We present new findings in our drug discovery effort to develop an anticomplement therapeutic. We have designed several active analogues of compstatin by altering its amino acid composition at positions 4 and 9. The most effective analogues have tryptophan or fused-ring non-natural amino acids at position 4 and alanine or an unbranched single-methyl amino acid at position 9. Twenty-one of these analogues have 2-99-fold higher activities compared to the parent peptide compstatin. The analogue Ac-V4(2Nal)/H9A-NH(2) has the highest inhibitory activity with IC(50) 500 nM. NMR data, through NOE and chemical shift analysis, suggest the presence of interconverting conformers spanning the extended and helical regions of the Ramachandran plot, and they detect a predominant averaged conformer with coil structure and at least one flexible beta-turn, of type I. The fused-ring non-natural amino acids at position 4 contribute to the formation of the hydrophobic cluster of compstatin, which has been previously proposed, together with the beta-turn and a disulfide bridge, to be essential for binding to the target of compstatin, complement component C3. We propose that additional mechanisms may contribute to the structural stability of the analogues and to binding to C3, involving intra- and intermolecular electrostatic interactions of the pi-electron system of side chain aromatic rings. The presence of pi-pi interactions for Trp4-Trp7 was confirmed with a molecular dynamics simulation for the most active analogue with natural amino acids, Ac-V4W/H9A-NH(2). Alanine or aminobutyric acid at position 9 contribute to the weak propensity for helical structure of the residue segment 4-10 of the analogues, which may also play a role in increased activity.
The formation of micelles by dodecylphosphocholine (DPC) is modeled by treating the surfactants in atomic detail and the solvent implicitly, in the spirit of the EEF1 solvation model for proteins. The solvation parameters of the DPC atoms are carried over from those of similar atoms in proteins. A slight adjustment of the parameters for the headgroup was found necessary for obtaining an aggregation number consistent with experiment. Molecular dynamics simulations of 960 DPC molecules at different concentrations are used to obtain the aggregation number, the micelle size distribution, and the CMC. At 20 mM concentration we obtain an aggregation number of 53-56 and a CMC of 1.25 mM, values close to the experimental ones. At 100 mM the aggregation number increases to 90. Simulations of individual micelles of varying size show that the effective energy per surfactant molecule is initially a decreasing function of aggregation number but stabilizes at about 60 molecules. The van der Waals term and the desolvation of nonpolar groups contribute to micellization, whereas the desolvation of polar groups opposes it. From the difference between the effective energy and the free energy (calculated from the CMC), the translational and rotational entropy contributions to the free energy are estimated at about 7 kcal/mol per monomer. The micelles obtained here are more irregular than those obtained in explicit water simulations. This modeling approach allows the study of larger surfactant aggregates for longer times and the extraction of thermodynamic in addition to structural information.
We judge the energetic sequence of spin states in substituted methylenes by ab initio multiconfigurational computations and, where feasible, density functional modeling techniques. The best of these calculations reproduce well-established singlet−triplet gaps in X−C−Y species, in which X can be phenyl and Y can be H, methyl, or chloro. Similar computations on p-phenylene-coupled Y−methylenes and meta-coupled Y−methylenes support the suggestion by Zuev and Sheridan that bis(chloromethylene)-p-phenylene has a singlet diradical ground state. However, despite the density functional computations' support for those authors' suggestion that bis(chloromethylene)-m-phenylene has a singlet ground state, we find that our best MCSCF calculations place the quintet ground state suggested by the simplest theory almost equal in energy to that singlet.
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