RESUMEN
Tyrosine aminotransferase is a well-characterized enzyme in the Leishmania parasite, but the role of TAT in the parasite functioning remains largely unknown. In this study, we attempt to gain a better understanding of the enzyme's role in the parasite by gene knockout and overexpression of the TAT gene. The overexpression of TAT protein was well tolerated by the parasites in two independent repeats. Single knockout of TAT gene by homologous recombination, LdTAT+/- displayed distinct retardation in the proliferation rates and entered the death phase immediately. Morphology of LdTAT+/- parasites had important structural defects as they rounded up with elongated flagella. Gene regulation studies suggested the upregulation of key apoptotic and redox metabolism genes in LdTAT+/-. Moreover, LdTAT+/- cells accumulated higher ROS, thiols, intracellular Ca2+ concentrations, and mitochondrial membrane depolarization signifying the onset of apoptosis. Tocopherol levels were reduced by 50% in LdTAT+/- suggesting the involvement of TAT in tocopherol biosynthesis in the parasite. Overall, our results provide the first evidence that gene knockout of TAT results in apoptosis and that TAT is required for the survival and viability of Leishmania donovani.
Asunto(s)
Leishmania donovani , Parásitos , Animales , Productos del Gen tat/genética , Productos del Gen tat/metabolismo , Homeostasis , Recombinación Homóloga , Oxidación-Reducción , Parásitos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Tocoferoles/metabolismo , Tirosina Transaminasa/química , Tirosina Transaminasa/genética , Tirosina Transaminasa/metabolismoRESUMEN
Most neurodegenerative diseases such as Alzheimer's disease, type 2 diabetes, Parkinson's disease, etc. are caused by inclusions and plaques containing misfolded protein aggregates. These protein aggregates are essentially formed by the interactions of either the same (homologous) or different (heterologous) sequences. Several experimental pieces of evidence have revealed the presence of cross-seeding in amyloid proteins, which results in a multicomponent assembly; however, the molecular and structural details remain less explored. Here, we discuss the amyloid proteins and the cross-seeding phenomena in detail. Data suggest that targeting the common epitope of the interacting amyloid proteins may be a better therapeutic option than targeting only one species. We also examine the dual inhibitors that target the amyloid proteins participating in the cross-seeding events. The future scopes and major challenges in understanding the mechanism and developing therapeutics are also considered. Detailed knowledge of the amyloid cross-seeding will stimulate further research in the practical aspects and better designing anti-amyloid therapeutics.
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Amiloidosis , Diabetes Mellitus Tipo 2 , Amiloide/química , Péptidos beta-Amiloides/metabolismo , Proteínas Amiloidogénicas , Amiloidosis/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , HumanosRESUMEN
The H1N1 influenza virus causes a severe disease that affects the human respiratory tract leading to millions of deaths every year. At present, certain vaccines and few drugs are used to control the virus during seasonal outbreaks. However, high mutation rates and genetic reassortment make it challenging to prevent and mitigate outbreaks, leading to pandemics. Thus, alternate therapies are required for its management and control. Here, we report that a bacterial protein, azurin, and its peptide derivatives p18 and p28 target critical proteins of the influenza virus in an effective manner. The molecular docking studies show that the p28 peptide could target C-PB1, NS1-ED, PB2-CBD, PB2-RBD, NP, and PA proteins. These complexes were further subjected to the simulation of molecular dynamics and binding free energy calculations. The data indicate that p28 has an unusually high affinity and forms stable complexes with the viral proteins C-PB1, PB2-CBD, PB2-RBD, and NP. We suggest that the azurin derivative p28 peptide can act as an anti-influenza agent as it can bind to multiple targets and neutralize the virus. Additional experimental studies need to be conducted to evaluate its safety and efficacy as an anti-H1N1 molecule.
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Antivirales/química , Azurina/química , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Fragmentos de Péptidos/química , Proteínas Virales/química , Antivirales/farmacología , Azurina/farmacología , Sitios de Unión , Dominio Catalítico , Descubrimiento de Drogas , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Conformación Molecular , Fragmentos de Péptidos/farmacología , Unión Proteica , Relación Estructura-Actividad , Proteínas Virales/antagonistas & inhibidoresRESUMEN
Leishmaniasis is a zoonotic disease in humans caused by the bite of a parasite-infected sandfly. The disease, widely referred to as "poor man's disease," affects millions of people worldwide. The clinical manifestation of the disease depends upon the species of the parasite and ranges from physical disfigurement to death if left untreated. Here, we review the past, present, and future of leishmaniasis in detail. The life cycle of Leishmania sp., along with its epidemiology, is discussed, and in addition, the line of therapeutics available for treatment currently is examined. The current status of the disease is critically evaluated, keeping emerging threats like human immunodeficiency virus (HIV) coinfection and post kala-azar dermal leishmaniasis (PKDL) into consideration. In summary, the review proposes a dire need for new therapeutics and reassessment of the measures and policies concerning emerging threats. New strategies are essential to achieve the goal of leishmaniasis eradication in the next few decades.
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Leishmania donovani/patogenicidad , Leishmaniasis Cutánea/epidemiología , Leishmaniasis Cutánea/patología , Leishmaniasis Visceral/epidemiología , Leishmaniasis Visceral/patología , Animales , Coinfección/patología , Femenino , Infecciones por VIH/patología , Humanos , Masculino , Psychodidae/parasitología , Zoonosis/patologíaRESUMEN
Leishmaniasis is a neglected tropical disease with no effective vaccines to date. Globally, it affects around 14 million people living in undeveloped and developing countries. Leishmania, which is the causative eukaryotic organism, possesses unique enzymes and pathways that deviates from its mammalian hosts. The control strategy against leishmaniasis currently depends on chemotherapeutic methods. But these chemotherapeutic therapies possess several side effects, and therefore, the identification of potential drug targets has become very crucial. Identification of suitable drug targets is necessary to design specific inhibitors that can target and control the parasite. These unique enzymes can be used as possible drug targets after biochemical characterization and understanding the role of these enzymes. In this review, the authors discuss various metabolic pathways that are essential for the survival of the parasite and can be exploited as potential drug targets against leishmaniasis.
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Antiparasitarios , Leishmania/metabolismo , Leishmaniasis/tratamiento farmacológico , Redes y Vías Metabólicas/efectos de los fármacos , Terapia Molecular Dirigida , Animales , Antiparasitarios/farmacología , Antiparasitarios/uso terapéutico , Sistemas de Liberación de Medicamentos , Humanos , Leishmania/efectos de los fármacosRESUMEN
Leishmaniasis is caused by Leishmania genus parasites and has a high mortality rate. The available drugs to treat leishmaniasis fail due to acquired resistance in parasites. Several enzymes of the Leishmania parasite have been used to design new therapeutic molecules against leishmaniasis. This study uses a pharmacophore-guided approach to design the drug candidate by targeting Leishmania N-Myristoyl transferase (LdNMT). From the initial sequence analysis of LdNMT, we have identified a unique 20 amino acid stretch exploited for screening and designing the small molecules. The pharmacophore for the myristate binding site on LdNMT was elucidated, and a heatmap was constructed. The leishmanial NMT pharmacophore has similarities with other pathogenic microorganisms. Moreover, substituting alanine in pharmacophoric residues elevates the affinity of myristate with NMT. Furthermore, a molecular dynamics (MD) simulation study was conducted to ascertain the stability of the mutants and or wild type. The wild-type NMT has a comparatively low affinity to myristate compared to alanine mutants, indicating that hydrophobic residues favor the myristate binding. The molecules were initially designed by using pharmacophore as a sieving mechanism. In subsequent steps, the selected molecules screened against leishmanial unique amino acid stretch and subsequently with human, leishmanial full-size NMTs. The compounds BP5, TYI, DMU, 3PE and 4UL were the top hits and chemical features similar to the myristate. The molecule 4UL was found to be highly specific towards leishmanial NMT over human NMT, suggesting the molecule is a strong leishmanial NMT inhibitor. The molecule can be taken further to assess it in in-vitro conditions.
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Leishmania , Leishmaniasis , Humanos , Transferasas , Farmacóforo , Miristatos , Leishmaniasis/tratamiento farmacológico , Diseño de Fármacos , Alanina , AminoácidosRESUMEN
Pancreatic cancer is a highly aggressive and often lethal malignancy with limited treatment options. Its late-stage diagnosis and resistance to conventional therapies make it a significant challenge in oncology. Immunotherapy, particularly cancer vaccines, has emerged as a promising avenue for treating pancreatic cancer. Multi-epitope vaccines, designed to target multiple epitopes derived from various antigens associated with pancreatic cancer, have gained attention as potential candidates for improving therapeutic outcomes. In this study, we have explored transcriptomics and protein expression databases to identify potential upregulated proteins in pancreatic cancer cells. After examining a total of 21,054 proteins from various databases, it was discovered that 143 proteins expressed differently in malignant and healthy cells. The CTL, HTL and BCE epitopes were predicted for the shortlisted proteins. 51,840 vaccine constructs were created by concatenating CTL, HTL, and B-cell epitopes in the respective sequences. The best 86 structures were selected from a set of 51,840 designs after they were analyzed for vaxijenicity, allergenicity, toxicity, and antigenicity scores. In further simulation of the immune response using constructs, it was found that 41417, 37961, and 40841 constructs could produce a strong immune response when injected. Further, it was found that construct 37961 showed stronger interaction and stability with TLR-9 as determined from the large-scale molecular dynamics simulations. Moreover, the 37961 construct has shown interactions with TLR-9 suggests its potential in inducing immune response. In addition, construct 37961 has shown 100% predicted solubility in the E. coli expression system. Overall, the study indicates the designed construct 37961 has the potential to induce an anti-tumor immune response and long-standing protection pending further studies.
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Vacunas contra el Cáncer , Neoplasias Pancreáticas , Humanos , Epítopos/genética , Proteoma , Escherichia coli , Receptor Toll-Like 9 , Neoplasias Pancreáticas/genéticaRESUMEN
Leishmaniasis is a major infectious disease having high mortality which could be attributed to lack of a suitable vaccine candidate. We propose a novel approach to design multiepitope vaccine to leishmaniasis exploiting specific membrane proteome from infected macrophage from host. The MHC-I, MHC-II and BC epitopes predicted for unique proteins from the infected macrophages and Leishmania and a MEV designed in various combinations (1a-1m). The epitope arrangements 1a, 1k, 1l, and 1 m showed a strong antigenicity profile and immune response. The molecular dynamics simulation indicate the 1k, 1l, and 1 m constructs have strong affinity toward TLR-2, TLR-3, and TLR-4. Overall the structural and immunogenicity profile suggests 1k is top candidate. Further, a computational model system with TLR-2, TLR-3, TLR-4, BCR, MHC-I and MHC-II was generated for 1k construct to understand the MEV interactions with immune components. Dihedral distribution and distance was enumerated to understand the movement of immune components towards 1k. The results indicate 1k has strong affinity for the immune response molecules especially TLR-3, BCR and MHC-II are coming in close contact with the MEV through the simulation. The study suggests that designed multi-epitope vaccine 1k has potential to induce proper immune response but warrants further studies.
RESUMEN
World health organization has called for academic research and development of new chemotherapeutic strategies to overcome the emerging resistance and side effects exhibited by the drugs currently used against leishmaniasis. Diospyrin, a bis-naphthoquinone isolated from Diospyros montana Roxb., and its semi-synthetic derivatives, were reported for inhibitory activity against protozoan parasites including Leishmania. Presently, we have investigated the antileishmanial effect of a di-epoxide derivative of diospyrin (D17), both in vitro and in vivo. Further, the safety profile of D17 was established by testing its toxicity against normal macrophage cells (IC50â¼20.7 µM), and also against normal BALB/c mice in vivo. The compound showed enhanced activity (IC50â¼7.2 µM) as compared to diospyrin (IC50â¼12.6 µM) against Leishmania donovani promastigotes. Again, D17 was tested on L. donovani BHU1216 isolated from a sodium stibogluconate-unresponsive patient, and exhibited selective inhibition of the intracellular amastigotes (IC50â¼0.18 µM). Also, treatment of infected BALB/c mice with D17 at 2mg/kg/day reduced the hepatic parasite load by about 38%. Subsequently, computational docking studies were undertaken on selected enzymes of trypanothione metabolism, viz. trypanothione reductase (TryR) and ornithine decarboxylase (ODC), followed by the enzyme kinetics, where D17 demonstrated non-competitive inhibition of the L. donovani ODC, but could not inhibit TryR.
Asunto(s)
Antiprotozoarios/farmacología , Leishmania donovani/efectos de los fármacos , Naftoquinonas/farmacología , Ornitina Descarboxilasa/efectos de los fármacos , Animales , Antiprotozoarios/toxicidad , Línea Celular , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Concentración 50 Inhibidora , Riñón/efectos de los fármacos , Riñón/enzimología , Leishmania donovani/enzimología , Leishmania donovani/genética , Hígado/efectos de los fármacos , Hígado/enzimología , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Naftoquinonas/química , Naftoquinonas/toxicidad , Ornitina Descarboxilasa/genética , Ornitina Descarboxilasa/metabolismo , Inhibidores de la Ornitina Descarboxilasa , Distribución AleatoriaRESUMEN
The treatment for leishmaniasis is currently plagued by side effects such as toxicity and the emergence of drug resistance to the available repertoire of drugs, as well as the expense of these drugs. Considering such rising concerns, we report the anti-leishmanial activity and mechanism of a flavone compound 4',7-dihydroxyflavone (TI 4). Four flavanoids were initially screened for anti-leishmanial activity and cytotoxicity. The results showed that the compound TI 4 exhibited higher activity and selectivity index at the same time as maintaining low cytotoxicity. Preliminary microscopic studies and fluorescence-activated cell sorting analysis reported that the parasite underwent apoptosis on TI 4 treatment. Further in-depth studies revealed high reactive oxygen species (ROS) production and thiol levels in the parasites, suggesting ROS-mediated apoptosis in the parasites upon TI 4 treatment. Other apoptotic indicators such as intracellular Ca2+ and mitochondrial membrane potential also indicated the onset of apoptosis in the treated parasites. The mRNA expression levels signified that the redox metabolism genes were upregulated by two-fold along with the apoptotic genes. In summary, the use of TI 4 on Leishmania parasites induces ROS-mediated apoptosis; therefore, the compound has immense potential to be an anti-leishmanial drug. However, in vivo studies would be required to ascertain its safety and efficacy before we can exploit the compound against the growing leishmaniasis crisis.
Asunto(s)
Antiprotozoarios , Leishmania , Leishmaniasis , Parásitos , Animales , Leishmania/metabolismo , Parásitos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Leishmaniasis/tratamiento farmacológico , Apoptosis , Antiprotozoarios/farmacologíaRESUMEN
One of the global problems of rising concern is the spread of the neglected tropical disease, leishmaniasis. There are several drugs used for the treatment of the disease but the repertoire of drugs has drawbacks like toxicity and low therapeutic value. Considering the need for new drugs, we studied the synthesis of 4',7-dihydroxyflavone conjugated multi-walled carbon nanotubes (47DHF-MWCNTs) and evaluated their anti-leishmanial activity against Leishmania donovani. The compound 47DHF was conjugated to the acid oxidized MWCTNs by Steglich esterification. The synthesized 47DHF-MWCNTs were characterized by UV spectroscopy, and, from the zeta value of 35 mV, they were found to be stable. 47DHF-MWCNTs possessed 84% drug loading efficiency and 63% cumulative drug release at intra-macrophage pH of 5.8. Moreover, the evaluation of 47DHF-MWCNTs for activity showed an IC50 value of 0.051 ± 0.01 µg/ml and 0.072 ± 0.01 µg/ml against the promastigote and amastigote form, respectively. 47DHF-MWCNTs exhibited an infectivity index of 42 and selectivity index of 95, suggesting the activity of 47DHF-MWCNTs against intracellular amastigotes in the study. The 47DHF-MWCNTs also had low cytotoxicity towards macrophage cells. Fascinatingly, the 47DHF-MWCNTs treatment causes a high accumulation of ROS in the promastigotes suggesting the mechanism of anti-leishmanial activity to be ROS mediated. Summarizing from our results, we propose for the first time a novel 47DHF conjugated MWCNTs capable of anti-leishmanial activity with lower cytotoxicity that has a huge potential to be a formulation against leishmaniasis.
Asunto(s)
Antiprotozoarios , Leishmania donovani , Leishmaniasis , Nanotubos de Carbono , Parásitos , Animales , Especies Reactivas de Oxígeno , Antiprotozoarios/farmacología , Antiprotozoarios/química , Leishmaniasis/tratamiento farmacológicoRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a pandemic that has devastated the lives of millions. Researchers around the world are relentlessly working in hopes of finding a cure. Even though the virus shares similarities with reported SARS-CoV and MERS-CoV at the genomic and proteomic level, efforts to repurpose already known drugs against SARS-CoV-2 have resulted ineffective. In this succinct review, we discuss the different potential targets in SARS-CoV-2 at both the genomic and proteomic levels. In addition, we analyze the compounds inhibiting individual target protein as well as multiple targets of SARS-CoV-2. ACE-2 receptor in humans has also been considered a target, keeping the role of the receptor in mind. The mechanism of action of these compounds has also been highlighted along with their clinical manifestation. Towards the end of the review, a brief note on the drugs currently in clinical trials and the current status of the vaccines are also examined. In conclusion, compounds targeting multiple targets of the virus hold the key in putting an end to the coronavirus malady.Communicated by Ramaswamy H. Sarma.
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COVID-19 , Coronavirus del Síndrome Respiratorio de Oriente Medio , Humanos , SARS-CoV-2 , Proteómica , Antivirales/uso terapéutico , Coronavirus del Síndrome Respiratorio de Oriente Medio/metabolismoRESUMEN
The CRISPR-Cas9 system is becoming an imperative tool to edit the genome of various organisms. The gene-editing study by the CRISPR-Cas9 system has revolutionized the diverse field of biomedical research, genome engineering, and gene therapy. CRISPR-Cas9 system has been modified to induce genome editing by small-guide RNAs, which function together with Cas9 nuclease for sequence-specific cleavage of target sequences. Here, we describe the simplified protocol of CRISPR-Cas9-mediated DNA editing in multicellular eukaryotes, including the construction of gRNA plasmids into vectors, screening of positive clones, transfections into 293FT cell line, and transduction into Jurkat cells. We also describe different bioinformatic tools to design suitable gRNAs with increased efficiency and decreased off-target events. Further, we describe the assessments of DNA editing by indel mutations and sequencing in transduced cells.
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Sistemas CRISPR-Cas , Eucariontes , Humanos , Sistemas CRISPR-Cas/genética , Eucariontes/genética , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , Edición Génica/métodos , ADN/genéticaRESUMEN
Genome alteration results in several diseases for which therapeutics are limited. Gene editing provides a strong and potential alternative for the treatment of rare and genetic diseases. CRISPR-Cas9-based system is now being envisaged as a potential tool for the cure of genetic diseases. The RNA-guided nuclease, SaCas9 enzyme, along with its HF versions is widely employed for in vivo gene editing because of its small size and high efficiency. The current work summarizes the widely used and improved methods for in vivo manipulation of genes. The potential of CRISPR-Cas9-based systems can be harnessed to treat genetic diseases and holds great promise for therapeutic interventions in gene therapy. The in vivo gene editing poses a caveat in the form of delivery systems, the tissue in question, and several other factors. This work describes the methods which have been optimized to offer high efficiency, delivery, and gene editing in vivo.
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Sistemas CRISPR-Cas , Staphylococcus aureus , Sistemas CRISPR-Cas/genética , Staphylococcus aureus/metabolismo , Edición Génica/métodos , Terapia Genética , Endonucleasas/genéticaRESUMEN
Several studies have been reported linking the role of polyglutamine (polyQ) disease-associated proteins with altered gene regulation induced by an unstable trinucleotide (CAG) repeat. Owing to their dynamic nature of expansion, these DNA repeats form secondary structures interfering with the normal cellular mechanisms like replication and transcription and, thereby, have become the underlying cause of numerous neurodegenerative disorders involving mental retardation and/or muscular or neuronal degeneration. Despite the widespread expression of the disease-causing protein, specific subsets of neurons are susceptible to specific patterns of inheritance and clinical symptoms. Although this cell-type selectivity is still elusive and less understood, it has been found that aberrant transcriptional regulation is one of the primary causes of polyQ diseases where the functions of histone-modifying complexes are disrupted. Besides, epigenetic modifications play a critical role in the pathogenesis of these diseases. In this chapter, we will be delving into how these polyQ repeats induce the self-assembly and aggregation of altered carrier proteins based on gene alterations, causing neuronal toxicity and cellular deaths. Besides, genomic instability in CAG repeats due to altered chromatin-related enzymes will be highlighted, along with epigenetic changes present in many polyQ disorders. Understanding the underlying molecular mechanisms in the root cause of these disorders will culminate in identifying therapeutic approaches for the treatment of these neurodegenerative disorders.
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Glutamina , Enfermedades Neurodegenerativas , Humanos , Glutamina/genética , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/terapia , Estructura Secundaria de Proteína , Histonas/genética , Epigénesis Genética , Expansión de Repetición de Trinucleótido , Repeticiones de TrinucleótidosRESUMEN
Tumors are usually associated with oxygen-deficient regions (hypoxia) which results from reduced and disorganized intratumoral vasculature, increased diffusion distances, and growing tumor masses. The proteomic and metabolomic landscape of the hypoxic cells is reprogrammed through hypoxia-induced transcription factor 1 which is activated in hypoxic conditions and is inactive when oxygen is abundant. This transcription factor has also been shown to inhibit or even reverse cell differentiation. Hypoxia impedes chemotherapy as it hampers the formation of cytotoxic free radicals due to the lesser availability of molecular oxygen. The metastatic and invasive attributes of cancer cells in hypoxic conditions are exacerbated, which results in poor therapeutic outcomes. Various cell-based assays for measuring hypoxia have been developed which give an estimate of the hypoxic state of cancer cells. Prior knowledge of these assays will improve the efficacy of the treatment regimens for cancers. This article provides exhaustive information on the hypoxia-based assays which are sensitive, robust, reliable, and give easy readout with choice of cell type for these assays may be dictated by the procedural or endpoint selection.
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Neoplasias , Oxígeno , Humanos , Oxígeno/farmacología , Oxígeno/metabolismo , Hipoxia de la Célula/fisiología , Proteómica , Línea Celular , Neoplasias/terapia , Factores de Transcripción/metabolismo , Hipoxia , Línea Celular TumoralRESUMEN
Estrogen-related receptor gamma (ERRγ), the latest member of the ERR family, does not have any known reported natural ligands. Although the crystal structures of the apo, agonist-bound, and inverse agonist-bound ligand-binding domain (LBD) of ERRγ have been solved previously, their dynamic behavior has not been studied. Hence, to explore the intrinsic dynamics of the apo and ligand-bound forms of ERRγ, we applied long-range molecular dynamics (MD) simulations to the crystal structures of the apo and ligand-bound forms of the LBD of ERRγ. Using the MD trajectories, we performed hydrogen bond and binding free energy analysis, which suggested that the agonist displayed more hydrogen bonds with ERRγ than the inverse agonist 4-OHT. However, the binding energy of 4-OHT was higher than that of the agonist GSK4716, indicating that hydrophobic interactions are crucial for the binding of the inverse agonist. From principal component analysis, we observed that the AF-2 helix conformation at the C-terminal domain was similar to the initial structures during simulations, indicating that the AF-2 helix conformation is crucial with respect to the agonist or inverse agonist for further functional activity of ERRγ. In addition, we performed residue network analysis to understand intramolecular signal transduction within the protein. The betweenness centrality suggested that few of the amino acids are important for residue signal transduction in apo and ligand-bound forms. The results from this study may assist in designing better therapeutic compounds against ERRγ associated diseases.
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Agonismo Inverso de Drogas , Simulación de Dinámica Molecular , Ligandos , Furilfuramida , Receptores de Estrógenos/metabolismoRESUMEN
Interleukin-1 receptor-associated kinase 4 (IRAK4) is a vital protein involved in Toll-like and interleukin-1 receptor signal transduction. Several studies have reported regarding the crystal structure, dynamic properties, and interactions with inhibitors of the phosphorylated form of IRAK4. However, no dynamic properties of inhibitor-bound unphosphorylated IRAK4 have been previously studied. Herein, we report the intrinsic dynamics of unphosphorylated IRAK4 (uIRAK4) bound to type I and type II inhibitors. The corresponding apo and inhibitor-bound forms of uIRAK4 were subjected to three independent simulations of 500 ns (total 1.5 µs) each, and their trajectories were analyzed. The results indicated that all three systems were relatively stable, except for the type II inhibitor-bound form of uIRAK4, which exhibited less compact folding and higher solvent surface area. The intra-hydrogen bonds corroborated the structural deformation of the type-II inhibitor-bound complex, which could be attributed to the long molecular structure of the type-II inhibitor. Moreover, the type II inhibitor bound to uIRAK4 showed higher binding free energy with uIRAK4 than the type I inhibitor. The free energy landscape analysis showed a reorientation of Phe330 side chain from the DFG motif at different metastable states for all the systems. The intra-residual distance between residues Lys213, Glu233, Tyr262, and Phe330 suggests a functional interplay when the inhibitors are bound to uIRAK4, thereby hinting at their crucial role in the inhibition mechanism. Ultimately, the intrinsic dynamics study observed between type I/II inhibitor-bound forms of uIRAK4 may assist in better understanding the enzyme and designing therapeutic compounds.
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Quinasas Asociadas a Receptores de Interleucina-1 , Transducción de Señal , Quinasas Asociadas a Receptores de Interleucina-1/química , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Leishmaniasis is a neglected tropical disease that has been burdening the world for over a century. Though there are drugs to treat leishmaniasis, the repertoire suffers several drawbacks like toxicity and low therapeutic value. Therefore, there is a rising concern to develop new anti-leishmanial strategies. In this study, we report, for the first time, the one-pot synthesis method and functionalization of gold and silver nanoparticles with 4',7-dihydroxyflavone (Au-47DHF and Ag-47DHF)) and their anti-leishmanial activity. Oval and spherical-shaped Au-47DHF nanoparticles were obtained with an average size of 5.8 ± 0.1 nm and while synthesized dodecahedron-shaped Ag-47DHF had an average size of 25.1 ± 1 nm. The zeta potential of Au-47DHF and Ag-47DHF were measured to be stable with values of 40 mV and 60 mV, respectively. The functionalization of nanoparticles with 4',7-dihydroxyflavone was confirmed by FTIR spectra. Both Au-47DHF and Ag-47DHF exhibited promising anti-leishmanial activity against the promastigote forms with IC50 values of 0.1226 ± 0.02 µg/ml and 0.8483 ± 0.14 µg/ml, respectively. The nanoparticles were also capable of anti-intracellular amastigote activity with 0.121 ± 0.36 µg/ml and 0.215 ± 0.85 µg/ml for Au-47DHF and Ag-47DHF, respectively. Interestingly, the treatment with Au-47DHF and Ag-47DHF nanoparticles generated high ROS concentrations in the parasites suggesting a ROS-mediated anti-leishmanial activity of Au-47DHF and Ag-47DHF. Concluding from the results, we present here a novel synthesis method of Au-47DHF and Ag-47DHF nanoparticles that have immense potential to be anti-leishmanial agents.
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Leishmania donovani , Nanopartículas del Metal , Flavonoides , Oro/farmacología , Especies Reactivas de Oxígeno , Plata/farmacologíaRESUMEN
Current treatments for leishmaniasis involve various drugs, including miltefosine and amphotericin B, which are associated with several side effects and high costs. Long-term use of these drugs may lead to the development of resistance, thereby reducing their efficiency. Chrysin (CHY) is a well-known, non-toxic flavonoid with antioxidant, antiviral, anti-inflammatory, anti-cancer, hepatoprotective, and neuroprotective properties. Recently we have shown that CHY targets the MAP kinase 3 enzyme of Leishmania and neutralizes the parasite rapidly. However, CHY is associated with low bioavailability, poor absorption, and rapid excretion issues, limiting its usage. In this study, we developed and tested a novel CHY-gold nanoformulation with improved efficacy against the parasites. The reducing power of CHY was utilized to reduce and conjugate with gold nanoparticles. Gold nanoparticles, which are already known for their anti-leishmanial properties, along with conjugated CHY, exhibited a decreased parasite burden in mammalian macrophages. Our findings showed that this biofunctionalized nanoformulation could be used as a potential therapeutic tool against leishmaniasis.