Substrate mapping and inhibitor profiling of falcipain-2, falcipain-3 and berghepain-2: implications for peptidase anti-malarial drug discovery.

The Plasmodium falciparum cysteine peptidases FP-2 (falcipain-2) and FP-3 (falcipain-3), members of the papain-like CAC1 family, are essential haemoglobinases and are therefore potential anti-malarial drug targets. To facilitate a rational drug discovery programme, in the current study we analysed the synthetic substrate and model inhibitor profiles of FP-2 and FP-3 as well as BP-2 (berghepain-2), an orthologue from the rodent parasite Plasmodium berghei. With respect to substrate catalysis, FP-2 exhibited a promiscuous substrate profile based around a consensus non-primeside motif, FP-3 was somewhat more restricted and BP-2 was comparatively specific. Substrate turnover for FP-2 was driven by a basic or acidic P1 residue, whereas for FP-3 turnover occurred predominately through a basic P1 residue only, and for BP-2, turnover was again mainly through a basic P1 residue for some motifs and surprisingly a glycine in the P1 position for other motifs. Within these P1 binding elements, additional recognition motifs were observed with subtle nuances that switched substrate turnover on or off through specific synergistic combinations. The peptidases were also profiled against reversible and irreversible cysteine peptidase inhibitors. The results re-iterated the contrasting kinetic behaviour of each peptidase as observed through the substrate screens. The results showed that the substrate and inhibitor preferences of BP-2 were markedly different from those of FP-2 and FP-3. When FP-2 and FP-3 were compared to each other they also displayed similarities and some significant differences. In conclusion, the in vitro data highlights the current difficulties faced by a peptidase directed anti-malarial medicinal chemistry programme where compounds need to be identified with potent activity against at least three peptidases, each of which displays distinct biochemical traits.

A single-step method for the production of sugar hydrazides: intermediates for the chemoselective preparation of glycoconjugates.

The ability to selectively conjugate carbohydrate molecules to a protein is a key step in the preparation of conjugate vaccines, while facile methods for linking carbohydrates to polymers or solid surfaces to produce diagnostic probes and functional microarrays are also sought. Here, we describe a simple, single-step method of producing glycosylhydrazides from unprotected sugars, which were then linked in a controlled manner to a desired carrier, through an appropriate linker. The method was chemoselective and did not require coupling reagents, and the native pyranose form of the reducing end residue was retained. Initially, mono- and disaccharide hydrazides were produced from the corresponding reducing sugars and linked to BSA through a bifunctional linker. Final exemplification of the procedure was demonstrated by the preparation of a LewisY tetrasaccharide protein conjugate, which was recognized by a LewisY monoclonal antibody indicating the preservation of the natural conformation of the tetrasaccharide in the final construct. It is envisaged that this method will have general applicability to a variety of functionally diverse reducing sugars and provide a route to highly defined glycoconjugates, without the need for elaborate synthetic strategies.

Novel chemoselective linkage chemistry toward controlled loading of ligands to proteins through in situ real-time quantification of conjugate formation.

'Linkage chemistry', which encompasses the science of chemical attachment of a ligand molecule to a carrier moiety, plays a crucial role in a wide range of biochemical and biophysical disciplines. In particular, the production of synthetic vaccines, where quality assurance criteria are an essential part of the approvals procedure for development of medicines, is reliant upon reproducible linkage chemistries. Herein, we describe novel 2-hydroxybenzaldehyde-based quaternary amine containing chemoselective linkers that provide a simple and robust linkage process that overcomes the deficiencies present in state-of-the-art linkage chemistries. The 2-hydroxybenzaldehyde groups undergo a pH-dependent absorbance change that enabled its nondestructive quantification, even when covalently attached to a wide range of proteins. Additionally, formation of a hydrazone bond between the benzaldehyde group and a range of ligand hydrazides resulted in a second reversible absorbance change enabling the forward (ligand loading) and reverse (ligand release for analysis) reactions of ligand-loaded proteins to be monitored in situ and quantified in real time. Incorporation of the quaternary amine moiety into our improved linkage chemistries was found to increase the relative solubility of protein conjugates and enabled significantly higher loading of proteins with linker and subsequent ligands, while retaining aqueous solubility, when compared to standard methods.