Recombinant therapeutic proteins are a rapidly growing category of the prescription drug market, with sales projected to reach $50 billion worldwide by 2010, according to BCC Research. These products, especially antibodies, tend to be highly specific for their targets and possess a long half-life in serum.
Yet they suffer from severe disadvantages. Their developmental path is long and costly, they are difficult to manufacture, and they lack oral bioavailability. Their relative inability to cross the cell membrane confines them to extracellular interventions.
While they were initially believed to be rather benign in terms of side effects, especially when compared to harsh interventions, such as chemotherapy, a recent catastrophic failure of an antibody trial has shattered this serene sense of confidence. This trial, in which four patients in the U.K. were given an anticancer antibody reactive against an important T-cell receptor, CD28, resulted in severe and life-threatening responses; the cause is at present not understood.
But there is another choice that has recently gained momentum. Protein-protein interactions are pivotal in most biological processes and thus represent an appealing target for innovative drug development. They can be targeted by small molecule inhibitors and by peptides and peptidomimetics, which represent an alternative to protein therapeutics and avoid many of their disadvantages.
Peptides serve as active regulators and participate in molecular cross talk, which drives metabolic processes. They are extremely potent, show high specificity, and have few toxicological problems. Moreover, they do not accumulate in organs or suffer from drug-drug interactions as many small molecules do. They can be used as therapeutic agents, or as a starting point for developing peptidomimetics and small molecular weight inhibitors.
However, therapeutic development of inhibitors of protein-protein interactions has so far proven difficult, and progress has been slow. Challenges include the lack of small molecule starting points for drug design, the apparent nondescript nature of the target area on the surface of the molecule, the difficulty of distinguishing artifactual binding from real associations, and the insufficiency of small molecule libraries.
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.
Mimicking the Alpha Helix
Johnson & Johnson Pharmaceutical Research & Development is investigating how to take advantage of protein surface alpha helices as targets for peptidomimetics. Globular proteins have a 30% alpha helical content suggesting a vast repertory of such surfaces that can participate in protein-protein interactions.
As pointed out above, the HDM2-p53 protein-protein interaction is an important target for the design of anticancer agents, and this interaction is known to involve a p53 alpha helix. Since HDM2 downregulates the tumor suppressor p53, a disruptor of this complex could serve as an antitumor agent.
Maxwell D. Cummings, Ph.D., is a senior scientist in molecular design and informatics at J&JPRD. According to Dr. Cummings, his team evaluated its in-house screening collection and ultimately determined that the 1,4 benzodiazepine-2,5-dione scaffold mimics the key binding features of the natural p53 alpha helix ligand.
This approach allows us to combine discovery through screening, followed by optimization of the candidate molecules, Dr. Cummings said.
Benzodazepines comprise a widely used class of drugs, so there is a long history on the health effects of extended administration of these substances.
While this does not guarantee the safety of new compounds, said Dr. Cummings, in general such history allows you to bootstrap, and provides some confidence that toxicology issues will be manageable.
The discovery of an antagonist of the HDM2-p53 binding interaction we believe is the first report of a benzodiazepine compound showing pharmaceutically relevant activity through its action as an alpha-helix proteomimetic, Dr. Cummings said. The design of these molecules represents the exploitation of a number of tools in our discovery toolbox. In this case we used discovery through high-throughput screening, followed by protein crystallography and structural analysis using computational techniques.
What we’re seeing at this time is a lot of tactics being brought to bear on each new target. No doubt new approaches that we haven’t yet imagined will be brought into play, combined with genomics, proteomics, and other traditional tools of drug discovery, he predicted.
The development of protein-protein inhibitors has been received with great interest within the biotech community, as there are many potential applications for peptides, peptidomimetics, and small peptide-derived molecules.
Using improved smart-throughput screening methods it is possible to isolate high-affinity binders with potential therapeutic properties, Dr. Arkin stated. Indeed, several improved screening methods, such as fragment discovery but also structure-based design and other approaches, can be used simultaneously.
However, it must be cautioned that good binders do not necessarily translate into effective drugs, and much work remains before these promising substances can be moved forward into workable therapeutic agents.