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Custom Drug Screening

 Custom Drug Screening:

Anti-mitotic/Anti-Cancer/

Anti-Microtubule Drugs


Correct regulation of mitotic spindle formation during mitosis is critical to successful cellular reproduction in all eukaryotes. The bipolar mitotic spindle is a complex structure consisting of centrosomes, structural and motor proteins, enzymes and microtubules. Inducing aberrant mitosis in tumor cells by targeting any of the components of mitotic spindle leads to mitotic arrest, the consequence of which can be, but not always, cell death. The success of cancer therapies which target microtubules is driving the demand for better anti-microtubule compounds/drugs.  Microtubules (MTs)
are composed primarily of the protein tubulin and some microtubule associated proteins (MAPS). The
a-, b- and g-tubulins constitute a family of proteins of ~450 residue, with characteristics of GTP binding motifs. The a- and b- tubulins, each with a molecular mass of 55-kDa, form the heterodimers of 110-kDa. These heterodimers assemble reversibly to form cellular MTs. The g -tubulin is a centrosomal protein whose expression is necessary for MT organization and cell division. Both a and b tubulin exist in several isotypic forms. In addition, the isotypes are subjected to several posttranslational modifications. The tubulin dimers, assembled from isotypically pure subunits, especially b-, differ from each other in their conformational flexibility and stability, ligand binding properties and polymerization. Towards the goal of discovering an ideal anti-mitotic/anti-cancer drug, we offer our customers the identification of the lead compounds with an anti-microtubule/anti-tubulin activity. This service is offered as six different tests:

 

Test 1: Sensitivity of the Microtubules to perspective drugs/compounds: This basic screening utilizes drug-induced inhibition of the assembly of the mammalian MTs (mixture of tubulin and MAPs) to assess the potency of the drug. Unless specified, we will test the drugs at 1 and 100 mM concentrations. From this test, one should be able to identify those drugs from a given library, which are active against at least one of the constituents of microtubules (tubulin or one of the MAPs).

 

Test 2: Anti-tubulin (MAP-free) Activity of test- compounds: Our high thorough-put screening utilizes the drug-induced inhibition of polymerization of the purified mammalian tubulin (MAP-free), to assess the potency of the lead compound. Unless specified, we will test the drugs at 1 and 100 mM concentrations. The results from this screening can resolve whether or not the anti-mitotic activity of the compounds will arise from their interaction with tubulin.

 

Test 3: Screening of the Drugs/Compounds for the Tubulin Isotype Specific Reactivity: The screening at Tests 1 and 2 utilizes tubulin, which is a mixture of at least 7 isotypes, each of the a and b-tubulins. Depending on the choice of customers, we can test the effect of a compound on the assembly of all or one of the following isotypes: abII, abIII and abIV. Unless specified, we will test the drugs at 300 nM and 30 mM concentrations. From this test, one should be able to identify whether or not, a given drug can differentiate among the isotypes.  

 

Test 4: Determination of IC50 of the Drugs for the Microtubule Assembly: This service is an extension of the Tests-1 through 3. The inhibitory constant for the drug (s) will be determined using the tubulin polymerization assay. ALAMO LABS will test the effect of a compound (1 nM to 1 mM) on the assembly of either microtubule protein, pure tubulin or one of the isotypes of tubulin chosen by the customer. From this test, one should be able to appraise the potency of a given drug relative to other drugs in the same library or to those reported in literature.

 

Test 5: Determination of Binding Constant (Ka) of the Drug(s)/compounds for tubulin: The apparent affinity constant (Ka) of the drug(s)/compound(s) for purified mammalian tubulin or its isotypes is measured. The interaction is measured at 20 different concentrations (1 nM to 30 mM) of the drug/compound. The Ka values, which are invariably related to the anti-microtubule potency of the drug (measured at Tests-1 through-5) will assist the customer to sort out the lead compounds/drugs from a library for in vivo evaluation of their anti-microtubule potency.

 

Test 6: Cell Toxicity of the drug(s): Most of the anti-tubulin/anti-microtubule compounds inhibit the normal dynamic organization of the MTs that is necessary for the cell division. We can evaluate the effect of a given drug/compound on any cell line of customer’s choice, using a cell proliferation/ viability assay to appraise the anti-proliferative/viability potency of a given drug relative to the other drugs in the same library or to those reported in literature.

1. Screening of Anti-mitotic/Microtubule Compounds.

 Test ID

 Service

 Price*/
Compound
 MSCR-01  Screening of Anti-cancer/Anti-mitotic compounds using microtubules (Tubulin plus MAPs).  $125
 MSCR-02  Screening of Anti-cancer/Anti-mitotic compounds using Tubulin (without MAPs).  $175
 MSCR-03  Screening of Anti-cancer/Anti-mitotic compounds using Beta-II tubulin.  $285
 MSCR-04  Screening of Anti-cancer/Anti-mitotic compounds using Beta-III tubulin.  $285
 MSCR-05  Screening of Anti-cancer/Anti-mitotic compounds using Beta-IV tubulin.  $285
 MSCR-06  Binding Constant (Ka) of the Anti-cancer/Anti-mitotic compounds or tubulin.  Enquire
 MSCR-07  Cell Toxicity of Anti-cancer/Anti-mitotic compound.  Enquire

 *Price is based on a minimum of 10 test-compounds. A set-up fee of $500 will be added to the price of screening Anti-mitotic/Microtubule compounds.

Test Set-up for MSCR-01 thru 05:

Screening of the Drugs for the Anti-microtubule or Anti-tubulin Activity: Drug-induced inhibition
of the assembly of the mammalian microtubule protein or tubulin or tubulin isotypes is measured
to assess the potency of the drug. From this test, one should be able to identify those drugs
from a given library, which are active against microtubules, tubulin or one of its isoforms.
Microtubule Protein (5 mg/ml) or Tubulin Protein (1 mg/ml) will be polymerized in the presence
/absence of the test-compound and the polymerization will be followed by turbidimetry (change
 in absorbance at 340 nm). Total amount of the polymers in the steady state (where microtubule
 polymers are in equilibrium with the free tubulin), formed in the presence or absence of the test- compound, will be compared. All the experiments will be run in duplicate.

 Result-format for Tests MSCR-01 thru 05:

(1) The ORIGINAL DA340 values of control samples (in the absence of the test- compound).

(2) The ORIGINAL DA340 values of test samples (in the presence of the test- compound).

(3) The percent inhibition/stimulation of microtubule polymerization by the test- compound.