Target validation

Some questions that target validation has to answer are the following  

Which disease pathway does the target regulate  

What is the expected therapeutic index if a drug is to interact with the target  

To use the microarray, a known sequence of short DNA is printed onto a solid support of membrane or glass slide. From healthy and diseased cells, mRNAs are isolated. The mRNAs are used to generate complementary DNAs (cDNAs). Fluorescent tags are attached to the cDNAs, and the cDNAs are then mixed and incubated with the microarray supports (slides). 

Through a process called hybridization probing, the genes from the samples pair up with their complementary counterparts on the solid supports. When the hybridization step is completed, a scanner (laser beam and camera) is used to capture the fluorescence image of the array. 

When bacteria multiply through cell division, FtsZ is the first protein recruited to the division site and thereby defines the geometry of binary fission. Moreover, it directs the production of a new cell wall between the separating parts of the cell and is thus absolutely essential to bacterial reproduction. A temperature-sensitive mutant strain devoid of FtsZ above a certain temperature showed a filamenta-tion phenotype exactly as observed with ADEP-treated cells. In summary, ADEP-overactivated CIpP degrades FtsZ, which prevents cell division, causes the formation of massively elongated bacterial cells, and ultimately leads to cell death [7]. 

In the second section of this chapter, we turn to a different class of compounds. While ADEPs can be comprehended as large cyclic esters, fi-lactones are among 

Intact protein mass spectrometry allows the molecular mass determination of either proteins or complexes of proteins and covalently bound ligands/other proteins. In a first step, the sample is desalted to detach from buffer components and small ions that would interfere through noncovalent complexes in the gas phase. Next, the isolated protein is ionized, for example, by electrospray ionization (ESI). The acid in the eluent causes protonation of the protein at basic sites, particularly lysine and arginine residues, so that m/z values of multiple species with different charges can be measured in a mass spectrometer. These data are then combined during the deconvolution process to yield the mass of the protein or complex. 

Kohanski, M.A., Dwyer, D.J., and Collins, J.J. (2010) How antibiotics kill bacteria from targets to networks. Nat. Rev. Microbiol, 8, 423-435. 

Clatworthy, A.E., Pierson, E., and Hung, D.T. (2007) Targeting virulence a new paradigm for antimicrobial therapy. Nat. Chem. Biol, 3, 541-548. 

---

However, the target validation step is unique to target-based drug discovery. 

Inhibitors of the Angiopoietin/Tie-2 and Ephrin/Eph systems are in preclinical development which parallels the biological target validation of these molecules. As vascular assembly, maturation, and homeostasis regulating molecules, therapeutic interference with these molecular systems may hold promise for a number of vascular indications. 

Target validation, to determine that a gene product is causative of disease symptoms or that activation of the target protein ameliorates disease symptoms. Agonist/activator or an inhibitor which may be therapeutic could be identified using microarrays... 

Marton MJ et al. Drug target validation and identification of secondary drug tar-... 

INTERCONNECTING DISEASE, TARGET VALIDATION MODELS, AND PHARMACOLOGICAL RESPONSE... 

Strachan, R.T., Ferrara, G. and Roth, B.L. (2006) Screening the receptorome an efficient approach for drug discovery and target validation. Drug Discovery Today, 11, 708-716. 

Drug-discovery process is a highly complex, multidisciplinary and time-consuming procedure, which typically starts from the identification of appropriate drug targets (biomolecules, enzymes, ion channels, receptors) to target validation, where it was established whether the target was of relevance for the disease under study. 

Xin, H., Bernal, A., Amato, F.A., Pinhasov, A., Kauffman, J., Brenneman, D.E., Derian, C.K., ndrade-Gordon, P., Plata-Salaman, C.R., and Ilyin, S.E., High-throughput siRNA-based functional target validation, /. Biomol. Screen., 9, 286, 2004. 

Sundberg, S.A., Chow, A., Nikiforov, T., and Wada, H.G., Microchip-based systems for target validation and HTS, Drug Discov. Today, 5, 92, 2000. 

---