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Cystatin M/E is a member of a superfamily of evolutionarily-related cysteine protease inhibitors that provide regulatory and protective functions against uncontrolled proteolysis by cysteine proteases. Although most cystatins are ubiquitously expressed, high levels of cystatin M/E expression are mainly restricted to the epithelia of the skin (epidermis, hair follicles, sebaceous glands, and sweat glands) and to a few extracutaneous tissues. The identification of its physiological targets and the localization of these proteases in skin have suggested a regulatory role for cystatin M/E in epidermal differentiation. In vitro biochemical approaches as well as the use of in vivo mouse models have revealed that cystatin M/E is a key molecule in a biochemical pathway that controls skin barrier formation by the regulation of both crosslinking and desquamation of the stratum corneum. Cystatin M/E directly controls the activity of cathepsin V, cathepsin L, and legumain, thereby regulating the processing of transglutaminases. Misregulation of this pathway by unrestrained protease activity, as seen in cystatin M/E-deficient mice, leads to abnormal stratum corneum and hair follicle formation, as well as to severe disturbance of skin barrier function. Here, we review the current knowledge on cystatin M/E in skin barrier formation and its potential role as a tumor suppressor gene.

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Target Publications Biology Mcq Pdf Download

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Fully profiled chemical probes are essential to support the unbiased interpretation of biological experiments necessary for rigorous preclinical target validation. We believe that by developing a 'chemical probe tool kit', and a framework for its use, chemical biology can have a more central role in identifying targets of potential relevance to disease, avoiding many of the biases that complicate target validation as practiced currently.

Medicinal chemistry design and synthesis can provide selective tool compounds to interrogate biology, thus illustrating the synergy between chemical biology and drug discovery1,2. A major issue with using small molecules to augment target validation before launching a full drug discovery program is having the confidence that we have effectively validated the target of interest in a relevant phenotypic assay. There are many widely used chemical probes that do not meet generally accepted potency and selection criteria, and the conclusions made from their use are suspect. Similarly, the use of selective chemical probes in heavily manipulated biological assays is less likely to generate information of relevance to human disease.

When considering this problem, we can directly draw from our experiences in clinical drug development. A retrospective analysis of 44 drug programs in phase 2 clinical trials at Pfizer revealed that most failures resulted from a lack of efficacy, whereas the successful programs achieved what is termed the 'three pillars of survival': pillar 1, sufficient exposure at the site of action; pillar 2, proof of target engagement; and pillar 3, expression of functional pharmacological activity3. Moreover, for the projects that failed, it was often impossible to ascertain whether the target had been effectively tested in the clinic owing to gaps in some or all of the three pillars, reinforcing the need for an integrated understanding of the pharmacokinetic and pharmacodynamic characteristics of a drug to be established4.

Here we suggest that the three pillars concept provides a framework that may also be useful in the exploratory process of cell-based target validation using chemical probes, with the addition of a 'fourth pillar': proof of phenotype perturbation (Fig. 1 and Box 1).

Pillar 2. Target engagement. This is the most technically challenging pillar, though it is essential to link exposure at the site of action (pillar 1) to pharmacology (pillar 3) and phenotype (pillar 4). Exciting advances in techniques such as activity-based proteomics have enabled the quantification of target engagement to be made. Sophisticated functional probes can measure occupancy inside the cell and facilitate unbiased selectivity determination in a more physiologically relevant environment.

Pillar 4. Proof of phenotype perturbation. The challenge for cell biology is to create assays that capture the most relevant phenotypic changes in the context of human disease and for which there is a high degree of confidence in their 'translatability'. Phenomena that may lead to false positive results, such as nonspecific cell death, should be ruled out at an early stage. The strongest rationale for target validation can be provided if all four pillars are built in the same pathophysiologically relevant cell system.

By applying the principles of the four pillars to the use of chemical probes in cell-based assays, we propose that a more confident link between target perturbation and disease-relevant pharmacological modulation might be made. Here, we highlight examples of enabling technologies that can be applied in this context and provide an example of the successful application of the four pillars philosophy.

Selectivity against close-family-member biological targets, chemical structure and mode of inhibition, cell permeability and biochemical activity are four essential considerations in designing successful chemical probes. Additional considerations may include aqueous solubility and potency in cellular assays, the value of building a chemical tool kit that includes probes from orthogonal active and selective chemical classes and the importance of having structurally related inactive controls (for example, an inactive enantiomer, if available). Threshold values for potency and selectivity are discussed in the literature2.

Another related aspect of pillar 1 is ensuring that exposures are commensurate with the on-target activity of the probe and not in great excess, such that selectivity windows over off-targets are eroded. This is important as the use of probes in cell biology experiments at concentrations in excess of their selectivity window could lead to erroneous links being made between on-target activity and phenotypes, when it may be the off-target activity that is actually driving the observed response. We would recommend this as a key consideration in target validation studies, even when using quality probes with high selectivity.

Simple techniques can be applied to measure in-cell concentrations, such as LC-MS analysis of cell extracts. Local concentrations in specific subcellular compartments may also vary (subcellular compartments can even be targeted in the chemical probe design), and therefore more stringent fractionation might be required, together with the application of sophisticated microscopic imaging, MS and radiometric methods of detection and quantification6. An accurate and quantifiable pillar 2 (measurement of target engagement inside the cell) also, by definition, provides pillar 1.

Simple biochemical assays, though useful to drive the drug discovery process through optimization of potency and selectivity, provide limited information regarding the performance of a chemical probe in a whole-cell or in vivo context. For cell biology applications, it is important to understand the 'true' on- and off-targets of chemical probes to appropriately interpret the observed phenotypes (pillar 4) and biochemical mechanisms (pillar 3). Only with in-depth molecular mode-of-action studies can one gain high confidence in new mechanisms of therapeutic intervention. Arguably, the selectivity criteria for chemical biology probes (Fig. 2) should be more stringent than those for drug candidates, where 'safe' promiscuity may be a desirable property to drive efficacy. Focused selectivity profiling against potential specific off-targets can also be facilitated by considering the known biological targets of small molecules that are structurally similar to the chemical probe. However, even with stringent biochemical selectivity criteria, it is always possible (and indeed likely) that a given probe has unknown off-target activity. Therefore, we also advocate the use of a structurally orthogonal chemical probe to enable cross-validation studies and the identification of an inactive close analog for use as a negative control (for example, an inactive enantiomer; Fig. 2).

Once an appropriate tool has been identified, it is worthwhile to create a bespoke chemical proteomics probe on the basis of that specific tool's chemical structure. This provides an orthogonal method to assess selectivity and target engagement in a cell and also allows an unbiased proteomic assessment (as illustrated in example below). When generating affinity probes for proteomics, it is preferable to link the molecule via several attachment points to the resin so that multiple interacting domains can be sampled in the cell lysate in an unbiased manner (as on- and off-target SARs may not be the same).

Probes and their biological targets: 1 and 2 (NS5A inhibitors); 3 and 4 (irreversible RSK inhibitors); 5 and 6 (irreversible BTK inhibitors); and 7 and 8 (irreversible FAAH inhibitors). Arrows indicate the protein-reactive warhead, and red circles highlight clickable handles for subsequent attachment of fluorescent dyes or biotin reporters.

Irreversible inhibition modalities can often provide exquisitely selective chemical tools that can be subsequently furnished with additional functionalities to report on target engagement. For example, one approach has used a fluoromethylketone-containing probe (3) to target a specific cysteine residue in the ribosomal RSK1/2 kinase active site, leading to very specific target modulation13. Moreover, by installing an alkyne click handle into the molecule (enabling subsequent biotinylation), a selective probe for isolation and enrichment of RSK was created (4) that, when applied in cells, led to further biochemical understanding of RSK14. Similar approaches have been used to quantify target occupancy (pillar 2) of irreversible BTK inhibitors (such as 5) using functional derivatives (6), allowing more accurate assessment of pharmacodynamic-pharmacokinetic correlations in early clinical trials that enabled efficacious dose projections15,16. 006ab0faaa