Abu Bakar Salleh

Ligand-based virtual screening for the discovery of inhibitors for protein arginine deiminase type 4 (PAD4)

Protein Arginine Deiminase type 4 (PAD4) is a new therapeutic target for the treatment of rheumatoid arthritis. In this study, ligand-based virtual screening with the integration with drug repurposing strategy was applied to the discovery of PAD4 inhibitors. Ultrafast Shape Recognition (USR) was used to search for compounds with similar shape to a previously reported inhibitor with harmful side-effects, i.e., streptonigrin. Thirty five lead-like compounds and two existing drugs were obtained from virtual screening and their inhibitory activity was tested at fixed concentration of 100 μM. Five lead-like compounds showed significant inhibition on the enzymatic activity of PAD4. The potency of the best compound was investigated by carrying out IC50 study. Importantly, the structure of the best of these new active molecules was strikingly different from that of streptonigrin. Furthermore, this new PAD4 inhibitor is the most potent to date found by a computational approach and its structure can be optimized in the future for the design of an even better inhibitor of PAD4.

Conformational Design and Characterisation of a Truncated Diamine Oxidase from Arthrobacter globiformis

A functional mini protein can be developed by miniaturising its size. The minimisation technique provides an excellent model system for studying native enzymes, especially in creating an alternative novel biocatalyst. Miniaturised proteins may have enhanced stability, a crucial characteristic for large-scale production and industrial applications. In this study, a huge enzyme molecule, known as diamine oxidase (DAO, comprising 700 amino acids), was selected to undergo the process. By retaining the arrangement of the original functional sites of DAO in the fourth domain, a mini DAO can be designed via homology modelling. After several downsizing processes, a final configuration of 220 amino acids displayed high binding affinity towards histamine, a short-chain substrate that was catalysed by the parental DAO. The configuration also showed enhanced affinity towards a long-chain substrate known as spermidine. The gene for the designed protein was cloned and expressed in pET102/TOPO vector and overexpressed in E. coli BL21 (DE3). The new mini DAO had similar temperature tolerance and versatile substrates specificity characteristics as its parental protein. An active mini-protein with these characteristics is potentially useful for several applications such as detecting biogenic amines in the biological fluids and the environment that may give rise to health issues.

Biomolecular Design and Receptor-Ligand Interaction of a Potential Industrial Biocatalsyt: A Thermostable Thermolysin-Phosphoethanolamine-Ca2+ Protein Complex

Protein structures are prone to modification based on the fundamental rules of design and function. Calculations of free binding energies ( G) of chemical molecules (effectors) that bind to proteins are important in molecular signaling processes and catalytic mechanisms of certain key enzymes. These calculations can be obtained via in silico and theoretical approaches. A series of 48 pockets were identified in thermolysin (KEI) and the four biggest pockets were selected for their suitable sites for modification. Application of molecular docking on phosphoethanolamine (PSE) and 1,10-phenanthroline (PHN) that act as intermediate ligands in the designated protein complex showed favorable final docked energy at different pockets (-8.49 to -4.80 kcal/mol). Analysis on docking of a divalent metal ion (Ca) to ligand (PSE) produced a final docked energy of -4.15 kcal/mol within acceptable distance (1.5 Å M 3.0 Å). It was found that the thermolysin-phosphoetanolamine-Ca represented the putative protein complex of semisynthetic metalloprotease. Combinatorial modeling methods were applied in order to determine the best metalloenzyme complex. The identification of the potential protein pocket was conducted using CASTp. Selected ligands and metal ions were docked into each pocket using AutoDock 3.05. Analyses on their docking energy, non-covalent interaction as well as their geometry were conducted in order to determine the best metalloenzyme complex. This complex displayed the lowest docking energy with the additional Ca suitably docked. It was hypothesized that metal ions can add new functionality to proteins and catalyze some of the challenging biological reactions, particularly in the pharmaceutical and fine chemicals industries.