Obtaining bioactive conformations

Conformational analysis can be performed using a variety of methods.1161 Most important are X-ray analysis and NMR spectroscopic techniques, which allow detailed insights into the topology of the peptides. NMR combined with molecular dynamic calculations provides the spatial structure of the peptide, but also its dynamics in solution.13643491 Additional information can be obtained from FTIR,15,4521 CD,151,53-551 or Raman spectroscopy. 56,571 The results derived from such conformational analysis of cyclic peptides have a considerable impact on the study of the bioactive conformations of peptides and on the design of cyclic peptides as proteinomimetics.124,58,59,6301... 

The design of new drugs with improved activity is frequently based on the introduction of conformational constraints in order to freeze bioactive conformations of the molecules. For this purpose, a variety of conformationally constrained scaffolds, that can be functionalized with different pharmacophores, have been developed. In this context, carbohydrates appear to be ideal substrates due to their conformational rigidity and offer the possibility of different functionalization of their hydroxyl groups. (/) On the other hand, the introduction of an amino and a carboxylic group into the rigid structure of the carbohydrate, allows to obtain conformationally constrained peptidomimetics,... 

There are three NMR-based approaches suitable to propose the bioactive conformation of ligands in the absence of the coordinates of the whole complex 1. conformation analysis of free ligand in solution has a high chance to find with some population the bound conformation, although it may be solvent dependent (refer to Sect. 2.2.2) 2. determination of bound ligand conformation by means of transferred NMR methods if the dissociation constant is not smaller than approximately 50 pM (refer to Sects. 2.1 and 2.3) 3. solid-state NMR of the complex if the ligand can be obtained with 13C labels (Sect. 2.2.3). 

There has been long controversy regarding the bioactive conformation of PTX due to the difficulties to obtain high-resolution structural data from PTX bound to MT. Before the EC structure of PTX-stabilized tubulin polymer sheets [11] was available, solution NMR spectroscopy was used to derive candidate conformations for the tubulin-bound state of PTX. Early solution NMR studies on PTX have been reviewed by Jimenez-Barbero et al. [51] and only the fundamental results are summarized here. 

The tubulin-bound conformation of DDM in solution has been determined from tr-NOE data [112], Sample conditions were similar to those used previously to determine the bioactive conformation of EpoA. Distance restraints were obtained from a series of tr-NOE spectra recorded at several mixing times and were used in the structure calculation based on the complete relaxation matrix methodology [37], The NMR-derived bioactive conformation is quite similar to the crystal structure except for the conformation of the 8 lactone ring, that is close to a flattened chair in solution but a twisted boat in the crystal (Fig. 18). 

The work of Veber et al. established that valuable information about the bioactive conformation of a flexible peptide could be obtained by applying the principles of conformational restriction, and several additional examples soon were reported that followed this strategy. Conformationally restricted enkephalin analogs, e.g., 02-13), were formed by cyclizing between positions 2 and 5 of enkephalins (4a-b) (39). Cyclization of a-mela-notropin (14) gave the unusually active analog... 

Recently, we proposed a new bioactive conformation of paclitaxel, RKDOR-Taxol [50], based on (i) the 19F-13C distances obtained by the REDOR experiment [49], (ii) the photoaffinity labeling of microtubules [51], (iii) the crystal structure (PDB code 1TUB) of the Zn2+-stabilized aP-tubulin dimer model determined by cryo-electron microscopy (cryo-EM) [52], and (iv) molecular modeling (Monte Carlo Macromodel) [50], In this computational biology analysis, we first docked a paclitaxel-photoaffinity label molecule to the position identified by our photoaffinity labeling study and then optimized the... 

Recently, the possibility of obtaining dihedral angle information from a ligand in the bound state by exchange-transferred cross-correlation spectroscopy has been reported. This method has also been employed in the carbohydrate field with partial success.305 More examples are still required to further validate this approach to get bound bioactive conformations. 

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