicated that carfilzomib was better tolerated and could be administered daily to provide extensive proteasome inhibition.

The development premise was that carfilzomib would be efficacious at its maximum tolerated dose. Early dose-progression studies bore that out, according to Proteolix. “At the maximum tolerated dose, we are getting substantial inhibition of the target enzyme—up to 85%,” Dr. Molineaux reports. Consequently, more intensive dosing appears possible; up to daily doses for five consecutive days.

Carfilzomib also appears less toxic than bortezomib, he adds, noting that of “more than 50 patients treated, some for more than one year, none have developed naturopathic pain.” The reason for lower toxicity remains speculative but “may be related to off-target inhibition of some other enzyme by bortezomib.” Dr. Molineaux believes that this compound may have applications in solid tissue tumors.

Currently, the company is scaling up production and completing the FDA approval process. “We worked with manufacturers early in development, using the most straightforward manufacturing process we could put together,” Dr. Molineaux states. Although he knew it would have to change to accommodate large-scale manufacturing processes, the goal was to initiate clinical development to get proof of concept for carfilzomib as soon as possible.

Genentech’s ( approach to anticancer treatments uses a peptide to inhibit cyclinA, according to Daniel Sutherlin, Ph.D., scientist, medicinal chemistry. Dr. Sutherlin, speaking at the “Protein-Protein Interactions as Drug Targets” conference explained that cyclinA is integral in the cell cycle and is considered an important cancer target.

Unlike most small molecule drug discovery programs, Genentech is focusing on “inhibitors that bind to a substrate recognition groove on the surface of cyclin A,” Dr. Sutherlin says. “Peptides that mimic endogenous substrates and inhibitors of the kinase complex bind to this site,” and using peptides to inhibit cyclinA is selectively killing cancer cells both in vitro and in vivo, he explains. Genentech currently is validating this site as a small molecule target.

7TM Pharma ( is in Phase I and II trials for obesity therapies. TM30339 works through the Y4 receptor and is in Phase I/II development. TM30338 targets the Y2 and Y4 receptors and is being evaluated in Phase II. Considered first-in-class compounds, they have a mechanism of action similar to that of the natural satiety hormone pancreatic polypeptide and mimic natural satiety, thus helping regulate food intake, explains Paul Little, Ph.D., senior chemist. Dr. Little will be presenting this research at “TIDES”.

As its name implies, the company focuses on seven transmembrane receptors. Rather than launching a high-throughput screening campaign, 7TM has developed its Site Directed Drug Discovery® platform to provide targeted libraries.

Progress in Metabolic Diseases

The program doesn’t rely on crystallization and x-ray structure determination like other structure-based programs, because the 7TM receptors are difficult membrane proteins that are hard to produce in sufficient amounts and quality, according to the company. Instead, it identifies receptors already associated with ligand information that are closely related to the target receptor. It then relates those receptor proteins to the physiochemical properties of the binding site for their ligands, prior to in silico screening.

This approach lets 7TM complete the cycle of receptor identification to hit identification within three to four months.

Existing obesity drugs have limited efficacy and significant side effects, according to the company. The two lead compounds have the promise of being more effective without side effects. So far, “the safety and tolerability study for TM30338 demonstrated the compound to be safe and well-tolerated, but no long-term study has been performed yet,” notes Dr. Little. Early results indicate TM30339 will prove more effective than the pancreatic polypeptide in long-term body weight reduction, but details are confidential at this stage, the company states.

Although 7TM is focusing on metabolic diseases, it also has a discovery program under way for inflammation and vascular diseases. It plans to move two or three candidates into clinical development within the next few years.

Activating the EPO Receptor

AplaGen Biopharmaceuticals ( is developing potent, erythropoietin (EPO) mimetic peptides that are yielding improved pharmacodynamics and pharmacokinetic properties. The first compound in preclinical trials, AGEM 40, targets anemia and central nervous system conditions by activating the natural EPO receptor.

According to Hans-George Frank, M.D., scientific officer, “We used hydroxyethyl starch, a plasma expander, rather than polyethylene glycol (PEG) to improve pharmacokinetic and pharmacodynamics properties.” The reason, he explains, is that PEG isn’t filtered by the kidneys. The starch, in contrast, has a longer half-life and, in animal models, is three times better than the competition, Dr. Frank points out.

This compound binds symmetrically to its natural receptor, getting rid of side effects and allowing the compound to be eliminated. In contrast, EPO does not. In fact, it has no homologies to endogenous EPO, thus avoiding the issues of antibody formation and manufacturing standardization that are inherent with synthetic EPO.

“It’s comparable to EPO but has no side effects,” Dr. Frank says, noting that the compound is still in preclinical trials. “The toxicological studies are fine.” AGEM40 is being developed for parenteral and intravenous application but eventually may be adapted to other administration modes.

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