Integrated cellular, proteomic and metabolite analysis of argyrin B with rational design approaches for immunoproteasome-selective inhibitors

The ubiquitin-proteasome system plays a critical role in cellular protein degradation and homeostasis. Current proteasome inhibitors (PI) present great efficacy against haematologic cancers due to the high secretory load creating an increased dependency on proteostasis, yet resistance and adverse effects persist. The immunoproteasome has recently emerged as an attractive and selective target due to its preferential expression in disease states. Here the constitutive proteasome (CP) active sites β1c, β2c and β5c are replaced with immunoproteasome isoform containing β1i, β2i and β5i. Many ongoing efforts are focused on studying immunoproteasome-selective inhibitors, characterising their active site binding specificity and biochemical impact.

Argyrin analogues are naturally derived cyclic peptides showcasing antibacterial, immunosuppressive and anti-tumour effects. Efficacy and mechanisms vary by analogues, many of which are not characterised nor assessed in a variety of models. Despite this, there is demonstrable therapeutic potential through proposed targeting of the proteasome and mitochondrial elongation factor G; both integral to proteostasis. Further to this, argyrin B analogue has shown potential for selective inhibition of the β1i site of the immunoproteasome. This thesis extents studies on argyrin analogues as potential proteasome inhibitors through in silico analysis. Alongside biological studies of argyrin B cellular impact, proteomic effect and phase I metabolism.

Potential interactions of 16 argyrin analogues at CP and immunoproteasome active sites were studied by molecular docking using Autodock 4.2. Results showed striking preferential binding at β1i site over β1c site (p<0.05) in 75% of analogues, with analogues K, J, I and B additionally exhibiting improved β5i over β5c binding (p<0.05). For further SAR insights, a panel of naphthyl-azotricyclic-urea-phenyl scaffold-based compounds were docked using the same methods, corroborated through in vitro purified enzymatic assays. The key reasons for observed selectivity of these compounds are differences in subunit protein sequences, including substitutions G97H and M95S in β1c-β1i and T21S, A46S and S116E β5c-β5i, alongside differences in hydrophobic motifs that may have broader applications in rational drug design.

Argyrin B was further characterised for in vitro cellular impact in RPMI8226 multiple myeloma (MM) cells and noncancerous B-lymphocyte derived RPMI1788 cells and compared with the established PI, carfilzomib. From MTT assays and flow cytometry analysis, argyrin B exposure revealed cytotoxicity at low micromolar IC50 values. Interestingly, an increased sensitivity was shown towards RPMI8226 compared to RPMI1788, in contrast to carfilzomib. Quantitative proteomics experiments at IC50 doses, using UHPLC coupled to a Q-exactive Orbitrap mass spectrometer were employed to study the effects of argyrin B and carfilzomib on protein expression. Indicative of PIs, both compounds and cells triggered an unfolded protein response with upregulation of heat shock proteins, autophagy and proteasome bounce-back. Distinct regulation and putative selective MM targets identified in RPMI8226 included: STHM1, API5, PARK7 and MIF. Compared to carfilzomib, argyrin B exhibited greater differential protein expression between the cell types, yet overall at both cell types fewer biological pathways were impacted (FDR<0.005), together suggesting a more targeted action. Finally, the microsomal degradation of argyrin B followed by detection of metabolite compounds, exhibited oxidation and demethylation transformations, providing links back to SAR applications and analogue differences.

In summary, this study identified site-selective mechanisms for IP inhibition through in-silico investigation of argyrin analogues and analogues based on a naphthyl-azotricyclic-urea-phenyl scaffold. Further to this, the unique biochemical impacts of argyrin B on MM and B-lymphocyte cells were identified. With distinct impacts from carfilzomib, this can help to overcome current issues of resistance and off-target effects. Combined, the results from this study will advance the development of next generation PIs and understanding of the therapeutic applications of argyrins.