Posts classified under: Aside

Interest in using peptide molecules as therapeutic agents due to high selectivity and efficacy is increasing within the pharmaceutical industry. However, most peptide-derived drugs cannot be administered orally because of low bioavailability and instability in the gastrointestinal tract due to protease activity. Therefore, structural modifications peptides are required to improve their stability. For this purpose, several in-silico software tools have been developed such as PeptideCutter or PoPS, which aim to predict peptide cleavage sites for different proteases. Moreover, several databases exist where this information is collected and stored from public sources such as MEROPS and ExPASy ENZYME databases. These tools can help design a peptide drug with increased stability against proteolysis, though they are limited to natural amino acids or cannot process cyclic peptides, for example.

We worked to develop a new methodology to analyze peptide structure and amide bond metabolic stability based on the peptide structure (linear/cyclic, natural/unnatural amino acids). This approach used liquid chromatography / high resolution, mass spectrometry to obtain the analytical data from in vitro incubations. We collected experimental data for a set (linear/cyclic, natural/unnatural amino acids) of fourteen peptide drugs and four substrate peptides incubated with different proteolytic media: trypsin, chymotrypsin, pepsin, pancreatic elastase, dipeptidyl peptidase-4 and neprilysin. Mass spectrometry data was analyzed to find metabolites and determine their structures, then all the results were stored in a chemically aware manner, which allows us to compute the peptide bond susceptibility by using a frequency analysis of the metabolic-liable bonds. In total 132 metabolites were found from the various in vitro conditions tested resulting in 77 distinct cleavage sites. The most frequent observed cleavage sites agreed with those reported in the literature. The main advantages of the developed approach are the abilities to elucidate metabolite structure of cyclic peptides and those containing unnatural amino acids, store processed information in a searchable format within a database leading to frequency analysis of the labile sites for the analyzed peptides. The presented algorithm may be useful to optimize peptide drug properties with regards to cleavage sites, stability, metabolism and degradation products in drug discovery. 

More than 150 peptide therapeutics are globally in clinical development. Many enzymatic barriers should be crossed by a successful drug to be prosperous in such a process. Therefore, the new peptide drugs must be designed preventing the potential protease cleavage to make the compound less susceptible to protease reaction. We present a new data analysis tool developed in WebMetabase, an approach that stores the information from liquid chromatography mass spectrometry-based experimental data or from external sources such as the MEROPS database. The tool is a chemically aware system where each peptide substrate is presented as a sequence of structural blocks (SBs) connected by amide bonds and not being limited to the natural amino acids. Each SB is characterized by its pharmacophoric and physicochemical properties including a similarity score that describes likelihood between a SB and each one of the other SBs in the database. This methodology can be used to perform a frequency analysis to discover the most frequent cleavage sites for similar amide bonds, defined based on the similarity of the SB that participate in such a bond within the experimentally derived and/or public database. 

Aldehyde oxidase (AOX) is a metabolic enzyme catalyzing the oxidation of aldehyde and aza-aromatic compounds and the hydrolysis of amides, moieties frequently shared by the majority of drugs. Despite its key role in human metabolism, to date only fragmentary information about the chemical features responsible for AOX susceptibility are reported and only “very local” structure-metabolism relationships based on a small number of similar compounds have been developed.

This study reports a more comprehensive coverage of the chemical space of structures with a high risk of AOX phase I metabolism in humans. More than 270 compounds were studied to identify the site of metabolism and the metabolite(s). Both electronic [supported by density functional theory (DFT) calculations] and exposure effects were considered when rationalizing the structure-metabolism relationship. 

Aldehyde oxidase (AOX) is a molibdo-flavoenzyme that has raised great interest in recent years, since its contribution in xenobiotic metabolism has not always been identified before clinical trials, with consequent negative effects on the fate of new potential drugs. The fundamental role of AOX in metabolizing xenobiotics is also due to the attempt of medicinal chemists to stabilize candidates toward cytochrome P450 activity, which increases the risk for new compounds to be susceptible to AOX nucleophile attack.

Therefore, novel strategies to predict the potential liability of new entities toward the AOX enzyme are urgently needed to increase effectiveness, reduce costs, and prioritize experimental studies. In the present work, we present the most up-to-date computational method to predict liability toward human AOX (hAOX), for applications in drug design and pharmacokinetic optimization. The method was developed using a large data set of homogeneous experimental data, which is also disclosed as Supporting Information.