GPCR Druggability

The potential to create a potent and selective human therapeutic agent for a GPCR can also influence the selection of that receptor as a target. Biological factors to consider prior to advancing any screening effort for a GPCR target include a number of those already discussed above such as the localization and distribution of the GPCR, the nature of the receptor family type, structure of the receptor, endogenous ligands (i.e., small molecule versus large peptide), and type of the pharmacological effect (agonist or antagonist) desired for the compound. Receptor-binding models can be established from this type of information to allow one to conduct "virtual screening"4950 — a computational method that selects from an electronic database the compounds that best fit three-dimensional models of a known pharmacophore and/or receptor, generally based on the protein structure of bovine rhodopsin.51

Such approaches have proven useful in reducing the number of compounds required for screening, selecting chemical hits for the dopamine D3,52 neurokinin NK1,53 and somatostatin SST254 receptors that were verified as active by biological screening. The chemical nature of the hits obtained from a chemical library screen can also be used to determine whether a GPCR target is worthy of further investigation from a drug discovery standpoint. Favorable attributes of a collection of screening hits for a target including structural diversity, minimal representation by known toxic structures, low micromolar affinity, and structure amenable to chemical modification would favor advancement of a GPCR target.

3.4.2 Lead Optimization

Lead optimization is an iterative process involving the rational design and synthesis of chemical entities followed by the screening of these compounds for in vitro binding and functional potencies at the GPCR target (see Figure 3.2). Results from these in vitro studies, which are generally higher throughput assays, are then used to build structure-activity relationships for hundreds to thousands of compounds in order to optimize potency and selectivity. Compounds that exhibit acceptable in vitro potency and selectivity are then screened for in vivo GPCR target-related activity and tested for other in vivo properties such as pharmacokinetics, absorption, distribution, metabolism, excretion, and toxicity (ADMET).

There are a number of important criteria to consider when establishing in vitro screens for a GPCR target. Binding assays are useful in quickly establishing potencies of compounds for the target receptor and can also provide information about the nature of the binding (competitive, noncompetitive, number of binding sites the compound recognizes). It is important to screen against both the human form of the GPCR and also the species (often rodent) used in in vivo studies to account for any potential species differences. In addition, multiple splice isoforms exist for a number of GPCRs that could influence the pharmacological profile of the receptor.

Ancillary binding screens are often established for closely related receptor subtypes or other targets found to have affinity for early lead compounds in order to further direct rational drug design. A wide host of functional assays measuring a variety of signaling activation pathways are available (see Chapter 9) for determining pharmacological activities (agonist, partial agonist, antagonist, neutral antagonist, inverse agonist) of new compounds at the recombinant or native GPCR.

It is also advantageous to establish in vitro functional assays measuring the activity of the natively expressed target GPCR in order to determine whether the results correlate with those from the recombinant GPCR systems. Once in vitro activity, potency, and selectivity have been demonstrated for new compounds, they are advanced to in vivo animal studies designed to assess their efficacy and potency as drug candidates in disease state models. As with the in vitro assays, multiple models, preferably across several species, should be employed to assure efficacy for the therapeutic indication. Testing of the compounds in transgenic animal models of the target GPCR can also help assess the validity of the receptor for the proposed therapeutic indication.55

A number of considerations from a chemical perspective also contribute to the synthesis of novel compounds targeting GPCRs. Some of these are basic rules applicable to all drug synthesis operations and some are more specific to designing drugs for GPCRs. The term "privileged structures"56 referring to structural chemical motifs that serve to provide small molecule compounds that recognize a diverse group of receptor targets seems to apply to a number of classes of GPCR ligands as well and can be employed to optimize lead compound development. Some common GPCR privileged structure motifs include 4-arylpiperidines, 4-arylpipera-zines, benzodiazepines, biphenyls, 1,1-diphenylmethane, indoles, spiropiperidines, and xanthines.57-60

Focused compound libraries targeting GPCRs can be designed and synthesized based upon such privileged structures using parallel synthesis and higher throughput synthetic methods.57 Both potency and intrinsic activity (positive for agonists and negative for inverse agonists) must be designed into the chemical leads.

Many recombinantly expressed GPCRs are active in the absence of agonists (constitutively active) and over 60 wild-type GPCRs such as the histamine H3 receptor61 exhibit this behavior. Many known antagonists of GPCRs block the effects of agonists at the receptors and also reverse constitutive activity. Based on the large number of GPCRs that are constitutively active in native conditions, designing inverse agonist activity into compounds may optimize the therapeutic potential of chemical leads.

Beyond the efficacy, potency, and selectivity of the compound at the GPCR itself, other factors such as ADMET that influence the drug-like properties of the compound must be considered during the design of a molecule. Models such as the "Rule of Five"62 (which predicts a compound will be poorly absorbed if it does not possess at least two chemical properties such as a molecular weight below 500 Da, fewer than 5 hydrogen bond donors, fewer than 10 hydrogen bond acceptors, or a logP below 5) have aided chemical optimization of lead compounds.

Some ADMET testing can be done in higher throughput in vitro assays such as predictions of metabolic liability with cytochrome P450 inhibition and metabolism profiling, absorption predictions examining Caco-2 cell permeability, and geno-toxicity testing utilizing bacterial reverse mutation assays. Broad-based animal screens, generally across several species, are also used to test the ADMET properties of compounds that demonstrate the best in vitro profiles and most drug-like properties. These include pharmacokinetic testing, cardiovascular profiling, acute and chronic toxicity, and in vivo metabolism. For example, induction of QT prolongation, a potential precursor to cardiac arrhythmia,63 is now routinely investigated preclinically because it was observed for several drugs targeting GPCRs, such as the serotonin agonist cisapride, and the histamine H1 receptor antagonist terfenadine that have been removed from the market. These in vivo tests are often conducted in parallel. As with many of the other screens involved in the optimization process, the results help synthetic chemists improve upon the drug-like properties of the lead chemical series.

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