RESUMEN
PADI4 is one of the human isoforms of a group of enzymes intervening in the conversion of arginine to citrulline. It is involved in the development of several types of tumors, as well as other immunological illnesses, such as psoriasis, multiple sclerosis, or rheumatoid arthritis. PADI4 auto-citrullinates in several regions of its sequence, namely in correspondence of residues Arg205, Arg212, Arg218, and Arg383. We wanted to study whether the citrullinated moiety affects the conformation of nearby regions and its binding to intact PADI4. We designed two series of synthetic peptides comprising either the wild-type or the relative citrullinated versions of such regions - i.e., a first series of peptides comprising the first three arginines, and a second series comprising Arg383. We studied their conformational properties in isolation by using fluorescence, far-ultraviolet (UV) circular dichroism (CD), and 2D1H NMR. Furthermore, we characterized the binding of the wild-type and citrullinated peptides in the two series to the intact PADI4, by using isothermal titration calorimetry (ITC), fluorescence, and biolayer interferometry (BLI), as well as by molecular docking simulations. We observed that citrullination did not alter the local conformational propensities of the isolated peptides. Nevertheless, for all the peptides in the two series, citrullination slowed down the kinetic koff rates of the binding reaction to PADI4, probably due to differences in electrostatic effects compared to the presence of arginine. The affinities of PADI4 for unmodified peptides were slightly larger than those of the corresponding citrullinated ones in the two series, but they were all within the same range, indicating that there were no relevant variations in the thermodynamics of binding due to sequence effects. These results highlight details of the self-citrullination of PADI4 and, more generally, of possible auto-catalytic mechanisms taking place in vivo for other citrullinating enzymes or, alternatively, in proteins undergoing citrullination passively.
Asunto(s)
Citrulinación , Arginina Deiminasa Proteína-Tipo 4 , Desiminasas de la Arginina Proteica , Arginina Deiminasa Proteína-Tipo 4/metabolismo , Arginina Deiminasa Proteína-Tipo 4/química , Humanos , Desiminasas de la Arginina Proteica/metabolismo , Desiminasas de la Arginina Proteica/química , Conformación Proteica , Péptidos/química , Péptidos/metabolismo , Citrulina/química , Citrulina/metabolismo , Unión Proteica , Secuencia de AminoácidosRESUMEN
Polycomb groups (PcGs) are transcriptional repressors, formed by a complex of several proteins, involved in multicellular development and cancer epigenetics. One of these proteins is the E3 ubiquitin-protein ligase RING1 (or RING1B), associated with the regulation of transcriptional repression and responsible for monoubiquitylation of the histone H2A. On the other hand, PADI4 is one of the human isoforms of a family of enzymes implicated in the conversion of arginine to citrulline, and it is also involved in the development of glioblastoma, among other types of cancers. In this work, we showed the association of PADI4 and RING1B in the nucleus and cytosol in several cancer cell lines by using immunofluorescence and proximity ligation assays. Furthermore, we demonstrated that binding was hampered in the presence of GSK484, an enzymatic PADI4 inhibitor, suggesting that RING1B could bind to the active site of PADI4, as confirmed by protein-protein docking simulations. In vitro and in silico findings showed that binding to PADI4 occurred for the isolated fragments corresponding to both the N-terminal (residues 1-221) and C-terminal (residues 228-336) regions of RING1B. Binding to PADI4 was also hampered by GSK484, as shown by isothermal titration calorimetry (ITC) experiments for the sole N-terminal region, and by both NMR and ITC for the C-terminal one. The dissociation constants between PADI4 and any of the two isolated RING1B fragments were in the low micromolar range (~2-10 µM), as measured by fluorescence and ITC. The interaction between RING1B and PADI4 might imply citrullination of the former, leading to several biological consequences, as well as being of potential therapeutic relevance for improving cancer treatment with the generation of new antigens.
Asunto(s)
Unión Proteica , Arginina Deiminasa Proteína-Tipo 4 , Desiminasas de la Arginina Proteica , Humanos , Línea Celular Tumoral , Núcleo Celular/metabolismo , Citrulinación , Simulación del Acoplamiento Molecular , Neoplasias/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Complejo Represivo Polycomb 1/química , Complejo Represivo Polycomb 1/genética , Arginina Deiminasa Proteína-Tipo 4/metabolismo , Desiminasas de la Arginina Proteica/metabolismo , Desiminasas de la Arginina Proteica/genéticaRESUMEN
The development of specific antiviral therapies targeting SARS-CoV-2 remains fundamental because of the continued high incidence of COVID-19 and limited accessibility to antivirals in some countries. In this context, dark chemical matter (DCM), a set of drug-like compounds with outstanding selectivity profiles that have never shown bioactivity despite being extensively assayed, appears to be an excellent starting point for drug development. Accordingly, in this study, we performed a high-throughput screening to identify inhibitors of the SARS-CoV-2 main protease (Mpro) using DCM compounds as ligands. Multiple receptors and two different docking scoring functions were employed to identify the best molecular docking poses. The selected structures were subjected to extensive conventional and Gaussian accelerated molecular dynamics. From the results, four compounds with the best molecular behavior and binding energy were selected for experimental testing, one of which presented inhibitory activity with a Ki value of 48 ± 5 µM. Through virtual screening, we identified a significant starting point for drug development, shedding new light on DCM compounds.
Asunto(s)
Antivirales , Proteasas 3C de Coronavirus , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Inhibidores de Proteasas , SARS-CoV-2 , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/enzimología , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/química , Proteasas 3C de Coronavirus/metabolismo , Antivirales/farmacología , Antivirales/química , Humanos , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , COVID-19/virología , Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Evaluación Preclínica de Medicamentos/métodos , Unión Proteica , LigandosRESUMEN
Plakophilin 1 (PKP1), a member of the p120ctn subfamily of the armadillo (ARM)-repeat-containing proteins, is an important structural component of cell-cell adhesion scaffolds although it can also be ubiquitously found in the cytoplasm and the nucleus. RYBP (RING 1A and YY1 binding protein) is a multifunctional intrinsically disordered protein (IDP) best described as a transcriptional regulator. Both proteins are involved in the development and metastasis of several types of tumors. We studied the binding of the armadillo domain of PKP1 (ARM-PKP1) with RYBP by using in cellulo methods, namely immunofluorescence (IF) and proximity ligation assay (PLA), and in vitro biophysical techniques, namely fluorescence, far-ultraviolet (far-UV) circular dichroism (CD), and isothermal titration calorimetry (ITC). We also characterized the binding of the two proteins by using in silico experiments. Our results showed that there was binding in tumor and non-tumoral cell lines. Binding in vitro between the two proteins was also monitored and found to occur with a dissociation constant in the low micromolar range (~10 µM). Finally, in silico experiments provided additional information on the possible structure of the binding complex, especially on the binding ARM-PKP1 hot-spot. Our findings suggest that RYBP might be a rescuer of the high expression of PKP1 in tumors, where it could decrease the epithelial-mesenchymal transition in some cancer cells.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Placofilinas , Unión Proteica , Humanos , Placofilinas/metabolismo , Placofilinas/genética , Placofilinas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Represoras/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas del Dominio Armadillo/metabolismo , Proteínas del Dominio Armadillo/química , Proteínas del Dominio Armadillo/genética , Dominios Proteicos , Dicroismo CircularRESUMEN
MeCP2 is a general regulator of transcription involved in the repression/activation of genes depending on the local epigenetic context. It acts as a chromatin regulator and binds with exquisite specificity to gene promoters. The set of epigenetic marks recognized by MeCP2 has been already established (mainly, cytosine modifications in CpG and CpA), as well as many of the constituents of its interactome. We unveil a new set of interactions for MeCP2 with the four canonical nucleosomal histones. MeCP2 interacts with high affinity with H2A, H2B, H3 and H4. In addition, Rett syndrome associated mutations in MeCP2 and histone epigenetic marks modulate these interactions. Given the abundance and the structural/functional relevance of histones and their involvement in epigenetic regulation, this new set of interactions and its modulating elements provide a new addition to the 'alphabet' for this epigenetic reader.
Asunto(s)
Epigénesis Genética , Histonas , Proteína 2 de Unión a Metil-CpG , Nucleosomas , Proteína 2 de Unión a Metil-CpG/metabolismo , Proteína 2 de Unión a Metil-CpG/genética , Nucleosomas/metabolismo , Histonas/metabolismo , Humanos , Unión Proteica , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Mutación , AnimalesRESUMEN
MDM2 is an E3 ubiquitin ligase which is crucial for the degradation and inhibition of the key tumor-suppressor protein p53. In this work, we explored the stability and the conformational features of the N-terminal region of MDM2 (N-MDM2), through which it binds to the p53 protein as well as other protein partners. The isolated domain possessed a native-like conformational stability in a narrow pH range (7.0 to 10.0), as shown by intrinsic and 8-anilinonapthalene-1-sulfonic acid (ANS) fluorescence, far-UV circular dichroism (CD), and size exclusion chromatography (SEC). Guanidinium chloride (GdmCl) denaturation followed by intrinsic and ANS fluorescence, far-UV CD and SEC at physiological pH, and differential scanning calorimetry (DSC) and thermo-fluorescence experiments showed that (i) the conformational stability of isolated N-MDM2 was very low; and (ii) unfolding occurred through the presence of several intermediates. The presence of a hierarchy in the unfolding intermediates was also evidenced through DSC and by simulating the unfolding process with the help of computational techniques based on constraint network analysis (CNA). We propose that the low stability of this protein is related to its inherent flexibility and its ability to interact with several molecular partners through different routes.
Asunto(s)
Pliegue de Proteína , Proteína p53 Supresora de Tumor , Desnaturalización Proteica , Conformación Proteica , Dicroismo Circular , Concentración de Iones de Hidrógeno , Espectrometría de Fluorescencia , Rastreo Diferencial de CalorimetríaRESUMEN
PADI4 is one of the human isoforms of a family of enzymes implicated in the conversion of arginine to citrulline. MDM2 is an E3 ubiquitin ligase which is crucial for down-regulation of degradation of the tumor suppressor gene p53. Given the relationship between both PADI4 and MDM2 with p53-signaling pathways, we hypothesized they may interact directly, and this interaction could be relevant in the context of cancer. Here, we showed their association in the nucleus and cytosol in several cancer cell lines. Furthermore, binding was hampered in the presence of GSK484, an enzymatic PADI4 inhibitor, suggesting that MDM2 could bind to the active site of PADI4, as confirmed by in silico experiments. In vitro and in silico studies showed that the isolated N-terminal region of MDM2, N-MDM2, interacted with PADI4, and residues Thr26, Val28, Phe91 and Lys98 were more affected by the presence of the enzyme. Moreover, the dissociation constant between N-MDM2 and PADI4 was comparable to the IC50 of GSK484 from in cellulo experiments. The interaction between MDM2 and PADI4 might imply MDM2 citrullination, with potential therapeutic relevance for improving cancer treatment, due to the generation of new antigens.
Asunto(s)
Neoplasias , Proteína p53 Supresora de Tumor , Humanos , Proteína p53 Supresora de Tumor/química , Ubiquitina-Proteína Ligasas/química , Desiminasas de la Arginina Proteica/metabolismo , Línea Celular , Unión Proteica , Proteínas Proto-Oncogénicas c-mdm2/metabolismoRESUMEN
RYBP (Ring1 and YY 1 binding protein) is a multifunctional, intrinsically disordered protein (IDP), best described as a transcriptional regulator. It exhibits a ubiquitin-binding functionality, binds to other transcription factors, and has a key role during embryonic development. RYBP, which folds upon binding to DNA, has a Zn-finger domain at its N-terminal region. By contrast, PADI4 is a well-folded protein and it is one the human isoforms of a family of enzymes implicated in the conversion of arginine to citrulline. As both proteins intervene in signaling pathways related to cancer development and are found in the same localizations within the cell, we hypothesized they may interact. We observed their association in the nucleus and cytosol in several cancer cell lines, by using immunofluorescence (IF) and proximity ligation assays (PLAs). Binding also occurred in vitro, as measured by isothermal titration calorimetry (ITC) and fluorescence, with a low micromolar affinity (~1 µM). AlphaFold2-multimer (AF2) results indicate that PADI4's catalytic domain interacts with the Arg53 of RYBP docking into its active site. As RYBP sensitizes cells to PARP (Poly (ADP-ribose) polymerase) inhibitors, we applied them in combination with an enzymatic inhibitor of PADI4 observing a change in cell proliferation, and the hampering of the interaction of both proteins. This study unveils for the first time the possible citrullination of an IDP, and suggests that this new interaction, whether it involves or not citrullination of RYBP, might have implications in cancer development and progression.
Asunto(s)
Neoplasias , Factores de Transcripción , Humanos , Factores de Transcripción/genética , Línea Celular , Neoplasias/genética , Epigénesis Genética , Proteínas Represoras/genéticaRESUMEN
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.
RESUMEN
(1) Background: About 50% of prescribed colonoscopies report no pathological findings. A secondary screening test after fecal immunochemical test positivity (FIT+) would be required. Considering thermal liquid biopsy (TLB) as a potential secondary test, the aim of this work was to study possible interferences of colonoscopy bowel preparation on TLB outcome on a retrospective study; (2) Methods: Three groups were studied: 1/514 FIT(+) patients enrolled in a colorectal screening program (CN and CP with normal and pathological colonoscopy, respectively), with blood samples obtained just before colonoscopy and after bowel preparation; 2/55 patients from the CN group with blood sample redrawn after only standard 8-10 h fasting and no bowel preparation (CNR); and 3/55 blood donors from the biobank considered as a healthy control group; (3) Results: The results showed that from the 514 patients undergoing colonoscopy, 247 had CN and 267 had CP. TLB parameters in these two groups were similar but different from those of the blood donors. The resampled patients (with normal colonoscopy and no bowel preparation) had similar TLB parameters to those of the blood donors. TLB parameters together with fluorescence spectra and other serum indicators (albumin and C-reactive protein) confirmed the statistically significant differences between normal colonoscopy patients with and without bowel preparation; (4) Conclusions: Bowel preparation seemed to alter serum protein levels and altered TLB parameters (different from a healthy subject). The diagnostic capability of other liquid-biopsy-based methods might also be compromised. Blood extraction after bowel preparation for colonoscopy should be avoided.
RESUMEN
Hydroxymethylated cytosine (5hmC) is a stable DNA epigenetic mark recognized by methyl-CpG binding protein 2 (MeCP2), which acts as a transcriptional regulator and a global chromatin-remodeling element. Because 5hmC triggers a gene regulation response markedly different from that produced by methylated cytosine (5mC), both modifications must affect DNA structure and/or DNA interaction with MeCP2 differently. MeCP2 is a six-domain intrinsically disordered protein (IDP) with two domains responsible for dsDNA binding: methyl-CpG binding domain (MBD) and intervening domain (ID). Here we report the detailed thermodynamic characterization of the interaction of hmCpG-DNA with MeCP2. We find that hmCpG-DNA interacts with MeCP2 in a distinctly different mode with a particular thermodynamic signature, compared to methylated or unmethylated DNA. In addition, we find evidence for Rett syndrome-associated mutations altering the interaction of MeCP2 with dsDNA in a cytosine modification-specific manner which may correlate with disease onset time and clinical severity score.
Asunto(s)
Cromatina , ADN , Citosina , Epigenómica , TermodinámicaRESUMEN
Plakophilin 1 (PKP1), a member of the armadillo repeat family of proteins, is a key structural component of cell-cell adhesion scaffolds, although it can also be found in other cell locations, including the cytoplasm and the nucleus. PADI4 (peptidyl-arginine deiminase 4) is one of the human isoforms of a family of enzymes engaged in the conversion of arginine to citrulline, and is present in monocytes, macrophages, granulocytes, and in several types of cancer cells. It is the only family member observed both within the nucleus and the cytoplasm under ordinary conditions. We studied the binding of the armadillo domain of PKP1 (ARM-PKP1) with PADI4, by using several biophysical methods, namely fluorescence, far-ultraviolet (far-UV) circular dichroism (CD), isothermal titration calorimetry (ITC), and molecular simulations; furthermore, binding was also tested by Western-blot (WB) analyses. Our results show that there was binding between the two proteins, with a dissociation constant in the low micromolar range (â¼ 1 µM). Molecular modelling provided additional information on the possible structure of the binding complex, and especially on the binding hot-spot predicted for PADI4. This is the first time that the interaction between these two proteins has been described and studied. Our findings could be of importance to understand the development of tumors, where PKP1 and PADI4 are involved. Moreover, our findings pave the way to describe the formation of neutrophil extracellular traps (NETs), whose construction is modulated by PADI4, and which mediate the proteolysis of cell-cell junctions where PKP1 intervenes.
Asunto(s)
Placofilinas , Arginina Deiminasa Proteína-Tipo 4 , Humanos , Western Blotting , Hidrolasas , Neoplasias , Arginina Deiminasa Proteína-Tipo 4/metabolismoRESUMEN
Bacteroides fragilis is an abundant commensal component of the healthy human colon. However, under dysbiotic conditions, enterotoxigenic B. fragilis (ETBF) may arise and elicit diarrhea, anaerobic bacteremia, inflammatory bowel disease, and colorectal cancer. Most worrisome, ETBF is resistant to many disparate antibiotics. ETBF's only recognized specific virulence factor is a zinc-dependent metallopeptidase (MP) called B. fragilis toxin (BFT) or fragilysin, which damages the intestinal mucosa and triggers disease-related signaling mechanisms. Thus, therapeutic targeting of BFT is expected to limit ETBF pathogenicity and improve the prognosis for patients. We focused on one of the naturally occurring BFT isoforms, BFT-3, and managed to repurpose several approved drugs as BFT-3 inhibitors through a combination of biophysical, biochemical, structural, and cellular techniques. In contrast to canonical MP inhibitors, which target the active site of mature enzymes, these effectors bind to a distal allosteric site in the proBFT-3 zymogen structure, which stabilizes a partially unstructured, zinc-free enzyme conformation by shifting a zinc-dependent disorder-to-order equilibrium. This yields proBTF-3 incompetent for autoactivation, thus ablating hydrolytic activity of the mature toxin. Additionally, a similar destabilizing effect is observed for the activated protease according to biophysical and biochemical data. Our strategy paves a novel way for the development of highly specific inhibitors of ETBF-mediated enteropathogenic conditions.
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Infecciones Bacterianas , Toxinas Bacterianas , Antibacterianos/metabolismo , Toxinas Bacterianas/metabolismo , Bacteroides fragilis/metabolismo , Precursores Enzimáticos/metabolismo , Humanos , Metaloendopeptidasas/metabolismo , Factores de Virulencia/metabolismoRESUMEN
PADI4 is a peptidyl-arginine deiminase (PADI) involved in the conversion of arginine to citrulline. PADI4 is present in macrophages, monocytes, granulocytes, and several cancer cells. It is the only PADI family member observed within both the nucleus and the cytoplasm. PADI4 has a predicted nuclear localization sequence (NLS) comprising residues Pro56 to Ser83, to allow for nuclear translocation. Recent predictors also suggest that the region Arg495 to Ile526 is a possible NLS. To understand how PADI4 is involved in cancer, we studied the ability of intact PADI4 to bind importin α3 (Impα3), a nuclear transport factor that plays tumor-promoting roles in several cancers, and its truncated species (ΔImpα3) without the importin-binding domain (IBB), by using fluorescence, circular dichroism (CD), and isothermal titration calorimetry (ITC). Furthermore, the binding of two peptides, encompassing the first and the second NLS regions, was also studied using the same methods and molecular docking simulations. PADI4 interacted with both importin species, with affinity constants of ~1-5 µM. The isolated peptides also interacted with both importins. The molecular simulations predict that the anchoring of both peptides takes place in the major binding site of Impα3 for the NLS of cargo proteins. These findings suggest that both NLS regions were essentially responsible for the binding of PADI4 to the two importin species. Our data are discussed within the framework of a cell mechanism of nuclear transport that is crucial in cancer.
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Carioferinas , Señales de Localización Nuclear , Arginina Deiminasa Proteína-Tipo 4 , Núcleo Celular/metabolismo , Humanos , Carioferinas/metabolismo , Simulación del Acoplamiento Molecular , Señales de Localización Nuclear/metabolismo , Unión Proteica , Arginina Deiminasa Proteína-Tipo 4/metabolismoRESUMEN
The work assesses the performance of nanocarriers from amphiphilic block copolymers with functional azobenzene or coumarin moieties for delivery of paclitaxel. Placlitaxel was encapsulated by the nanoprecipitation method. Characterisations were performed by DLS, TEM, Zeta potential and HPLC. Cell viability was investigated in HeLa and Huh-5-2-cell lines. Coumarin-containing polymeric micelles (Dh = 26 ± 2 nm, PDI = 0.28, ζ = â22.9 ± 3.6 mV) with 11.2 ± 0.5%w/w drug loading showed enhanced cytotoxicity in HeLa cells (IC50 < 0.02 nM) compared to free paclitaxel (IC50 = 0.17 ± 0.02 nM). Azobenzene-containing polymeric vesicles (Dh = 390 ± 20 nm, PDI = 0.24, ζ = â33.2 ± 5.0 mV) with a 6.8 ± 0.4%w/w drug loading showed increased cytotoxicity under 530 nm light (IC50 = 0.0114 ± 0.00033 nM) in HeLa cells due to a stimulated delivery of paclitaxel. Effectivity of these block copolymers as paclitaxel nanovectors and light stimulated release has been demonstrated.
Asunto(s)
Micelas , Paclitaxel , Compuestos Azo , Cumarinas , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Células HeLa , Humanos , Paclitaxel/química , Paclitaxel/farmacología , Cemento de Policarboxilato , Polietilenglicoles/química , Polímeros/químicaRESUMEN
PADI4 (protein-arginine deiminase, also known as protein l-arginine iminohydrolase) is one of the human isoforms of a family of Ca2+-dependent proteins catalyzing the conversion of arginine to citrulline. Although the consequences of this process, known as citrullination, are not fully understood, all PADIs have been suggested to play essential roles in development and cell differentiation. They have been found in a wide range of cells and tissues and, among them, PADI4 is present in macrophages, monocytes, granulocytes and cancer cells. In this work, we focused on the biophysical features of PADI4 and, more importantly, how its expression was altered in cancer cells. Firstly, we described the different expression patterns of PADI4 in various cancer cell lines and its colocalization with the tumor-related protein p53. Secondly, we carried out a biophysical characterization of PADI4, by using a combination of biophysical techniques and in silico molecular dynamics simulations. Our biochemical results suggest the presence of several forms of PADI4 with different subcellular localizations, depending on the cancer cell line. Furthermore, PADI4 could have a major role in tumorigenesis by regulating p53 expression in certain cancer cell lines. On the other hand, the native structure of PADI4 was strongly pH-dependent both in the absence or presence of Ca2+, and showed two pH-titrations at basic and acidic pH values. Thus, there was a narrow pH range (from 6.5 to 8.0) where the protein was dimeric and had a native structure, supporting its role in histones citrullination. Thermal denaturations were always two-state, but guanidinium-induced ones showed that PADI4 unfolded through at least one intermediate. Our simulation results suggest that the thermal melting of PADI4 structure was rather homogenous throughout its sequence. The overall results are discussed in terms of the functional role of PADI4 in the development of cancer.
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Biomarcadores de Tumor/metabolismo , Desiminasas de la Arginina Proteica/metabolismo , Arginina/metabolismo , Carcinogénesis/metabolismo , Catálisis , Diferenciación Celular , Línea Celular Tumoral , Citrulina/metabolismo , Regulación de la Expresión Génica , Humanos , Simulación de Dinámica Molecular , Unión Proteica , Procesamiento Proteico-Postraduccional , Arginina Deiminasa Proteína-Tipo 4/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
SARS-CoV-2 is a type of coronavirus responsible for the international outbreak of respiratory illness termed COVID-19 that forced the World Health Organization to declare a pandemic infectious disease situation of international concern at the beginning of 2020. The need for a swift response against COVID-19 prompted to consider different sources to identify bioactive compounds that can be used as therapeutic agents, including available drugs and natural products. Accordingly, this work reports the results of a virtual screening process aimed at identifying antiviral natural product inhibitors of the SARS-CoV-2 Mpro viral protease. For this purpose, ca. 2000 compounds of the Selleck database of Natural Compounds were the subject of an ensemble docking process targeting the Mpro protease. Molecules that showed binding to most of the protein conformations were retained for a further step that involved the computation of the binding free energy of the ligand-Mpro complex along a molecular dynamics trajectory. The compounds that showed a smooth binding free energy behavior were selected for in vitro testing. From the resulting set of compounds, five compounds exhibited an antiviral profile, and they are disclosed in the present work.
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Productos Biológicos , COVID-19 , Antivirales/farmacología , Productos Biológicos/farmacología , Humanos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Péptido Hidrolasas , Inhibidores de Proteasas/farmacología , SARS-CoV-2RESUMEN
Intrinsically disordered proteins (IDPs) are emerging as attractive drug targets by virtue of their physiological ubiquity and their prevalence in various diseases, including cancer. NUPR1 is an IDP that localizes throughout the whole cell, and is involved in the development and progression of several tumors. We have previously repurposed trifluoperazine (TFP) as a drug targeting NUPR1 and, by using a ligand-based approach, designed the drug ZZW-115 starting from the TFP scaffold. Such derivative compound hinders the development of pancreatic ductal adenocarcinoma (PDAC) in mice, by hampering nuclear translocation of NUPR1. Aiming to further improve the activity of ZZW-115, here we have used an indirect drug design approach to modify its chemical features, by changing the substituent attached to the piperazine ring. As a result, we have synthesized a series of compounds based on the same chemical scaffold. Isothermal titration calorimetry (ITC) showed that, with the exception of the compound preserving the same chemical moiety at the end of the alkyl chain as ZZW-115, an increase of the length by a single methylene group (i.e., ethyl to propyl) significantly decreased the affinity towards NUPR1 measured in vitro, whereas maintaining the same length of the alkyl chain and adding heterocycles favored the binding affinity. However, small improvements of the compound affinity towards NUPR1, as measured by ITC, did not result in a corresponding improvement in their inhibitory properties and in cellulo functions, as proved by measuring three different biological effects: hindrance of the nuclear translocation of the protein, sensitization of cells against DNA damage mediated by NUPR1, and prevention of cancer cell growth. Our findings suggest that a delicate compromise between favoring ligand affinity and controlling protein function may be required to successfully design drugs against NUPR1, and likely other IDPs.