To study the interaction of natural product MCs with their target proteins a set of X-ray co-crystal structures of such complexes was identified. Selection began by identifying Protein Data Bank (PDB) entries containing a bound ligand with a ring of 14 or more atoms, where ring atoms were defined as atoms in the continuous, sigma-bonded chain that defines the macrocyclic scaffold (see section 2.3.2 for details on defining the ring). The resulting set of complexes was then further filtered using a series of criteria intended to ensure a representative set and remove redundancies, as detailed below.
Only complexes containing natural product MC compounds or compounds derived from natural product MCs were retained, which excluded ligands that were discovered via cyclization of acyclic leads, even if the acyclic lead was a natural product. Because the goal was to identify properties that could inform synthetic MC inhibitor design,
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only MCs that had chemical structures likely to be useful in drugs and, more specif-ically, function as inhibitors of their target protein, were kept (eg. no substrates, coordinated metal cofactors, RNA binders, etc...). To minimize redundancy, if a compound had a series of closely related analogs bound to the same protein, one rep-resentative structure was selected, prioritizing the original compound— except where crystal resolution or B factors of the original compound complex indicated a poorer quality structure, but the mode and level of inhibition of the analog were near iden-tical. For cases where identical MCs bound orthologs of a protein, one representative was selected to avoid bias, prioritizing the human protein where possible. Structures of the same MC complexed to distinct proteins, or the same protein bound to dif-ferent MC ligands, were not considered to be duplicates, because a difdif-ferent binding partner usually effects a distinct binding mode. For instances of multiple versions of identical ligand-protein pairs, structures were again prioritized by a combination of overall resolution and resolution of the ligand atoms and protein binding site atoms (i.e. structures containing a dual occupancy for a residue near a ligand atom were ranked lower). To ensure only realistic inhibitor interactions and binding modes were studied, complexes containing a mutation in the binding site were excluded, as well as those with a mutation known or intended to change the topology of the binding site. Complexes where binding site conformation was known or determined to be dependent on crystal contacts were also excluded. No structures with a resolution
>3.0 ˚A were allowed, though <2.5 ˚A was preferred and many were <2 ˚A. It should be noted that for the case of actin, numerous MC co-crystal complexes were found (>10), but many of the MC ligands could be grouped by similarities in structure and/or targeted region (e.g ring composition, type of “tail” substituent, side of the binding site targeted, etc.). In this case a representative example of each related set of complexes was chosen so as not to bias set composition with many ligands of a
single protein. For actin binding MC groups with multiple complex structures having good resolution, no mutations, etc. available, we tried to select the MCs that were more thoroughly studied and validated as inhibitors, with a better understanding of the inhibitors’ mode of action.
2.2.2 Composition of the Final MC Test Set
Following application of the above selection criteria, our final MC test set was com-posed of 22 distinct MC-protein complexes, encompassing 19 distinct MCs and 13 distinct proteins (plus one ortholog, bound to two distinct MCs). The final set con-tained MC compounds ranging in ring sizes of 14–35 atoms, with molecular weights of 365–1291 Da. The complexes are compiled in Table 2.1, including their corresponding PDB ID code, and the MC structures can be seen in Figures 2·1 and 2·2. A full list of the PDB entries containing a protein-MC complex that were considered during compilation of the test, and their reasons for exclusion, can be found in Table A.1 in Appendix A.
To assess how well our relatively small test set represented natural product MCs generally, we compared it to the 3747 natural product MCs analyzed by Wessjohann and colleagues (Wessjohann et al., 2005), as well as to the 44 approved MC drugs (primarily obtained from the ChEMBL database (Gaulton et al., 2012) ). A compar-ison of ring sizes, depicted in Figure 2·3, shows that our set corresponds well with both reference sets, and can be considered fairly representative of the range of natural product MCs. The main exception is for ring sizes 13–15, which is under-represented in the test set compared to the Wessjohann set; the population of test set rings sized 13–15 is similar to that of the approved drugs set. Wessjohann suggests this differ-ence in the smallest ring sizes is a result of an abundance of diterpenoids and their derivatives in nature. Moreover, many of these smallest ring MCs contain structures not relevant to medicinal chemistry, therefore making them unlikely to be considered
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Figure 2·1: The chemical structures of the large MCs.
Figure 2·1: The large MC structures, continued.
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Table 2.1: A list of the macrocycle-protein complexes in the test set.
Macrocycle Abbrv. Protein PDB ID
in drug development scenarios. Another difference is that the test set slightly over-represents very large rings, 34–36 ring atoms, relative to the natural products set, though the relative amount of large ring compounds is similar to that for approved MC drugs.
2.3 Structural Features and Physicochemical Characteristics of the Final