. INHIBITION OF CysK
The inherent inhibition of CysK comes from its binding to the ?-aminoacrylate intermediate as seen in M. tuberculosis 14. As it has been thoroughly discussed, the de-novo synthesis of cysteine involves conversion of serine to O-acetylserine by CysE, followed by the conversion of this metabolite to cysteine by CysK with the addition of Hydrogen Sulfide. However, the formation of ?-aminoacrylate intermediate is associated with the inhibition of CysK as it results in the domain rotation in the active site which accompanies by the closure of the active site. Thus, it is safe to assume that ?-aminoacrylate intermediate is an inhibitor of CysK 14.
Since the role in cyteine biosynthesis and importance of CysK has already been well-understood in bacteria, it provides the scope of its treatment as a novel target. Emergence of several MDR strains of common pathogenic bacteria has created a clinical emergency for discovering the novel target to develop a novel therapeutic. In line to these events, many researches are going on to find novel inhibitors for inhibition of CysK.
To design the CysK inhibitors, the ineraction of CysK-CysE to form Cystine Synthase Complex can be taken as a model. The structures of CsyK-CysE reveal that a C-terminal decapeptide from CysE forms complex with CysK. For instance, CysK from H. influenza forms complex with the GIDDGMNLNI decapeptide of CysE, CysK from A. thaliana complexes with YLTEWSDYVI of CysE and CysK from M. tuberculosis complexes with the DFSI C-terminal peptide from CysE. It has been observed that CysE decapeptide penetrates into the active site of CysK and competes with the substrate. However, only the last four aminoacids interact with the active site, contributing predominantly to the binding free energy. Also, the conserved C-terminal Ile276 is observed to be essential for the binding 15.
In quest to find inhibitor peptides for CysK from H.influenzae, Salsi et. al. determined the binding free energy of 400 pentapeptides, MNXXI, interacting with the HiCysK active site using a combined docking-scoring procedure based on GOLD and HINT. The free energy predictions were verified by the experimental determination of the binding affinity of 14 of these pentapeptides, selected for spanning a large range of predicted binding affinity and presenting charged, polar or apolar residues at mutation sites. Moreover, docked poses of three pentapeptides were compared to the conformations determined by X-ray crystallography. The crystallized peptide MNWNI was found to be the best binder (experimentally) among the tested peptides and in decreasing order, MNYDI, MNENI, MNETI and MNKGI.
A similar study about the pentapeptide inhibitor for CysK inhibition was done by Benoni et al, in which they used MNYDI peptide as an inhibitor of CysK from H. influenza. They observed that MNYDI peptide interacts with the HiCysK active site mainly through H-bonds involving its C-terminal carboxylate and hydrophobic interactions involving the side chains of Ile5 and Tyr3. They also concluded that the side chain of Ile is nearest to the active site residues and thus dominates the binding. This conclusion is given in the regard of the fact that the mutagenesis studies where removal and/or substitution of the C-terminal Ile of CysE consistently prevented the complex formation.
Along with the synthesis of inhibitors that mimic the C-terminal pentapeptide of CysE, a new array of novel synthetic/non-natural inhibitors are also in development which can provide better efficacy and potent binding to CysK. These non-natural molecules display better stability and pharmacokinetics than the natural peptide inhibitors in-vivo. Some of those synthetic inhibitors are discussed below:
6.1 UPAR40: It is a class of 2-substituted-cyclopropane-1-carboxylic acids which is synthesized on the basis of the knowledge grasped by analyzing synthetic peptides known to bind CysK from H. influenzae efficiently. Two molecular determinants were selected to design the new potential inhibitors. The carboxylic moiety and C2 lipophilic side chain of Ile267 was incorporated into the new structures. A cylcopropane ring was chosen as a spacer, anticipating that 3-carbon ring can block the two anchoring arms of Ile267 into a favorable conformation for binding to CysK. To increase the lipophilicity and efficacy, a series of 2-substituted cyclopropanecarboxylic acids were synthesized 17. Comparative efficacy and potency studies were conducted using MD simulations to determine the potent inhibitor from UPAR40, synthetic natural peptide MNWNI and inhibitor-free HiCysK. MD simulations reveal that UPAR40 seems to stabilize the PLP, whereas inhibitor-free and MNWNI bound HiCysK shows PLP fluctuations. UPAR40 stabilizes a conformational state of HiOASS-A different from that stabilized by the peptide inhibitor. The observations from MD simulations show that HiCysK behaves differently to a differently bound ligand (UPAR40 and MNWNI). The particular behavior of UPAR40 can explain the high potency of this compound (Kdiss=1.46_0.26 mM), which makes UPAR40 one of the most potent OASS-A inhibitors 18.
6.2 ??Substituted-2-Phenylcyclopropane Carboxylic Acids: UPAR40 as being an efficient inhibitor had its own limitations, for instance, poor chemical feasibility and stability. Therefore, to eliminate these limitations, the 2-phenylcyclopropane carboxylic acids were prepared. Many variations and modifications were done to produce different chemical compounds with enhanced affinity towards both the isomers of CysK. The docking of these compounds gave many useful insights into the structural mechanism for inhibition and how to modify them. The binding of the 2-phenylcyclopropane carboxylic acid left some void volume in the active site which can be exploited to increase the affinity of the molecule. In order to use that available volume, a compound was synthesized bearing a benzyl group at the ?-carbon. The addition of a lipophilic substituent into the benzyl ring in para position seems to remarkably enhance the affinity ratio with the two isomers 19.