fragments that are to be simulated. An illustrative protocol for the
same is given below [65]- Generating a random number (RanX) with values ranging from
0to1. - If RanX<0.5, the fragment is inserted into the protein simu-
lation and if Ran X>0.5, the fragment is deleted. Both these
cases are trial moves with a probability assigned to each
attempted insertion and attempted deletion.
The calculation of the probability involves energy of the
system (depends on insertion or deletion of the fragment),
chemical potential of the simulation cell (the only parameter
which can be adjusted), gas constant, and the temperature.
Adjustment of high value of the chemical potential dramatically
enhances the chances of inserting a fragment, whereas low
value tends to evacuate the fragments from the simulation
cell. For each value of the chemical potential approximately
five million simulation steps are performed. - Independent running of SACP on a panel of fragments is done
which gives distinct binding pattern for each fragment on the
protein. - The individual simulations are combined together to predict
fragment binding with high affinity, clustering of the fragments
and exclusion of water.
The simulation results of SACP predicted that diphenylether
and its derivatives will bind in the hinge region without forming a
hydrogen bond at the ATP site. On the basis of SACP results,
Locus performed experiments and found that p38 is blocked by
two closely related diphenylether molecules having different modes
of binding in ATP site although their affinity was comparable. This
made Locus sure of not targeting the ATP site, as none of the
compounds had isoform specificity.
The fragment-based protocols finally shifted their focus to
develop compounds that binds purely to DFG motif. Hence, the
goal was to create a low molecular weight (<450 Da), soluble,
nonplanar, DFG binding compound. The compound must also
have the potential to inhibit p38 in cell assays with submicromolar
efficacy. To achieve the same, they tried replacing one of the func-
tional groups to reduce the molecular weight of the Bayer com-
pound. The phenyl moiety in Bayer was found to mimic the
phenylalanine of the conserved DFG triplet which binds in the
p38 allosteric site. Predictions of the SACP simulations showed
that free binding of energy for some aliphatic heterocyclic com-
pounds were comparable to that of phenyl groups. They confirmed
the predictions of substituting the phenyl group by dioxothiomor-
pholine and diazepanone using inhibition assays and
cocrystallography.138 Shashank P. Katiyar et al.