Structure–activity relationship (SAR) studies on a molecule nominated as a viable “hit” are essential to explore the chemical features that can improve the molecules’ potency, physiochemical and pharmaceutical drug-like properties. While a distinct strength of the unbiased high-content cell-based screening approach described herein is that knowledge of the target is not required and hence use of this approach can embrace numerous targets simultaneously, a complete understanding of the mode of action of the “hit” and a thorough understanding of the prospects of a lead to become a drug candidate include an appreciation of the molecular target. This is because the mechanism of action and target of the molecule can shed light on safety and clinical efficacy, as well as aid medicinal chemistry development of the lead as a drug (overview in ). Identification of the molecular target of a lead molecule even in a nebulous system such as the cardiac differentiation paradigm is becoming feasible because of recent advances in mass spectrometry and other bioanalytical procedures. Determination of the ultimate mechanism of action of the lead may be more challenging, but considerable progress in this area of research has been made as well.
Fig. 3 Target identification of hits. Once a “hit” has been validated, the extent of understanding of the target determines the development of a drug candidate. If a molecule with a known target is identified and the target is verified (branch (more ...)
The first step in characterizing the biology and identifying the target of a molecule is to understand the temporal developmental process that the lead molecules modulate. By carefully monitoring tissue-specific markers, it is possible to map a compound’s action to a particular step in the cardiogenic process. For the cardiac assays described above, gene expression, immunostaining, or fluorescent protein reporter analyses are used to visualize typical markers of cardiac fate such as Mesp1, Nkx2.5, Mef2c, or αMHC. Determining the temporal activity of a molecule in cardiac differentiation is crucial towards understanding whether a molecule would be suitable for stimulation of endogenous stem cells in vivo ().
A second step included in the identification of the direct target and mechanism of the lead is to examine the effect of the compound on candidate signaling pathways. “Hits” from screens of molecules with known targets (e.g., approved drugs) require minimal target analysis beyond validation and represent a relatively straightforward path to development of traditional target-based HTS or drug repurposing (, branch 1). Validation of the target is usually by either activation (e.g., over-expression of the target) or down-regulation (e.g., siRNA knock down). For “hits” with unknown targets (, branch 2), we take a candidate approach to determine if active compounds influence pathways known to modulate cardiac differentiation (discussed above). For all pathways examined, bioanalysis tools, such as the study of phosphorylation of downstream mediators and induction of direct targets as well as several candidate reporter systems in which specific gene promoter response elements drive luciferase expression, can shed light on the functional activity of the candidate molecule.
If known signaling pathways are not targeted, an informatics approach can be considered to reconstruct cellular signaling affected by the hit. Briefly, profiling of gene expression and phosphoproteins of small molecule-treated samples can be assembled into signaling networks by computer modeling [72
]. Gene expression profiling within a couple of hours after treatment of cells may provide clues, albeit indirect, on pathways that might be activated. Scrutiny of the phosphoproteome, in contrast, has the potential of directly unveiling the entire pathway. Given the central role played by protein kinases in controlling cell differentiation, interrogation of the phosphoproteome by mass spectrophotometry has been successful in helping unravel the control of cell behavior, including stem cell pluripotency [74
]. While this unbiased approach overcomes the limitations of Western blotting and other antibody-based approaches that probe a small target set limited by available reagents, there remain many challenges in generating samples and in the analysis of the total phosphoproteome dataset. Practical considerations that minimize throughput include: difficulty and cost of scaling up the assay to obtain large protein samples, sample processing time of about 1 month per sample, large variance between replicates and cost per sample. Additionally, the datasets generated require extensive statistical analysis and validation before signaling network reconstruction can proceed, but this can be accomplished through the use of protein–protein interaction databases, kinase prediction algorithms, and literature mining [74
Use of affinity reagents also has considerable utility in identifying the target of lead compounds. In this regard, extensive SAR studies of candidate compounds can provide an understanding of regions of the molecule that could be amenable to chemical manipulation and elaboration of linkers to affinity moieties. For example, a useful strategy is to attach biotin through a linker system to a region of the molecule that is non-essential from the standpoint of potency [76
]. Such affinity reagents aid in mapping the signaling activity of the molecule via biochemical analyses, including target pull downs and competition assays. In theory, pull down and competition assays seem very straightforward; however, several important drawbacks should be considered. For example, because libraries for screening often contain promiscuous compounds affecting several targets, multiple targets may be identified and this requires extra validation of each target. Moreover, one should be aware that such compounds may bind to different targets with different affinities, and that the actual target may not be identified due to strong binding to an abundant protein that is irrelevant to the mechanism of action.
Once candidate targets are obtained utilizing the above described strategies, additional studies are then required to verify the targets by inhibition of factors or pathways downstream of the small molecules discovered by implementing either siRNAs or well-known chemical inhibitors where available (for example, specific kinase inhibitors).
In summary, systems biology and affinity reagent technologies for target identification are beginning to show promise but still tend to be lengthy and tedious (and require more development) (, branch 2). Screening smaller scale libraries of well-characterized compounds that are highly selective for a specific target and in that way facilitate the target identification process immensely is an alternate approach that should be considered in the overall target identification strategy (, branch 1). After target identification, conventional approaches can be employed to validate the signaling pathway or protein as a target for consideration in a drug development pipeline, including validation in animal models and relevance to human disease.
An important early stage validation includes verification that the target or pharmacologically optimized tool compound is efficacious in a relevant animal disease model. Although a clinical endpoint, e.g., heart function, should be evaluated, endpoints more proximal to the effect of the compound or target give verifiable intended mechanism of action. As an example, cardiac stem cell progenitor differentiation or proliferation can be measured in transgenic mice harboring a reporter that allows visualization of progenitor cells, such as the Nkx2.5-eGFP mouse [77
]. Flow cytometry and immunocytochemistry can reveal the potential effects of candidate compounds on proliferation and differentiation of committed Nkx2.5+
progenitors in vitro as well as in vivo. In fact, a modest increase in GFP-positive cells was observed in the adult mouse heart in response to myocardial infarction (Sean Wu, Massachusetts General Hospital, Boston, personal communication), suggesting that this could be a valuable model to evaluate the activation of the endogenous repair response in the adult heart.