Since the Ahp cyclodepsipeptides had only recently been recognized as inhibitors that adopt the canonical conformation and thereby belong to the larger class of small molecule analogs of canonical-conformation proteinaceous serine protease inhibitors[18], the question arose if the Ahp residue is truly essential to form a canonical conformation. Indeed, it became apparent during this project that the synthesis of tailor-made Ahp cyclodepsipeptides is doable but nevertheless laborious. Consequently, synthetically easier accessible Ahp-mimics were sought.
To gain a quick and flexible access to the desired Ahp analogs, a new solid-phase synthesis was developed adapting some methodology from the previous synthesis of symplocamide A and using the specificity-determining P1 amino acid as the anchoring point to the solid
phase. Such an approach should enable access to a multitude of inhibitors for one protease by a combinatorial approach (Fig. 84).
Figure 84: General synthetic strategy for the synthesis of structure-activity analogs of Ahp cyclodepsipeptides,
A first set of symplocamide A analogs then proved that the Ahp unit could indeed be replaced by the - also hydroxylated - amino acid serine. On the other hand the hydroxylated amino acids threonine and homoserine did not provide active compounds.
The possibility of replacing the Ahp unit by serine then paved the way for the investigation of the elements that are required for the macrocycle to adopt the canonical conformation and thereby act as a non-covalent inhibitor of serine proteases. The different structure-activity analogs that were investigated are summarized in Table 5:
Table 5: Structure-activity analogs investigated to determine the crucial elements for the adoption of the
canonical conformation.
compound
(mimic of) replacementAhp substitutions further Ki
SAR-1 (Sym A) Ser - 3.8 μM
SAR-2 (Sym A) Hse - inactive
SAR-3 (Sym A) Thr - inactive
SAR-4 (Sym A) Ser 3-Br,4-OMe-mTyr→4-OMe-mTyr 4.8 μM
SAR-5 (Sym A) Ser 3-Br,4-OMe-mTyr→mPhe 56.1 μM
SAR-6 (Sym A) Ser 3-Br,4-OMe-mTyr→mPhe inactive
SAR-7 (Sym A) Ser 3-Br,4-OMe-mTyr→Pro 16.1 μM
SAR-8 (Sym A) Ser
3-Br,4-OMe-mTyr→mPhe
L-Thr→D-allo-Thr
inactive
SAR-9 (Sym A) Ser
3-Br,4-OMe-mTyr→mPhe
L-Thr→L-allo-Thr
inactive
SAR-10 (Scyp A) Ser - 19.1 μM
SAR-11 (Scyp A) Thr - 5.0 μM
SAR-12 (Scyp A) Val - inactive
As already expected from the works of Leatherbarrow et. al.[23] the most crucial element for the adoption of the canonical conformation is an amino acid that supports the formation of a cis amide bond in the P3’ position. In the Ahp cyclodepsipeptides this residue is a
proline residue or an even less active analog with a N-methyl phenylalanine residue. The possibility to substitute the conserved N-methylated amino acid with a proline clearly demonstrates that these analogs of Ahp depsipeptides are truly canonical-conformation inhibitors that require the reinforcement of the cis amide bond in the P3’ position.
Another structure element that was investigated and that has been suggested to play a role in the inhibition of serine proteases, is the threonine residue forming the depsipeptide linkage with the P4’ valine moiety. For the BBI inhibitors investigated by Leatherbarrow et. al.
a threonine in the P2 position is able to form a hydrogen bond to the P1’ serine and thereby
directs the aliphatic part of the threonine side chain towards a close contact with the imidazole part of the enzyme’s catalytic histidine. This contact is thought to interfere with a movement of the histidine that becomes necessary during the catalysis cycle and is proposed contribute to the inhibition of the protease.[18] Such a direction of the aliphatic part of the threonine side chain could indeed also be possible for Ahp cyclodepsipeptides and their structurally simplified analogs since the side chain is fixed by the ester bond and the direction of the methyl group is determined by the stereochemistry of the amino acid. Indeed, when the natural (L)-threonine residue was replaced by (D)-allo- or (L)-allo- threonine the resulting analogs were inactive against chymotrypsin. It has to be noted, however, that the requirement of a flexible histidine is discussed controversially in the scientific community. The results obtained here may yet be a humble contribution to this debate, hinting that indeed a blocking of the histidine’s movement may interfere with the processing of the substrate and thereby contribute to the inhibitory mode-of-action. On the other hand, the loss of activity may also result from an overall altered conformation of the macrocycle.
To finally prove that the concept of substituting the Ahp moiety in an Ahp cyclodepsipeptide with another amino acid is a global concept that affect other proteases than chymotrypsin, a different protease needed to be addressed. To achieve this, three different analogs of the natural product scyptolin A[27,28] were synthesized and tested for their inhibition of the target protease elastase. Fortunately, two of the three analogs were active against elastase, the expected serine analog and, surprisingly, also the analog with threonine as a replacement of the Ahp moiety.
The third analog incorporating a valine as a replacement for the Ahp moiety was inactive against elastase, pinpointing the requirement of a hydroxyl group in the side chain of the
Ahp replacement. In the Ahp residue, the hydroxyl group plays a crucial role forming one of the two intramolecular hydrogen bonds with the peptidic backbone, thereby reinforcing the rigid structure of the molecule that contributes to the inhibitory potency. The present data indicate that also serine- and threonine analogs can form such hydrogen bonds, thereby stabilizing the canonical conformation.
In summary, a general and flexible approach to the solid-phase combinatorial synthesis of Ahp cyclodepsipeptides was developed during this PhD work. The strategy was proven by the synthesis of the natural product symplocamide A which then served as starting point for the synthesis of more easily accessible analogs for structure-activity relationship studies. In these analogs the Ahp moiety was replaced by amino acids that are commercially available. For the synthesis of these analogs, a global, combinatorial solid-phase synthesis strategy was developed and several SAR analogs were synthesized.
As a result of the structure-activity-relationship studies serine and threonine could be determined as adequate replacements of the Ahp moiety. Furthermore, the crucial determinants that are required to adopt the canonical conformation could be studied as well as the mode-of-inhibition using the newly developed Ahp-mimics.
For future investigations, it would also be highly desirable to obtain a co-crystal structure or a NMR structure of one of the Ahp-mimicked analogs with their target protease in order to confirm the findings that so far are based only on enzyme inhibition assays. So far, all attempts to obtain a co-crystal structure with chymotrypsin or elastase were, however, not successful.
Another aspect that would be worthwhile to investigate in future studies is the structural basis of the different inhibitory activities of threonine- and serine-mimics of scyptolin A. The substitution of the Ahp moiety in these compounds with threonine resulted in a more potent species than the respective serine analog. A possible explanation for this unexpected finding could be a hindered rotation (and thus pre-organization) of the threonine side chain. Inhibitor binding would therefore result in less entropy loss (in comparison to the respective serine analog), thereby the binding may be favored energetically. A possible explanation for the absence of this effect in the symplocamide A analogs could be that here the threonine
investigations are however required and could involve on the one hand the synthesis of further analogs incorporating different P1 residues or Ahp substitutions with more bulky side
chain modifications. Finally, structural studies of the Ahp-mimics as well as the elucidation of the molecular binding mode via NMR or x-ray should provide the structural information required to prove the hypothesis.
Overall, the newly developed small molecule analogs of proteinaceous serine protease inhibitors may serve as tools in chemical biology to investigate serine proteases, their mode of action and their inhibition; furthermore, they could also serve as leads for the development of potent, selective and most importantly non-covalent drugs directed against serine proteases.
4.3 Development of a convergent synthesis of symplostatin 4 and derivatives for the