Accurate
and rapid crystal structure predictions have the potential to transform
the development of new materials, particularly in fields with highly
complex molecular structures (such as in drug development). In this
work we present a novel cloud-computing crystal structure prediction
(CSP) platform with the capability of scheduling hundreds of thousands
CPU cores and integrating cutting-edge computational chemistry algorithms.
This new cloud-computing based CSP platform has been applied to three
crystalline drug substances of increasing complexity. The lattice
energies of the experimental crystal structures are all within 4.0
kJ/mol of the lowest energy predicted structures. On the basis of
the results of this work, the algorithm improvement and the mass computational
power of cloud computing can reduce the whole CSP process to just
1–3 weeks for Z′ = 1 systems and less
than 5 weeks for significantly more complex systems. Furthermore,
it is possible to simultaneously perform calculations for multiple
molecules if desired. As a result of these improvements, CSP calculations
can potentially be applied in conjunction with state-of-the-art experimental
screening techniques to reduce the risk of finding new solid forms
after product launch provided that a sufficient number of stoichiometries
and space groups are explored.
Crystal
structure prediction (CSP) calculations can reduce risk
and improve efficiency during drug development. Traditionally, CSP
calculations use lattice energies computed through density functional
theory. While this approach is often successful in predicting the
low energy structures, it neglects the crucial role of thermal effects
on polymorph stabilities. In the present study, we develop a robust
and efficient protocol for predicting the relative stability of polymorphs
at different temperatures. The protocol is executed on a highly parallel
cloud computing infrastructure to produce results at time scales useful
for drug development timelines. We demonstrate this protocol on molecule
XXIII from the sixth crystal structure prediction blind test. Our
results predict that Form D is the most stable experimentally observed
polymorph at ambient temperature and Form C is the most stable at
low temperature consistent with experiments also conducted in the
present study.
Human soluble calcium-activated nucleotidase 1 (hSCAN-1) is the human homologue of soluble apyrases found in blood-sucking insects. This family of nucleotidases is unrelated in sequence to more well-studied nucleotidases, and very little is known about the enzymatic mechanism. By multiple sequence alignment, eight regions that are highly conserved in the hSCAN-1 family were identified and named. To identify amino acids important for catalytic activity and enzyme specificity, seven point mutations were constructed, expressed in bacteria, refolded, purified, and characterized. Substitution of glutamic acid 130 with tyrosine resulted in dramatically increased nucleotidase activities, while mutagenesis of aspartic acid 151 to alanine and aspartic acid 84 to alanine completely abolished activity. Mutagenesis of arginine 133 and arginine 271 resulted in enzymes with very little nucleotidase activity. Mutagenesis of aspartic acid 175 to alanine and glycine 122 to glutamic acid had smaller negative effects on enzyme activities. Previously, our laboratory showed that calcium triggers a conformational change in hSCAN-1 necessary for nucleotidase activity. Here we show that several mutants (D84A, R133A, and D151A) that lost most of their activity were unable to undergo the conformational change induced by Ca(2+), as shown by Cibacron blue binding, limited proteolysis, and tryptophan fluorescence. We conclude that aspartic acid residues 84 and 151, as well as arginine residue 133, are essential for the Ca(2+)-induced conformational change that is necessary for enzyme activity. Aspartic acid 175 and glutamic acid 130 are important for determining substrate specificity. In addition, we show that Sr(2+), unlike Mg(2+) and other divalent cations, can substitute for Ca(2+) to induce the conformational change necessary for enzyme activity. However, Sr(2+) cannot substitute for Ca(2+) to support nucleotide hydrolysis, presumably because Sr(2+) cannot substitute for Ca(2+) in its second role as a nucleotide cosubstrate. The ramifications of our results on the interpretation of a recently published crystal structure are discussed. This information will facilitate future engineering of this enzyme designed to enhance its ability to hydrolyze ADP and thus increase its potential for therapeutic use in the treatment of pathological ischemic events triggered via activation of platelets by ADP.
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