Autochthonous Peruvian Natural Plants as Potential SARS-CoV-2 Mpro Main Protease Inhibitors.

Over 750 million cases of COVID-19, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), have been reported since the onset of the global outbreak. The need for effective treatments has spurred intensive research for therapeutic agents based on pharmaceutical repositioning or natural products. In light of prior studies asserting the bioactivity of natural compounds of the autochthonous Peruvian flora, the present study focuses on the identification SARS-CoV-2 Mpro main protease dimer inhibitors. To this end, a target-based virtual screening was performed over a representative set of Peruvian flora-derived natural compounds. The best poses obtained from the ensemble molecular docking process were selected. These structures were subjected to extensive molecular dynamics steps for the computation of binding free energies along the trajectory and evaluation of the stability of the complexes. The compounds exhibiting the best free energy behaviors were selected for in vitro testing, confirming the inhibitory activity of Hyperoside against Mpro, with a Ki value lower than 20 µM, presumably through allosteric modulation.

[Clinicopathological profile of patients with hepatobiliary cancers at two main referral hospitals in Lima, Peru]. / Perfil clínico-patológico de pacientes con cáncer hepatobiliar en dos hospitales de referencia en Lima, Perú.

OBJECTIVE: The aim of the study was to describe the clinicopathological profile of patients diagnosed with liver, bile ducts or gallbladder cancer. MATERIALS AND METHODS: Between 2006 and 2017, 89 patients (57% female; mean age: 62 years-old) with these cancers were diagnosed at two national hospitals in Lima, Peru. RESULTS: Most patients (64%) had advanced stages of disease. Anemia was more frequent in patients with bile duct and liver cancer and in advanced stages. Hypertension (HTN) was frequent among liver cancer patients (32%). The analysis by age showed that HTN was more frequent in patients over 50 years. Likewise, people under 50 years had more frequent history of previous infections (50%), Hepatitis B (HBV) being the most common. CONCLUSIONS: This study describes the baseline clinicopathological characteristics of a malignancy poorly studied in Peru.

Perfil clínico-patológico de pacientes con cáncer hepatobiliar en dos hospitales de referencia en Lima, Perú / Clinicopathological profile of patients with hepatobiliary cancers at two main referral hospitals in Lima, Peru

RESUMEN Objetivo: El objetivo del estudio fue describir las características clínico-patológicas de individuos diagnosticados de cáncer de hígado, vías biliares o vesícula. Materiales y métodos: Entre el 2006 y 2017, se diagnosticaron 89 pacientes (57% mujeres; media: 62 años) con estos canceres en dos hospitales nacionales de Lima, Perú. Resultados: Los resultados mostraron que, independientemente del tipo de cáncer, 64% de los participantes habían sido diagnosticados en estadios avanzados. La anemia fue más frecuente en los pacientes con cáncer de vías biliares e hígado y en estadios avanzados. Se observó mayor frecuencia (32%) de hipertensión arterial (HTA) en el grupo con cáncer de hígado. El análisis por edad mostró que en los pacientes mayores de 50 años la HTA fue más frecuente. Asimismo, sujetos menores de 50 años reportaron antecedentes de infecciones previas en mayor frecuencia (50%), siendo Hepatitis B (HBV) la más común. Conclusiones: Este estudio describe las características clínico-patológicas de base de una neoplasia poco estudiada en el ámbito nacional.

ABSTRACT Objective: The aim of the study was to describe the clinicopathological profile of patients diagnosed with liver, bile ducts or gallbladder cancer. Materials and methods: Between 2006 and 2017, 89 patients (57% female; mean age: 62 years-old) with these cancers were diagnosed at two national hospitals in Lima, Peru. Results: Most patients (64%) had advanced stages of disease. Anemia was more frequent in patients with bile duct and liver cancer and in advanced stages. Hypertension (HTN) was frequent among liver cancer patients (32%). The analysis by age showed that HTN was more frequent in patients over 50 years. Likewise, people under 50 years had more frequent history of previous infections (50%), Hepatitis B (HBV) being the most common. Conclusions: This study describes the baseline clinicopathological characteristics of a malignancy poorly studied in Peru.

In silico prediction of secretory proteins of Opisthorchis viverrini, Clonorchis sinensis and Fasciola hepatica that target the host cell nucleus.

Liver flukes Fasciola hepatica, Opisthorchis viverrini and Clonorchis sinensis are causing agents of liver and hepatobiliary diseases. A remarkable difference between such worms is the fact that O. viverrini and C. sinensis are carcinogenic organisms whereas F. hepatica is not carcinogenic. The release of secretory factors by carcinogenic flukes seems to contribute to cancer development however if some of these target the host cell nuclei is unknown. We investigated the existence of O. viverrini and C. sinensis secretory proteins that target the nucleus of host cells and compared these with the corresponding proteins predicted in F. hepatica. Here we applied an algorithm composed by in silico approaches that screened and analyzed the potential genes predicted from genomes of liver flukes. We found 31 and 22 secretory proteins that target the nucleus of host cells in O. viverrini and C. sinensis, respectively, and that have no homologs in F. hepatica. These polypeptides have enriched the transcription initiation process and nucleic acid binding in O. viverrini and C. sinensis, respectively. In addition, other 11 secretory proteins of O. viverrini and C. sinensis, that target the nucleus of host cells, had F. hepatica homologs, have enriched RNA processing function. In conclusion, O. viverrini and C. sinensis have 31 and 22 genes, respectively, that may be involved in their carcinogenic action through a direct targeting on the host cell nuclei.

Potential Protective Effect from COVID-19 Conferred by Altitude: A Longitudinal Analysis in Peru During Full Lockdown.

Thomson, Timothy M., Fresia Casas, Harold Andre Guerrero, Rómulo Figueroa-Mujíca, Francisco C. Villafuerte, and Claudia Machicado. Potential protective effect from COVID-19 conferred by altitude: A longitudinal analysis in Peru during full lockdown. High Alt Med Biol. 22: 209-224, 2021. Background: The COVID-19 pandemic had a delayed onset in America. Despite the time advantage for the implementation of preventative measures to contain its spread, the pandemic followed growth rates that paralleled those observed before in Europe. Objectives: To analyze the temporal and geographical distribution of the COVID-19 pandemic at district-level in Perú during the full lockdown period in 2020. Methods: Analysis of publicly available data sets, stratified by altitude and geographical localization. Correlation tests of COVID-19 case and death rates to population prevalence of comorbidities. Results: We observe a strong protective effect of altitude from COVID-19 mortality in populations located above 2,500 m. We provide evidence that internal migration through a specific land route is a significant factor progressively overriding the protection from COVID-19 afforded by high altitude. This protection is independent of poverty indexes and is inversely correlated with the prevalence of hypertension and hypercholesterolemia. Discussion: Long-term adaptation to residency at high altitude may be the third general protective factor from COVID-19 severity and death, after young age and female sex. Multisystemic adaptive traits or acclimatization processes in response to chronic hypobaric hypoxia may explain the apparent protective effect of high altitude from COVID-19 death.

E6AP goes viral: the role of E6AP in viral- and non-viral-related cancers.

Since its discovery, the E3 ubiquitin ligase E6-associated protein (E6AP) has been studied extensively in two pathological contexts: infection by the human papillomavirus (HPV), and the neurodevelopmental disorder, Angelman syndrome. Vital biological links between E6AP and other viruses, namely hepatitis C virus and encephalomyocarditis virus, have been recently uncovered. Critically, oncogenic E6AP activities have been demonstrated to contribute to cancers of both viral and non-viral origins. HPV-associated cancers serve as the primary example of E6AP involvement in cancers driven by viruses. Studies over the past few years have exposed a role for E6AP in non-viral-related cancers. This has been demonstrated in B-cell lymphoma and prostate cancers, where oncogenic E6AP functions drive these cancers by acting on key tumour suppressors. In this review we discuss the role of E6AP in viral infection, viral propagation and viral-related cancer. We discuss processes affected by oncogenic E6AP, which promote cancers of viral and non-viral aetiology. Overall, recent findings support the role of oncogenic E6AP in disrupting key cellular processes, including tumour suppression and the immune response. E6AP is consequently emerging as an attractive therapeutic target for a number of specific cancers.

Screening the Pathogen Box for Identification of New Chemical Agents with Anti-Fasciola hepatica Activity.

Fascioliasis is an infectious parasitic disease distributed globally and caused by the liver fluke Fasciola hepatica or F. gigantica This neglected tropical disease affects both animals and humans, and it represents a latent public health problem due to the significant economic losses related to its effects on animal husbandry. For decades, triclabendazole has been the unique anti-Fasciola drug that can effectively treat this disease. However, triclabendazole resistance in fascioliasis has more recently been reported around the world, and thus, the discovery of novel drugs is an urgent need. The aim of this study was to investigate the fasciocidal properties of 400 compounds contained in the Pathogen Box. The first stage of the screening was carried out by measuring the fasciocidal activity on metacercariae at a concentration of 33 µM each compound (the standard dose). Subsequently, the activities of the most active compounds (n = 33) at their 50% inhibitory concentration (IC50) values against metacercariae were assayed, and the results showed that 13 compounds had IC50s of ≤10 µM. The second stage queried the activities of these compounds at 33 µM against adult flukes, with seven of the compounds producing high mortality rates of >50%. Four hit compounds were selected on the basis of their predicted nontoxic properties, and the IC50 values obtained for adult worms were <10 µM; thus, these compounds represented the best fasciocidal compounds tested here. A cytotoxicity assay on four types of cell lines demonstrated that three compounds were nontoxic at their most active concentration. In conclusion, three hit compounds identified in this proof-of-concept study are potential candidates in the discovery of new fasciocidal drugs. Further studies are warranted.

Negative serology of Fasciola hepatica infection in patients with liver cancer in Peru: a preliminary report.

INTRODUCTION: The etiology of several hepatocellular carcinoma (HCC) cases remains largely unknown. Although Fasciola hepatica has been associated with liver fibrosis in Latin America, it has not yet been associated with HCC. This study aimed to determine the existence of specific IgG antibodies against F. hepatica in the serum samples of HCC patients. METHODS: In total, 13 serum samples from 13 HCC patients were screened using Fas2-ELISA. RESULTS: Fas2-ELISA demonstrated negative results in all HCC patients included in this study. CONCLUSIONS: The pre-existence of F. hepatica infection in HCC patients needs to be further investigated in epidemiological and experimental studies.

Negative serology of Fasciola hepatica infection in patients with liver cancer in Peru: a preliminary report

Abstract INTRODUCTION: The etiology of several hepatocellular carcinoma (HCC) cases remains largely unknown. Although Fasciola hepatica has been associated with liver fibrosis in Latin America, it has not yet been associated with HCC. This study aimed to determine the existence of specific IgG antibodies against F. hepatica in the serum samples of HCC patients. METHODS In total, 13 serum samples from 13 HCC patients were screened using Fas2-ELISA. RESULTS Fas2-ELISA demonstrated negative results in all HCC patients included in this study. CONCLUSIONS The pre-existence of F. hepatica infection in HCC patients needs to be further investigated in epidemiological and experimental studies.

A computational assessment of the predicted structures of Human Macrophage Migration Inhibitory Factor 1 orthologs in parasites and its affinity to human CD74 receptor.

The human macrophage migration inhibitory factor 1 (Hu-MIF-1) is a protein involved in the inflammatory and immunology response to parasite infection. In the present study, the existence of Hu-MIF-1 from parasites have been explored by mining WormBase. A total of 35 helminths were found to have Hu-MIF-1 homologs, including some parasites of importance for public health. Physicochemical, structural, and biological properties of Hu-MIF-1 were compared with its orthologs in parasites showing that most of these are secretory proteins, with positive net charge and presence of the Cys-Xaa-Xaa-Cys motif that is critical for its oxidoreductase activity. The inhibitor-binding site present in Hu-MIF-1 is well conserved among parasite MIFs suggesting that Hu-MIF inhibitors may target orthologs in pathogens. The binding of Hu-MIF-1 to its cognate receptor CD74 was predicted by computer-assisted docking, and it resulted to be very similar to the predicted complexes formed by parasite MIFs and human CD74. More than 1 plausible conformation of MIFs in the extracellular loops of CD74 may be possible as demonstrated by the different predicted conformations of MIF orthologs in complex with CD74. Parasite MIFs in complex with CD74 resulted with some charged residues oriented to CD74, which was not observed in the Hu-MIF-1/CD74 complex. Our findings predict the binding mode of Hu-MIF-1 and orthologs with CD74, which can assist in the design of novel MIF inhibitors. Whether the parasite MIFs function specifically subvert host immune responses to suit the parasite is an open question that needs to be further investigated. Future research should lead to a better understanding of parasite MIF action in the parasite biology.

Association of Fasciola hepatica Infection with Liver Fibrosis, Cirrhosis, and Cancer: A Systematic Review.

BACKGROUND: Fascioliasis has been sporadically associated with chronic liver disease on previous studies. In order to describe the current evidence, we carried out a systematic review to assess the association between fascioliasis with liver fibrosis, cirrhosis and cancer. METHODOLOGY AND PRINCIPAL FINDINGS: A systematic search of electronic databases (PubMed, LILACS, Scopus, Embase, Cochrane, and Scielo) was conducted from June to July 2015 and yielded 1,557 published studies. Among 21 studies that met inclusion and exclusion criteria, 12 studies explored the association of F. hepatica with liver fibrosis, 4 with liver cirrhosis, and 5 with cancer. Globally these studies suggested the ability of F. hepatica to promote liver fibrosis and cirrhosis. The role of F. hepatica in cancer is unknown. Given the heterogeneity of the studies, a meta-analysis could not be performed. CONCLUSIONS: Future high-quality studies are needed to determine the role of F. hepatica on the development of liver fibrosis, liver cirrhosis, and cancer in humans.

Hypothetical granulin-like molecule from Fasciola hepatica identified by bioinformatics analysis.

Fasciola hepatica is considered an emergent human pathogen, causing liver fibrosis or cirrhosis, conditions that are known to be direct causes of cancer. Some parasites have been categorized by WHO as carcinogenic agents such as Opisthorchis viverrini, a relative of F. hepatica. Although these two parasites are from the same class (Trematoda), the role of F. hepatica in carcinogenesis is unclear. We hypothesized that F. hepatica might share some features with O. viverrini and to be responsible to induce proliferation of host cells. We analyzed the recently released genome of F. hepatica looking for a gene coding a granulin-like growth factor, a protein secreted by O. viverrini (Ov-GRN-1), which is a potent stimulator of proliferation of host cells. Using computational biology tools, we identified a granulin-like molecule in F. hepatica, here termed FhGLM, which has high sequence identity level to Ov-GRN-1 and human progranulin. We found evidence of an upstream promoter compatible with the expression of FhGLM. The FhGLM architecture showed to have five granulin domains, one of them, the domain 3, was homologue to Ov-GRN-1 and human GRNC. The structure of the FhGLM granulin domain 3 resulted to have the overall folding of its homologue the human GRNC. Our findings show the presence of a homologue of a potent modulator of cell growth in F. hepatica that might have, as other granulins, a proliferative action on host cells during fascioliasis. Future experimental assays to demonstrate the presence of FhGLM in F. hepatica are needed to confirm our hypothesis.

Carcinogenesis associated with parasites other than Schistosoma, Opisthorchis and Clonorchis: A systematic review.

Only three helminths (Schistosoma haematobium, Opisthorchis viverrini and Clonorchis sinensis) are directly associated with carcinogenesis in humans whereas the role of other parasites in cancer remains unclear. This study aimed to perform a systematic review to identify recent insights in the role of other parasite infections in carcinogenesis. We conducted systematic searches of MEDLINE and EMBASE on July 2015. Our primary outcome was the association between parasitic infections and carcinogenesis. Out of 1,266 studies, 19 were selected for detailed evaluation (eight for helminths and 11 for protozoa). The mechanisms of helminth-induced cancer included chronic inflammation, sustained proliferation, modulation of the host immune system, reprogramming of glucose metabolism and redox signaling, induction of genomic instability and destabilization of suppressor tumor proteins, stimulation of angiogenesis, resisting cell death, and activation of invasion and metastasis. In addition to the current knowledge, the following parasites were found in cancers or tumors: Echinococcus, Strongyloides, Fasciola, Heterakis, Platynosomum and Trichuris. Additional parasites were found in this systematic review that could potentially be associated with cancers or tumors but further evidence is needed to elaborate a cause-effect relationship.

Design of ligand binding to an engineered protein cavity using virtual screening and thermal up-shift evaluation.

Proteins could be used to carry and deliver small compounds. As a tool for designing ligand binding sites in protein cores, a three-step virtual screening method is presented that has been optimised using existing data on T4 lysozyme complexes and tested in a newly engineered cavity in flavodoxin. The method can pinpoint, in large databases, ligands of specific protein cavities. In the first step, physico-chemical filters are used to screen the library and discard a majority of compounds. In the second step, a flexible, fast docking procedure is used to score and select a smaller number of compounds as potential binders. In the third step, a finer method is used to dock promising molecules of the hit list into the protein cavity, and an optimised free energy function allows discarding the few false positives by calculating the affinity of the modelled complexes. To demonstrate the portability of the method, several cavities have been designed and engineered in the flavodoxin from Anabaena PCC 7119, and the W66F/L44A double mutant has been selected as a suitable host protein. The NCI database has then been screened for potential binders, and the binding to the engineered cavity of five promising compounds and three tentative non-binders has been experimentally tested by thermal up-shift assays and spectroscopic titrations. The five tentative binders (some apolar and some polar), unlike the three tentative non-binders, are shown to bind to the host mutant and, importantly, not to bind to the wild type protein. The three-step virtual screening method developed can thus be used to identify ligands of buried protein cavities. We anticipate that the method could also be used, in a reverse manner, to identify natural or engineerable protein cavities for the hosting of ligands of interest.

An intragenic suppressor in the cytochrome c oxidase I gene of mouse mitochondrial DNA.

We report here the identification of a cell line containing single and double missense mutations in cytochrome c oxidase (COX) subunit I gene of mouse mitochondrial DNA. When present in homoplasmy, the single mutant displays a normal complex IV assembly but a significantly reduced COX activity, while the double mutant almost completely compensates the functional defect of the first mutation. We discuss the potential structural consequences of those mutations based on the modeled structure of mouse complex IV. Based on genetic, biochemical and molecular analyses of cultured mouse cells we infer that: (i) deleterious mutations can arise and become predominant; (ii) cultured cells can maintain several mtDNA haplotypes at stable frequencies; (iii) the respiratory chain has little spare COX capacity; and (iv) the size of a cavity in the vicinity of Val421 in CO I of animal COX may affect the function of the enzyme.

Predicting the structure of protein cavities created by mutation.

To assist in the efficient design of protein cavities, we have developed a minimization strategy that can predict with accuracy the fate of cavities created by mutation. We first modelled, under different conditions, the structures of six T4 lysozyme and cytochrome c peroxidase mutants of known crystal structure (where long, hydrophobic, buried side chains have been replaced by shorter ones) by minimizing the virtual structures derived from the corresponding wild-type co-ordinates. An unconstrained pathway together with an all-atom atom representation and a steepest descent minimization yielded modelled structures with lower root mean square deviations (r.m.s.d) from the crystal structures than other conditions. To test whether the method developed was generally applicable to other mutations of the kind, we have then modelled eighteen additional T4 lysozyme, barnase and cytochrome c peroxidase mutants of known crystal structure. The models of both cavity expanding and cavity collapsing mutants closely fit their crystal structures (average r.m.s.d. 0.33 +/- 0.25 A, with only one poorer prediction: L121A). The structure of protein cavities generated by mutation can thus be confidently simulated by energy minimization regardless of the tendency of the cavity to collapse or to expand. We think this is favoured by the fact that the typical response observed in these proteins to cavity-creating mutations is to experience only a limited rearrangement.

Four-state equilibrium unfolding of an scFv antibody fragment.

The conformational stability of a single-chain Fv antibody fragment against a hepatitis B surface antigen (anti-HBsAg scFv) has been studied by urea and temperature denaturation followed by fluorescence and circular dichroism. At neutral pH and low protein concentration, it is a well-folded monomer, and its urea and thermal denaturations are reversible. The noncoincidence of the fluorescence and circular dichroism transitions indicates the accumulation in the urea denaturation of an intermediate (I(1)) not previously described in scFv molecules. In addition, at higher urea concentrations, a red-shift in the fluorescence emission maximum reveals an additional intermediate (I(2)), already reported in the denaturation of other scFvs. The urea equilibrium unfolding of the anti-HBsAg scFv is thus four-state. A similar four-state behavior is observed in the thermal unfolding although the intermediates involved are not identical to those found in the urea denaturation. Global analysis of the thermal unfolding data suggests that the first intermediate displays substantial secondary structure and some well-defined tertiary interactions while the second one lacks well-defined tertiary interactions but is compact and unfolds at higher temperature in a noncooperative fashion. Global analysis of the urea unfolding data (together with the modeled structure of the scFv) provides insights into the conformation of the chemical denaturation intermediates and allows calculation of the N-I(1), I(1)-I(2), and I(2)-D free energy differences. Interestingly, although the N-D free energy difference is very large, the N-I(1) one, representing the "relevant" conformational stability of the scFv, is small.

The 'relevant' stability of proteins with equilibrium intermediates.

Proteins perform many useful molecular tasks, and their biotechnological use continues to increase. As protein activity requires a stable native conformation, protein stabilisation is a major scientific and practical issue. Towards that end, many successful protein stabilisation strategies have been devised in recent years. In most cases, model proteins with a two-state folding equilibrium have been used to study and demonstrate protein stabilisation. Many proteins, however, display more complex folding equilibria where stable intermediates accumulate. Stabilising these proteins requires specifically stabilising the native state relative to the intermediates, as these are expected to lack activity. Here we discuss how to investigate the 'relevant' stability of proteins with equilibrium intermediates and propose a way to dissect the contribution of side chain interactions to the overall stability into the 'relevant' and 'nonrelevant' terms. Examples of this analysis performed on apoflavodoxin and in a single-chain mini antibody are presented.