Structural and mechanistic studies of porcine pancreatic elastase
Keywords: gamma-lactams
benzoxaboroles
elastase

Serine peptidases (SPs) were among the earliest discovered enzymes and have since become some of the best characterised of all enzyme classes. SPs play important roles in many physiological processes including the immune response, blood clotting, and digestion. Due to the inherent destructive power of SPs, many have been implicated in disease. Despite a generally accepted SP mechanism being proposed more than 50 years ago, many of its details remain a matter of debate. One aspect that remains elusive is the presence of (a) tetrahedral intermediate(s). Such intermediates have not been observed by spectroscopic methods in a catalytic reaction between a SP and a substrate, but are inferred from studies using inhibitors that form analogous tetrahedral complexes. There are, however, several proposed tetrahedral intermediates that have been observed by crystallographic methods. The work presented herein aimed to further characterise one such intermediate and to prepare for time-resolved studies with PPE.
In Chapter 2, a proposed tetrahedral intermediate complex, formed between PPE and the heptapeptide inhibitor BCM7 in crystallo following ‘pH-jump’ studies, was further investigated. The pH-jumps were reproduced under novel conditions. The results from these studies suggest that the proposed tetrahedral intermediate is not, at least solely, a tetrahedral species. Furthermore, preparations were made to probe the reaction mechanism of PPE using time-resolved serial femtosecond crystallographic studies.
In Chapter 3, alternative inhibitors for time-resolved crystallography with PPE were investigated. Two variants of BCM7 were explored, revealing a novel crystallisation condition and space group for PPE. However, both variants were found to be unsuitable for time-resolved studies. The reaction of PPE with Sivelestat was subsequently explored. A reaction intermediate was captured in crystallo, supporting the proposed mechanism of reaction for Sivelestat with PPE. However, the use of Sivelestat for further time-resolved studies was ruled out.
Chapter 4 describes the optimisation of expression conditions and the purification method for recombinant ProPPE from E. coli. Initial attempts at expression manifested near complete insoluble expression, but reconfiguration of the gene, the use of alternate expression strains, and optimisation of post-induction incubation time and temperature resulted in improved soluble expression of ProPPE. Initial purification attempts also produced poor results. Optimisation of the protocol enabled pure and catalytically active protein, although these were not obtained in a single purification. However, a method to achieve pure and active PPE is proposed, although further optimisation is required to improve the final yield.
In Chapter 5, three inhibitor scaffolds were screened for inhibitory potential against PPE as a model for HNE inhibition. Five novel thiophene γ-lactam scaffolds were identified with single-digit micromolar IC50 values. The oxindole and benzoxaborole scaffolds were both found to be poor inhibitors of PPE with less than 50% inhibition at 200 μM. Crystallographic analyses with the benzoxaboroles rationalised the poor inhibitory potential of the scaffold, but guided suggestions of how to improve the potency of the scaffold in the future.
Overall, the body of work presented herein provides insight into a proposed intermediate of the PPE deacylation reaction and prepares the ground work for time-resolved studies. Additionally, the work lays the foundations to enable site-directed mutagenesis studies with PPE to complement time-resolved studies.