In order to understand the pathophysiology of the disease, understanding the development of drug assays/screens that target essential and specific cellular and molecular processes that cause the disease. This in turn enhances the probability of discovering target- specific drug candidates that will be less toxic.
Developing a molecular-based assay
When a healthy cell is infected by a virus or bacterium there is cellular deterioration which occurs in different steps. This leads to the sick/dying cell. This then proliferates to form a disease. In the case of HIV patients where T cells are depleted they die not because of HIV but always due to secondary diseases.
Cancer as we all know is a disease of the older people; this is because over time there are genetic errors which lead to the formation of a tumor. There are ways to treat cancer to a certain extent and some of them are killing the cancer cells using chemotherapy, radiation or monoclonal antibodies. A new variety of drug class is angiogenesis where they disrupt the tumor's blood supply. Estrogen Inhibitors slows tumor growth in postmenopausal women with breast cancer. Metastasis stops cancer cells from spreading to other body locations.
Gleevac capsules created a sensation when they came into the market. Gleevac contains imatinib mesylate equivalent to 100mg of imatinib free base. The mechanism of action of gleevac is as follows:
Imatinib mesylate a protein-tyrosine kinase inhibitor that inhibits the Bcr-Abl tyrosine kinase, the constitutive abnormal tyrosine kinase created by the Philadelphia chromosome abnormality in chronic myeloid leukemia (CML). Gleevac knocks the functionality of Bcr-Abl, reduces cell division and reduces cancer but does not cure it.
The first step in assay development is to recognize your target and its activity, and then decide whether to inhibit the small or activate your target. If you possess an in vitro end-point enzymatic assay with a robust signal you would like to inhibit, this is usually the easiest type of assay to scale with appropriate positive and negative reference controls. Other types of assays, such as cell-based phenotypic assays, are more challenging to set up, scale and interpret since viable organisms must be uniformly deposited and maintained in microtitre plates and off target compound effects such as toxicity can confound the results. In addition, certain types of homogenous binding assays can be problematic because non-specific binding cannot be quantified and compound interference effects (such as autofluorescence) can swamp the specific signal.
The next step during the assay development phase is to identify suitable positive and negative reference controls to use in the assay. Traditionally, many biologists associate the term positive control with a maximum assay signal and the term negative control with a minimum signal. For purposes of consistency in screening, we define the positive control as being equivalent to what we would consider a "hit". For example, in a typical enzyme inhibition assay, we define the positive control as the enzyme + substrate + inhibitor, while the negative control is defined as the enzyme + substrate in the absence of inhibitor. For a typical enzyme inhibition screen, the ideal positive control would be a potent small molecule that inhibits the enzyme in the presence of substrate in a reliable fashion, while the negative control is usually the enzyme in the presence of substrate and DMSO (dimethyl sulphoxide.) Since our compound libraries are dissolved in DMSO, the reference controls should contain the same amount of DMSO solvent as the sample compounds being tested.
Because all samples and reference controls contain DMSO, it's very important to measure the DMSO tolerance of the assay. Whole cell assays normally tolerate no more than 1% DMSO, while biochemical assays can withstand higher DMSO concentrations. The recommended final working concentration of DMSO for biochemical assays is between 0.5%-5%.
One of the last major steps in assay development is to run entire plates each of the positive and negative controls using our bulk dispensers to aliquot reagents into plates. This step is necessary to evaluate the reproducibility and precision of your assay. Coefficients of variation can be calculated, in addition, one will easily spot any positional plate effects and systematic pipetting errors occurring in your methodology.
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