Abstract
Cancer remains one of the leading causes of death, with over 16 million cases worldwide. Several
therapeutic approaches have thus been developed over the years in the treatment of cancer all of
which display deficiencies in terms of specificity. New therapeutic approaches are therefore under
constant development, aimed at eliminating specific pathways exploited by cancerous cells. One
of these mechanisms to be investigated is the molecular interaction between the cancer associated
BAG2 protein and Heat shock protein 70.14 (Hsp70.14). Disruption of this interaction through the
discovery and development of protein-protein interaction (PPI) modulators serves as one of the
potential therapeutic approaches that can be used to reduce the development of cancer. This
present study was thus aimed at the recombinant expression, purification, and biochemical
characterization of the Hsp70.14~BAG2 complex, as well as using computational tools to identify
compounds that can disrupt the Hsp70.14~BAG2 interaction. The proteins were expressed in
competent E. coli cells, purified using affinity chromatography, and thereafter biophysically
characterized using FTIR, UV spectroscopy and Raman spectrometry. Additionally, Microscale
thermophoresis was used to assess the strength of the protein interaction. The molecular docking
and molecular dynamic simulations results reveal four potential compounds (CID:16260381,
CID:16391125, CID:16260381, and CID:42892779) displaying a high affinity to the BAG2
binding site. The FTIR results reveal a high number of carbonyl and hydroxyl groups present in
both proteins along with Hsp70.14~BAG2 complex, whilst the data from Raman spectroscopy of
the Hsp70.14~BAG2 complex displayed peaks representative of symmetric C-C stretching and
CH2 twisting vibrations. UV spectroscopy displayed a decrease in absorbance of the
Hsp70.14~BAG2 complex in comparison to the individual Hsp70.14 and BAG2 spectrums whilst
microscale thermophoresis quantitatively displayed a KD of 1.14 μM between the two proteins,
suggesting a moderate interaction. The interaction thus provides the basis for the structural
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determination of the protein complex towards the discovery and design of new anti-cancer
biopharmaceuticals.
Keywords: Cancer, chemotherapy, Hsp70.14, BAG2, protein-protein interaction, FTIR, Raman
spectrometry, anti-cancer biopharmaceuticals