Genome-wide characterisation of the binding repertoire of small molecule drugs

Genome-wide characterisation of the binding repertoire of small molecule drugs Lee Makowski 0 Diane J. Rodi 0 0 Biosciences Division, Argonne National Laboratory , Argonne, Illinois 60439 , USA Most, if not all, drugs interact with multiple proteins. One or more of these interactions are responsible for carrying out the primary therapeutic effects of the drug. Others are involved in the transport or metabolic processing of the drug or in the mediation of side effects. Still others may be responsible for activities that correspond to alternate therapeutic applications. The potential clinical impact of a drug and its cost of development are affected by the sum of all these interactions. The drug development process includes the identification and characterisation of a drug's clinically relevant interactions. This characterisation is presently accomplished by a combination of experimental laboratory techniques and clinical trials, with increasing numbers of patient participants. Efficient methods for the identification of all the molecular targets of a drug prior to clinical trials could greatly expedite the drug development process. Combinatorial peptide and cDNA phage display have the potential for achieving a complete characterisation of the binding repertoire of a small molecule. This paper will discuss the current state of phage display technology, as applied to the identification of novel receptors for small molecules, using a successful application with the drug Taxole as an example of the technical and theoretical benefits and pitfalls of this method. phage display; drug target; molecular recognition; drug receptor; Taxole; Bcl-2 - The concept of a receptor as the component of an organism with which a chemical agent interacts was based originally on the independent work of Paul Ehrlich and J. N. Langley during the late nineteenth century. Pharmaceutical agents are generally believed to exert their therapeutic effect by binding to a particular protein or nucleic acid target.1 Both natural product and synthetic chemical drugs, however, can and do have multiple macromolecule-binding partners (Table 1),2 48 such as metabolic breakdown enzymes (ie cytochrome P450 enzymes), side effect-mediating targets and, in some cases, secondary therapeutic targets (see aspirin and Taxole below as specific examples). Recognition of the binding promiscuity of drug molecules is reflected in the recent assembly of a publicly accessible web-based therapeutic target database.49 For a drug to reach the market, an average input of 14 years and US$359 million is required.50 The assessment of toxicity, side effects and metabolic breakdown pathways consumes a large percentage of this investment.51 53 The speed with which a drug passes through the various stages of the drug discovery process can be a critical factor for success. Novel strategies that provide information about the potential target proteins of a small molecule are badly needed to augment the current drug development process. A complete accounting of the repertoire of targets for a particular drug provides important information towards a complete understanding of the mode of action of a drug, as well as the prediction of potential complications from its use in the clinic. Detection of potential toxicity and side effect properties early on during drug development can translate into large financial savings and lowered patient risk, as efforts can be redirected towards drug leads that are more likely to be safe for patient use. Unfortunately, identifying macromolecules that bind to small molecule drugs can be a slow and laborious process, particularly if the target(s) are expressed at low levels in their respective tissue(s). Although computer methods for toxicity prediction offer a fast, attractive solution to this problem,54 56 even the best of this predictive software57 generates hypotheses based upon crystal structure-based rigid protein docking studies. In many cases, small molecules (ie non-peptide molecules of less than about 1,000 Daltons) appear to interact with their receptors through regions that are disordered prior to the interaction.58 This suggests that computer-based docking strategies may be limited in their success with a subset of potential drug/protein interactions. Topoisomerase I and II Oestrogen receptor b ParC (topoisomerase IV) gene product Multiple cellular effects Multiple cellular effects Partial agonist activity Note: 1 Implied means that there is no direct data to substantiate the direct binding of that drug to that particular target, merely indirect evidence. LCHAD: long chain 3-hydroxyacyl-CoA-dehydrogenase; nAChR: neuronal nicotinic acetylcholine receptor; COX: cyclooxygenase; PPAR: peroxisome proliferator-activated receptor; RRE: Rev response element; GSH: glutathione S-transferase; TAR: transactivation response; LPS: lipopolysaccharide; PMN: polymorpho-nuclear leukocyte. In this paper, we outline a strategy for identifying the targets of small molecules based on the screening of protein or combinatorial peptide libraries displayed on phage particles. The ultimate goal is the complete delineation of all the targets for a small molecule. Since known receptors are mainly proteins, the discussion will be limited to the identification of protein targets. These strategies have been validated using libraries of cDNA expression products59 61 and libraries of combinatorial peptides. The latter have been used to identify a novel target for Taxole,3 and carry out an in vitro approach to the identification of ATP-binding proteins within entire proteomes (Makowski et al., in preparation). The number of targets of a drug Most, if not all, drugs interact with multiple macromolecules. Some of these molecules conform to the concept of a receptor, in that they are responsible for mediating the primary therapeutic effects of the drug. Other target molecules would not classically be referred to as receptors because they are involved in transport, chemical processing or the side effects of the drug. Some of these interactions are non-specific, such as the interactions between hydrophobic drugs and serum proteins. These interactions are, however, critical elements in defining the clinical effect of the drug. Table 1 lists the known specific macromolecular targets for a few representative drugs. Some of the best drugs may be more effective than related molecules because they bind two or more targets relevant to their clinical action. Aspirin, the most potent anti-inflammatory agent identified to date, has at least three targets that mediate antiinflammatory activity. The anti-cancer activity of Taxole is greater than that of other mitotic spindle poisons, presumably because it interacts with the anti-apoptotic protein Bcl-2, as well as with b-tubulin.3 A small molecule that binds to no macromolecules (if such a thing exists) will be biologically inert. Conversely, a small molecule that binds to hundre (...truncated)