Bolsa Doutoramento FCT: Biointeration studies of recombinant HPV-16 oncoproteins to discover therapeutic inhibitors [2020.06792.BD]

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Biointeraction studies of recombinant HPV16 oncoproteins to discover therapeutic inhibitors

Publication . Gomes, Diana Vanessa Duarte ; Sousa, Ângela Maria Almeida de; Passarinha, Luís António Paulino

Cervical cancer (CC) remains a significant global health burden, particularly in low- and middle-income countries, where it is a leading cause of cancer-related mortality among women. Annually, more than 600,000 new cervical cancer cases are diagnosed worldwide, with a 53% mortality rate. The development of CC is strongly associated with a persistent infection with high-risk human papillomavirus (HR-HPV), particularly HPV16 and HPV18, which are responsible for 70% of cases. This persistent HPV infection leads to uncontrolled proliferation of infected cells via downregulation of epithelial differentiation, disruption of epidermal development, and evasion of innate immune responses. The oncogenic potential of HR-HPV types is primarily attributed to the expression of E6 and E7 oncoproteins, which are integral to cancer phenotype maintenance in HPV-infected cells. Despite the implementation of preventive strategies, such as prophylactic vaccination and screening, which have contributed to a reduction in disease incidence, there remains an urgent need for targeted therapeutic interventions aimed at addressing persistent HPV infection and advanced cervical lesions. The HR-HPV E6 protein is composed of 158 amino acids and 2 zinc-binding motifs, and its most important function is escaping cell death by the induction of p53 degradation. In fact, E6 interacts with the LxxLL motif of the E6-associated protein (E6AP), an E3 ubiquitin ligase, inducing p53 degradation through the proteasome pathway and, consequently, blocking p53-dependent apoptosis. The HR-HPV E7 protein has around 98 amino acids, with an N-terminal region intrinsically disordered, whereas the C-terminal features a well-structured zinc-binding site. One of the most important protein regulation mechanisms by the E7 protein is through the retinoblastoma (pRb)/E2F system. The E7 protein binds to the pRb through the motif LxCxE, displacing E2F complexes, allowing its release and, as a result, the transcription of S-phase genes. This ensures that the infected cells remain in an S-phase-competent state, leading to hyperproliferation. Considering their crucial role in driving cellular transformation and cancer maintenance, both proteins are primary targets for developing targeted HPV therapies. However, the rational design and optimization of therapeutic strategies may depend on the ability to obtain both proteins. Unfortunatelyss, isolating E6 and E7 proteins from human cells is not feasible, so recombinant technology is a great alternative. Nonetheless, challenges persist related to the low solubility and tendency of these proteins to aggregate. Overcoming these issues is essential for enabling structural studies, biophysical characterization, and high-throughput screening of potential inhibitors. Thus, this thesis proposes the implementation of a biotechnology platform, starting with the biosynthesis towards the purification of the HPV16 E6 and E7 proteins, to ultimately discover inhibitory drugs that will be able to block the oncogenic effects triggered by both targets through biophysical and in vitro approaches. Firstly, we established a biotechnological platform for the recombinant expression and purification of HPV16 E6 in Escherichia coli BL21, employing a dual-tagged construct to enhance yield and address solubility issues. A two-step chromatographic process: affinity chromatography, followed by immobilized-metal affinity or size exclusion chromatography, obtained highly pure protein fractions (~98%). Circular dichroism confirmed that the secondary structure of His6-MBP-E6 protein is preserved in both purification strategies. Additionally, thermal shift assay (TSA) suggested potential additives for protein stabilization, such as maltose, glycerol, and detergents that can be incorporated in further characterization studies. In parallel to obtain the E6 protein, we focused on identifying natural compounds that may disrupt the E6/E6AP interaction using virtual screening. Three candidates selected from in silico studies with favorable predicted binding affinities were evaluated in HPV-negative and HPV-positive cell lines. Taxifolin and lucidin protected p53 from E6-mediated degradation and reactivated its transcriptional activity, leading to apoptosis induction. Nevertheless, neither compound affected cell cycle progression; thus, we extended the search for E6 inhibitors targeting directly the p53 binding site by exploring four distinct families of small molecules (barbiturates, dihydropyrimidinones/thiones, acetanilides and steroid derivatives). The screening was achieved through a combination of computational strategies and biophysical assays using the recombinant E6 protein previously produced. Fluorescence-based studies suggested that 4 compounds displayed a moderate affinity for the E6 protein. These candidates (CMP44, CMP50, CMP10c and CMP11c) were further characterized in HPV-positive and HPV-negative cell lines. The results demonstrated that CMP50 and CMP11c effectively rescued p53 from E6 degradation, reducing the proliferation and migration of HPV-positive cancer cells. Additionally, both compounds reactivated p53-dependent transcriptional factors, activating an apoptotic pathway and inducing cell cycle arrest, which suggests their therapeutic potential against HPV. Finally, considering the aim is also to study the E7 protein and potential inhibitors, we explored and characterized the dual-tagged recombinant HPV16 E7 protein. Following a similar workflow to what we had built for the E6 protein, we explored several chromatographic approaches, including affinity, anion-exchange, mixed-mode, and sizeexclusion chromatography, to obtain the His6-MBP-E7 protein. A thorough analysis of buffer compositions and additives indicated that a pH between 6.0 and 7.0, as well as sugars and detergents, maximized E7 solubility and minimized sample heterogeneity. In this process, we obtained the target protein with high yield, a purity of around 98% as confirmed by LC-MS/MS analysis. Additionally, the inclusion of detergents in the final formulations was studied, where the inclusion of DM (η-Decyl-β-D-Maltopyranoside) or η-Dodecyl-β-D-Maltopyranoside (DDM) led to a monodisperse sample. The optimized protocol is suitable for downstream structural and functional characterization, as well as for future inhibitor screening studies. Collectively, the work presented in this thesis establishes a reproducible platform for the production and study of the HPV16 E6 and E7 oncoproteins, which can be used for expressing proteins from other HPV types. Additionally, by integrating computational, biophysical, and cellular approaches, this research identified specific anti-HPV drugs, which can be the starting point for structural studies aiming at obtaining structures of the protein-inhibitor complexes.

2026-02-09Tese de doutoramento

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Palavras-chave

Natural sciences , Natural sciences/Biological sciences

Entidade financiadora

Fundação para a Ciência e a Tecnologia, I.P.

Programa de financiamento

Bolsa de Investigação para Doutoramento

Número da atribuição

2020.06792.BD