Identification and Structural Analysis of Natural Product Inhibitors of Human Alpha-Amylase to Target Diabetes Mellitus
Authors: Ali Ameena Muhamed 
Published: July 2019

Introduction: Noninsulin dependent diabetes mellitus (NIDDM) or Diabetes Type 2 is a major health challenge worldwide. In 2014 the World Health Organization (WHO) revealed that the percentage of adults with fasting glucose ≥ 7.0 mmol/L accounts for 9% globally. Moreover, the highest percentage was in the Eastern Mediterranean Region with 26.8 ± 0.4%. Type 2 diabetes is a complex metabolic disorder associated with high level of glucose in blood (hyperglycemia) which lead to long-term pathogenic conditions such as: neuropathy, retinopathy, nephropathy and a consequent decrease in quality of life and increased mortality rate.Starch is the main source of energy for most living organisms; in humans it is digested over several stages that involve different amylolytic enzymes, such as α-Amylase and α-Glucosidase. Alpha Amylase is the major secretory product (about 5–6%) of the pancreas and salivary glands, playing a core role in starch and glycogen digestion. The control of postprandial hyperglycemia is an important strategy in the management of Type 2 diabetes; lifestyle modification and/or the use of medications such as insulin and α-Glucosidase inhibitors are the available treatments to date. Acrabose is a prominently used α-Glucosidase inhibitor for diabetes and obesity control, however it has many side effects and limitations. Numerous in vivo studies (REFS) have shown that many plant extracts inhibit the key enzymes of digestion (α-Amylase and α-Glucosidase) and the use of naturally occurring inhibitors is potentially the most effective and safest approaches for treating diabetes. Aims: Short-term aim: To clone, express and purify human α-Amylase protein using different yeast expression systems. Followed by protein (c0)crystallization and structural analysis.Long-term aim: Screening natural products (plant extracts) based on their traditional use followed by co-crystallisation of selected inhibitors. This will be complemented by inhibitors designed in silico based on the ANCHOR.QUERY approach (REF). Finally, identified compounds will be characterised by biophysical and kinetic studies. Methodology: Human α-Amylase has been cloned into two vectors pHIPZ-4 and pPIC9k, each with its own set of primers, restriction enzymes and dedicated expression host (pHIPZ-4: Hansenula polymorpha and pPIC9k: Pichia pastoris, respectively). After transformation, the grown colonies were tested for the presence of the target gene by colony PCR and digestion with cloning enzymes. Positive colonies were re-inoculated in growth media and recombinant plasmid was recovered. The plasmids were then transformed into competent yeast cells by electroporation. For H. polymorpha, cells were grown in minimal media with glucose (MM/G) for two days followed by induction of expression with 0.5% (v/v) MeOH. Protein was purified by lysing the cells and passing the lysate through Ni-NTA beads. Finally protein was identified with by Western blot using HisProbe-HRP antibody. Results: Human α-Amylase gene was successfully cloned in both vectors pHIPZ-4 and pPIC9k according to colony PCR and digestion judgment on Agarose gel. However, the expression of α-Amylase protein in H. polymorpha system is insufficient to support the downstream work. Analysis of secreted expression using Pichia pastoris is currently underway and the results will be reported at the meeting. Conclusion: Human α-Amylase was successfully cloned in both vectors and stably transformed to E. coli competent cells. The positive colonies were confirmed for the present of targeted gene then transformed to yeast cells.