1. How much inhibitor should I use? Top
The amount of inhibitor required depends on various factors, such as target accessibility, cell permeability, duration of incubation, type of cells used, etc. It is best to survey the literature to determine the initial concentration. If published K_i or IC₅₀ values are known, one should use 5 to 10 times higher than these values to maximally inhibit enzyme activity. If K_i or IC₅₀ values are unknown, then one should try a wide range of inhibitor concentrations and use Michaelis-Menten kinetics to determine the K_i value. It is not unusual to see either no inhibition or even a reverse effect when high concentrations of inhibitors are used. Researchers should always run an appropriate control to eliminate non-specific effects of the solvent used to solubilize the inhibitor.

2. Which protein kinase inhibitor is best suited for my experiment? Top
If the mechanism involved in phosphorylation is unknown, a broad range inhibitor, such as staurosporine should be used first to determine if indeed a protein kinase is involved. Secondly, a more specific inhibitor of PKA (H-89, CN 371963, or 8-Br-cAMP, Rp isomer, CN 116816), PKC (Bisindolylmaleimide, CN 203290), or PKG (KT5823, CN 420321; or PKG inhibitor, CN 370654) should be used to eliminate the possibility of more than one kinaseS. To elucidate the exact mechanism involved, isozyme specific inhibitors, such as for PKC isozymes, can be used (please see Protein Kinases section in CALBIOCHEM's 1998 Signal Transduction Catalog).

3. What is the difference between EC₅₀, ED₅₀, K_i, and IC₅₀? Top
In pharmacology and biochemistry, in order to determine the efficacy of a drug or inhibitor, the following terms are commonly used. Sometimes, confusion arises when researchers try to repeat experiments without considering the exact term used by the original investigators.

EC₅₀: Clinical efficacy of a drug (Concentration required) to produce 50% of the maximum effect (may be inhibitory or stimulatory effect). This term is used usually with pharmaceuticals.

ED₅₀: Median effective dose (as opposed to concentration) at which 50% of individuals exhibit the specified quantal effect.

IC₅₀: Concentration required to produce 50% inhibition.

K_i: Inhibitor concentration at which 50% inhibition is observed (it is calculated using Michaelis-Menten kinetics).

4. How can I determine if a caspase inhibitor is reversible or irreversible? Top
The C-terminal group determines the reversibility or the irreversibility of any caspase inhibitor. In general, caspase inhibitors with an aldehyde (CHO) group are reversible. The CMK, FMK, and FAOM groups are more reactive and form covalent bonds with the enzyme creating an irreversible linkage. FMK is slightly less reactive than CMK and therefore is considered more specific for the enzyme site being inhibited.

5. What determines the specificity of a particular caspase Inhibitor? Top
The peptide recognition sequence determines the specificity of the inhibitor for a particular caspase. Sometimes the aspartic acid residue is methylated (OMe) to increase cell permeability of the peptide. VAD is a general caspase inhibitor. Earlier it was considered to be specific for caspase-1 (ICE), however, now it is considered to inhibit even caspase-3 and caspase-4. Addition of a tyrosine residue (Y) to the sequence (YVAD) makes the inhibitor more specific for caspase-1. The sequence DEVD recognizes caspase-3 and also caspases 6, 7, 8 and 10.

6. What are the advantages of using FMK based caspase inhibitors and how do they differ from CHO-based inhibitors? Top
The FMK based caspase inhibitors are cell-permeable because of the fact that the carboxyl group of aspartic or glutamic acid is esterified. This makes them more hydrophobic. These inhibitors covalently modify the thiol group of the enzyme making them irreversible inhibitors. Generally, at the amine end of the inhibitor we have a Z, biotin, or Ac group. These groups increase hydrophobicity of the molecule which makes them more cell-permeable. Compared to the inhibitors with an Ac or a biotin group, those inhibitors with a Z-group are more cell permeable. Inhibitors with a biotin group can serve as a detection tool and are useful in tagging the enzyme-inhibitor site.

The CHO-based inhibitors are reversible due to the fact that the thiol group of the enzyme forms an adduct to the carbonyl group of the aldehyde that is reversible. As a general rule CHO-based inhibitors are hydrated and hence are slow binding. The extent of their reversibility depends on the pH, metal ion concentration, and other conditions. When the aldehyde group is attached to the aspartic acid (D-CHO), the product exists as a pseudo acid aldehyde in equilibrium. This makes it somewhat cell permeable.