Over the last decade progress in basic research has been made in the drug development of peptide inhibitors, leading to the development of candidate drugs that are well-advanced in clinical trials or have received FDA approval. A number of private companies are now beginning to see the fruits of their long and arduous efforts.
According to Frost and Sullivan, there are more than 40 peptides in the marketplace, close to 300 in clinical testing, and 400 in preclinical development.
While the appeal of protein-protein regulators as a new frontier of therapeutics is great, there is a significant downside to the technology. First, it has been difficult to identify compounds that bind to the hot spots, that is, the most important regions of the interfacial surfaces. These regions rarely form deep grooves or clefts for small molecules to bind.
Since they are often hydrophobic in nature, many of the molecular entities selected for binding will themselves be hydrophobic, thereby introducing properties of insolubility and aggregation into the molecules that hold the greatest promise as therapeutics.
Another problem posed by peptides is that on a per-gram basis they are significantly more expensive to produce than traditional small molecule drugs. For this reason it is unlikely that a peptide competitor to aspirin would make sense unless it were dramatically more effective and caused far fewer side effects. Other issues include peptides’ instability with a limited half-life in circulation, and, as with antibodies, their limited permeability.
So in many cases peptides represent both a compromise and a starting point for construction of small molecule mimics. Despite the formidable range of difficulties, the positive features offered by inhibitors of protein-protein interactions is so persuasive that both big pharm and small biotechs have moved aggressively to develop a new range of products.
Michelle Arkin, Ph.D., of Sunesis Pharmaceuticals (www.sunesis.com) has investigated small, organic molecules that inhibit protein-protein interactions, focusing on a variety of potential targets.
A number of research labs and pharma companies have investigated protein complexes that constitute potential drug objectives. Many of these possess protein interfaces in which a small, linear region of one protein binds into the hydrophobic cleft of the other.
Peptides derived from this region may also bind to their respective targets, thereby blocking a companion protein necessary for division of a cancer cell. The goal is to use the knowledge gained from the peptide ligand to design and develop nonpeptidic molecules for these targets. Such molecules have a greater chance of circumventing the standard mechanisms by which cancer cells acquire resistance.
The most ubiquitous means by which tumor cells mutate to escape the killing effects of anticancer agents is through amplification of the P-glycoprotein, a pumping mechanism that expels a large collection of different compounds from the cell.
There are a number of points in the apoptotic pathway in which proteins, such as tumor necrosis factor apoptosis inducing ligand (TRAIL), stimulate death receptors. In cancer cells, anti-apoptotic proteins are frequently upregulated, so the cells evade the apoptosis death spiral. Several groups have isolated peptides that block these proteins, and these peptides show antitumor effects in mouse tumor models.
Among the small molecules are drug-like inhibitors, known as Nutlins from Hoffman-La Roche, and benzodiazepinediones from Johnson & Johnsonson Pharmaceutical Research and Development (J&JPRD; www.jnjpharmarnd.com), which activate apoptosis in cells expressing wild type p53 tumor suppressor protein. Ordinarily the apoptotic action of p53 is suppressed by binding to MDM2, another protein. These small molecules bind to MDM2 at the p53 binding site, so in cancer cells over-expressing MDM2, they allow apoptosis to proceed. When dosed orally in mice such molecules inhibit tumor growth through inducing apoptosis.
Other protein complexes have been identified as possible anticancer targets for peptidomimetics and small molecule drugs. These include the Rac1, involved in cell adhesion and cell migration; beta-catenin, an activator of cell cycle proteins; and Sur-2-ESX, which regulates Her2, a growth factor receptor overexpressed in breast cancers XIAP and BCL-2 inhibitors.
Dr. Arkin discussed an interesting approach developed by Sunesis for isolating new compounds that bind effectively to protein targets. Our strategy, referred to as fragment assembly, uses collections of small organic molecules in order to probe a large chemical space, she explained.
Compounds that show some weak binding can be identified through a number of biophysical approaches. These small entities can be joined to other fragments and a larger, high affinity binder can be put together. We have developed a fragment-discovery method called Tethering, which uses a reversible disulfide bond between the protein and the fragment to select fragments. This protocol has been used successfully to isolate a high affinity inhibitor of interleukin 2, she stated.
Sunesis has broadened its focus from protein-protein interactions to include enzymes. Our group has found that the Tethering method is highly amenable to discovering novel, drug-like inhibitors for a range of important oncology targets, including kinases.