Why don't you try a dose-dependent assay with your molecule and then you could check the response of your organism using transcriptome, RNA-seq, etc. in a dose-dependent manner. That's just my opinion.

 Dear Zhiyuan li, 

Target identification can be approached by direct biochemical methods, genetic interactions, or computational inference. Combinations of these approaches may be required to fully characterize mechanisms of small-molecule action. Accordingly, a multi-faceted approach to the target identification problem in the context of genome-based drug discovery should include:

(i) quantitative proteomics based on mass spectrometry and (ii) genetic complementation of small-molecule effects using RNA interference

computational inference by connectivity analysis using reference compounds

Integrating these approaches depends on the ability to connect experimental data with annotations about both small molecule activities and the bioinformatics of candidate targets. An application of bioinformatic analyses to connect proteomics, RNAi knockdown, gene-expression, and other data to generate target hypotheses is considered a necessity to accomplish the goals of small molecules target. a development of a compound comparison method that uses small-molecule profiles based on historical screening data to assess ‘assay performance similarity’ of a pair of compounds is also a necessity. In combination with existing methods of hypothesis generation, such as the Connectivity Map, such approaches provide powerful insights into mechanism for small-molecules shown to be active in cells.

Whether it is an approved drug with an unknown mechanism of action, a natural product or a small molecule derived from a cell based phenotypic or biochemical screen the need to identify mechanism of action becomes a priority. Therefore, the use of a robust and unbiased method of probing the proteins that bind to the small molecule of interest in a biologically relevant setting should be a priority. It combines quantitative proteomics (SILAC) with biochemical affinity enrichment.

Generation of the affinity reagent (bait) is a critical first step, which involves tethering the small molecule of interest to a bead.  If there are sufficient structure-activity relationship (SAR) data available, a tether is placed at an unimportant position. If, however, there is no SAR, a position that can be modified without compromising activity must be found.  In either case, the modified compound must then be tested in the original system to ensure that the desired activity is retained. Once activity of the tethered compound has been confirmed it is attached to a solid support and the affinity reagent can be used in a pull-down studies. While generation of the affinity reagent is ongoing a test for the cell line of choice for SILAC labeling efficiency and best media formulation will be conducted.

For discovery of small molecules for cancer I suggest to read the following review article entitled "Discovery of small molecule cancer drugs: Successes, challenges and opportunities " published in Molecular Oncology Volume 6, Issue 2, April 2012, Pages 155–176.

Abstract

The discovery and development of small molecule cancer drugs has been revolutionised over the last decade. Most notably, we have moved from a one-size-fits-all approach that emphasized cytotoxic chemotherapy to a personalised medicine strategy that focuses on the discovery and development of molecularly targeted drugs that exploit the particular genetic addictions, dependencies and vulnerabilities of cancer cells. These exploitable characteristics are increasingly being revealed by our expanding understanding of the abnormal biology and genetics of cancer cells, accelerated by cancer genome sequencing and other high-throughput genome-wide campaigns, including functional screens using RNA interference. In this review we provide an overview of contemporary approaches to the discovery of small molecule cancer drugs, highlighting successes, current challenges and future opportunities. We focus in particular on four key steps: Target validation and selection; chemical hit and lead generation; lead optimization to identify a clinical drug candidate; and finally hypothesis-driven, biomarker-led clinical trials. Although all of these steps are critical, we view target validation and selection and the conduct of biology-directed clinical trials as especially important areas upon which to focus to speed progress from gene to drug and to reduce the unacceptably high attrition rate during clinical development. Other challenges include expanding the envelope of druggability for less tractable targets, understanding and overcoming drug resistance, and designing intelligent and effective drug combinations. We discuss not only scientific and technical challenges, but also the assessment and mitigation of risks as well as organizational, cultural and funding problems for cancer drug discovery and development, together with solutions to overcome the ‘Valley of Death’ between basic research and approved medicines. We envisage a future in which addressing these challenges will enhance our rapid progress towards truly personalised medicine for cancer patients.

Highlights

► Here we review small molecule cancer drug discovery and development. ► We focus on Target selection, hit identification, lead optimization and clinical trials. ► A particular emphasis of this article is personalized medicine.

http://www.sciencedirect.com/science/article/pii/S1574789112000166

References:

Kim, et al. (2011). Angew. Chem. Int. Ed., 50: 2761.

Shaw, et al. (2011). Proc. Natl. Acad. Sci. USA, 108: 492.

Fomina-Yadlin, et al. (2010). Proc. Natl. Acad. Sci. USA, 107: 15099.

Chou, et al. (2010). ACS Chem. Biol., 5: 729.

Dančík, et al. (2010). J. Am. Chem. Soc., 132: 9259.

Clemons, et al., eds. (2009). Cell-Based Assays for High-Throughput Screening; Humana Press.

Wagner, et al. (2008). J. Am. Chem. Soc. 130: 4208.

Seiler, et al. (2008). Nucl. Acids Res. 36: D351-D359.

Bender, et al. (2007). Comb. Chem. High Throughput Screen. 10: 719.

Lamb, et al. (2006). Science 313: 1929.

Clemons (2004). Curr. Opin. Chem. Biol. 8: 334.

Haggarty, et al. (2003). J. Am. Chem. Soc. 125: 10543.

Ong, S. E., M. Schenone, et al. (2009) "Identifying the proteins to which small-molecule probes and drugs bind in cells." Proc Natl Acad Sci U S A 106:4617-22. 

Margolin, A. A., S. E. Ong, et al. (2009) "Empirical Bayes analysis of quantitative proteomics experiments." PLoS One 4:e7454. 

Hoping this will be helpful,

Rafik

It would help to know what organism is being treated with the small molecule. For example, if you are treating bacteria or viruses, then it is practical to consider selecting for resistance mutants and doing whole genome sequencing to look for potential targets based on mutations, but this is less practical with mammals because of the much larger genome.

Another method is the pull-down assay coupled with mass spectrometry and proteomics, as already mentioned (SILAC). This requires an immobilized version of the small molecule that still has biological activity.

 Thanks for the good advice above by Rafik and Adam.

Unfortunately, the small molecule modified now is not work well as itself. So, pull-down assay may be not work through. However, we could try the resistant mutants by high-throughput sequencing.

Best

Zhiyuan

Hello

First of all the easiest way is selecting some molecular target in mechanism of the disease you plan to work. and simply do docking and see on which target your molecule has more affinity and then find out that target involves in which mechanism and go ahead with in vitro. best of all