The Gq, Gs, and Gi subcategories have the most relevance in pharmaceutical screening and drug discovery, and therefore, we discuss the signaling events associated with these three classes in more detail below.

Gaq. Signaling through Gaq is associated with the activation of phospholipase C (PLC) ß which results in activation of protein kinase C (PKC) which results in the cleavage of PLC into diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP₃). Increased intracellular IP₃ levels are associated with the release of calcium ions (Ca²⁺) from intracellular stores (such as the endoplasmic reticulum, ER). The increased intracellular Ca²⁺ concentration is associated with a large number of pleiotropic and effector functions that are part of the sequelae of responses associated with GPCR stimulation. The following Figure 11 depicts this schematically:

Gas. Signaling through this variety of G proteins is associated with activation of the enzyme adenylate cyclase, which cleaves ATP into cAMP and gives increased intracellular levels of cAMP. cAMP is a key second messenger in vivo with a large number of functions in the cell (i.e., activates protein kinase A). Also, signaling events through this G protein result in activation of calcium (Ca²⁺) and potassium (K⁺) ion channels in vivo. This ties together the GPCR target class with the ion channels target class.

Gai/o. Activation of this G protein class (via its cognate GPCR) results in the inhibition of cAMP production. Also resulting as parts of this signal transduction cascade are the inhibition of calcium (Ca2²⁺) channels and activation of GIRK potassium (K⁺) channels. Note the pleiotropic nature of the signal transduction events, and how diametrically opposed the signal transduction cascade can be driven by different effector—G protein—molecules.

Many Gq-coupled GPCRs upon stimulation with ligand result in the hydrolysis of phosphatidyl-4,5-bisphosphate (PIP₂) into IP₃ and DAG. The PLC enzyme catalyzes this reaction. Both IP₃ and DAG production are one of the earliest detectable events following the activation of this GPCR class, and hence production of these second messengers is a good screening proxy for this target class.

IP₃ mobilizes calcium ions from intracellular stores (through the IP₃ receptor), whereas DAG activates many isoforms of protein kinase C; both of these signaling intermediates activate a host of different effects in vivo.

In summary, this section has covered some central and essential signal transduction events that result from the activation of the GPCR target class and thereby serve to transduce signals resulting from the extracellular milieu (where GPCRs bind their ligands) into the intracellular contents.

NF-kB, I-kB, and Associated Signal Transduction Pathways
In the previous section of this article we addressed signal transduction events that occur at, or proximal to, the receptor (in this case, the GPCR). In this section, those signal transduction events that occur further downstream (distal) to receptor stimulation are discussed.

Perhaps one of the best-studied in vivo signal transduction pathways is the NF-kB pathway, a convergent pathway for a number of different stimuli that impact the cell. For instance, it is one of the pathways that is activated upon GPCR stimulation with cognate ligand and therefore is of interest in the pharmaceutical community in terms of drug discovery and development. Figure 14 presents a snapshot of the interconnections in this pathway and illustrates the central role this pathway dominates in cellular signaling. Ligand binding and other stimulatory events at the cell surface trigger activation of the cascade that results in the eventual translocation of NF-kB from the cytoplasm to the nucleus. In a non-stimulated cell, NF-kB is tightly complexed with I-kB, a molecule that serves to hold NF-kB in the cytoplasm (and keep it in a biologically inactive form). In this manner, I-kB serves as the brake on the NF-kB signal transduction cascade.

The upstream stimulation cascade (driven by ligand stimulation of receptor) results in the degradation of I-kB in the proteasome (the protein degradation machinery of the cell). This results in release of NF-kB, which then translocates into the nucleus and, by virtue of it being a transcription factor, transactivates a number of genes, thereby mediating effector function in vivo. In this manner, the signal from the cell surface is transduced to the interior of the cell together with concomitant display of biological activity. Note that the movement of NF-kB from the cytoplasm to the nucleus is a good example of how translocation events of proteins within the cell are associated with signal transduction.

There is much interest in the NF-kB signal transduction pathway as one of value in the pharmaceutical industry. Given the central effector role that this pathway occupies for a number of cell-surface receptors (cytokine receptors, GPCRs), it is an important potential drug target as well as a proxy for other effector molecules on the pathway. Figure 15 presents the connection of GPCR ligand-based activation and the NF-kB signaling pathway.

With the importance that NF-kB commands in this space, there is associated intellectual property with which commercial entities need to contend. ARIAD Pharmaceuticals, Inc. (Cambridge, MA) asserts intellectual property rights on the NF-kB pathway such that the deployment of NF-kB or associated molecules in the drug discovery process or direct targeting of NF-kB will result in infringement of patents that are exclusively licensed to ARIAD. ARIAD has currently embarked on a licensing program to enable pharmaceutical companies and drug discovery and development outfits to access this intellectual property portfolio. As of this writing, Bristol-Myers Squibb (New York, NY) and Genome Pharmaceuticals Corporation (Munich, Germany) had both executed drug discovery licensing agreements with ARIAD.

It is unclear which pharmaceuticals on the market or in R&D target NF-kB (or associated molecules). Pharmaceutical companies generally do not divulge the identity of the targets they are building drugs against for competitive reasons. However, thousands of papers have been published in the academic literature addressing NF-kB. Given that the majority of what ends up in Pharma is derived from academic research, it is reasonable to predict that NF-kB based target programs are of high priority and value within the pharmaceutical community.

Phospholipase C (PLC) Signaling Pathway
One of the key intracellular signaling pathways that is at a major crossroads and, therefore, impacts a number of distinct target classes, is the PLC pathway. PLC comes in multiple forms and plays a key role in the signal transduction process for many receptors. Its main function is to hydrolyze PIP₂ into DAG and IP₃. DAG is necessary for further activation of PKC while IP₃ leads to the release of intracellular calcium ions. Figure 16 presents a schematic of this pathway.

Note from this Figure (and Figure 15) that PKC activates the NF-kB signaling pathway. Therefore, the PLC pathway brings together intracellular calcium release as well as NF-kB pathway activation (in addition to activating the important signaling cascade—the mitogen associated protein [MAP] kinase pathway). Note that the pharmaceutical industry is focused upon interrogating distinct molecules within these pathways (as bona fide drug targets) as well as interrogating the pathway as a screening proxy.

In addition to proteins that are resident along a pathway (and thus offer a potential therapeutic targeting opportunity), small molecules as well as ions (most notably calcium ions) are key participants in the signal transduction pathways in vivo. Therefore, pharmaceutical agents such as calcium channel blockers (which affect calcium channels in the cell, such as in the endoplasmic reticulum) represent important pharmaceuticals with substantial clinical value.

Thus far, this article has provided an overview of some of the key signal transduction pathways that are of special interest to the pharmaceutical industry in terms of drug discovery and development. Furthermore, for obvious reasons we have focused on signaling events associated with the top standing pharmaceutical target class in this space, the GPCRs. In this next section, how pathway information is being deployed in the drug discovery paradigm will be discussed.

What are the Needs in the Drug Discovery Space Addressable via Pathway Maps?
There is a huge amount of interest currently in the drug discovery community in using small interfering RNA (variously termed RNAi or siRNA) molecules as a means to knock down the levels of proteins in cells. In this manner, researchers can attempt to simulate biological scenarios whereby cellular interactions with drug candidates, for example, can be studied in biologically relevant situations in vivo. The important point here is that RNAi-based gene knockdown is a way to gently perturb a biologically pathway in vivo without actually effecting gross changes in the biology of the system under study. Target validation, the concept of characterizing biological targets for their amenability to be druggable (i.e., for small molecule drug candidates to affect their function in vivo), is a key problem in the drug discovery space. Indeed, it is a bottleneck, given the large number of potential targets emanating from the human genome and the relatively small fraction thereof that are actually druggable. For this reason, pharmaceutical companies are looking to deploy RNAi technology to mildly perturb cellular signaling pathways in vivo and thereby assess the role of a given target in normal situations and disease processes.

Tools to address biological pathways are of potentially immense importance as they enable the pharmaceutical researcher to gently perturb one target in a pathway at a time and evaluate the effect on the biology of the system. Hopefully this is reminiscent of the in vivo situation where a pharmaceutical that is administered to the patient has a very specific effect on a given pathway rather than a broad pleiotropic effect on the individual.

Pathway mapping tools, therefore, are an important unmet need in the drug discovery industry. In this context are reagents and informatics. Cell-based assays where a given cell line is hard-wired to interrogate certain biological pathway(s) can be a precise means for addressing a given target in its natural state. Gene knockdowns via the RNAi approach is yet another way to achieve this end-result.

Informatics tools that enable the annotation, comparison, and analysis of biological pathways are important since the power of assembling individual components and targets into a pathway is in the ability to inspect and make predictions from the visualization of the ensemble.

In summary, this article has addressed a key pharmaceutical target—the GPCR target class—in terms of signaling pathways that emanate from their activation. Using this economically and clinically important example, it is demonstrated that indeed the study of biological pathways enables an in toto understanding of targets in vivo. The business opportunity for vendors in this space is to provide reagents (and specific cell-based assays) that enable the interrogation of key targets and associated targets, as well as informatics tools that enable researchers to analyze this data.

As more and more drug targets are addressed by pharmaceuticals, it will become more crucial than ever to have approaches that enable very selective modulation of signal transduction pathway parts in vivo in the attempt to find drug-like molecules effecting this modulation under physiological conditions in the body.