RESUMEN
Fucosylation of the innermost GlcNAc of N-glycans by fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can dramatically affect the activities and half-lives of glycoproteins, effects that are relevant to understanding the invasiveness of some cancers, development of mAb therapeutics, and the etiology of a congenital glycosylation disorder. The acceptor substrate preferences of FUT8 are well-characterized and provide a framework for understanding N-glycan maturation in the Golgi; however, the structural basis of these substrate preferences and the mechanism through which catalysis is achieved remain unknown. Here we describe several structures of mouse and human FUT8 in the apo state and in complex with GDP, a mimic of the donor substrate, and with a glycopeptide acceptor substrate at 1.80-2.50 Å resolution. These structures provide insights into a unique conformational change associated with donor substrate binding, common strategies employed by fucosyltransferases to coordinate GDP, features that define acceptor substrate preferences, and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also revealed how FUT8 dimerization plays an important role in defining the acceptor substrate-binding site. Collectively, this information significantly builds on our understanding of the core fucosylation process.
Asunto(s)
Fucosiltransferasas/química , Guanosina Difosfato/química , Simulación de Dinámica Molecular , Animales , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Humanos , RatonesRESUMEN
The circulating life span of blood platelets is regulated by the prosurvival protein BCL-XL It restrains the activity of BAK and BAX, the essential prodeath mediators of intrinsic apoptosis. Disabling the platelet intrinsic apoptotic pathway in mice by deleting BAK and BAX results in a doubling of platelet life span and concomitant thrombocytosis. Apoptotic platelets expose phosphatidylserine (PS) via a mechanism that is distinct from that driven by classical agonists. Whether there is any role for apoptotic PS in platelet function in vivo, however, is unclear. Apoptosis has also been associated with the platelet storage lesion (PSL), the constellation of biochemical deteriorations that occur during blood bank storage. In this study, we investigated the role of BAK/BAX-mediated apoptosis in hemostasis and thrombosis and in the development of the PSL. We show that although intrinsic apoptosis is rapidly induced during storage at 37°C, it is not detected when platelets are kept at the standard storage temperature of 22°C. Remarkably, loss of BAK and BAX did not prevent the development of the PSL at either temperature. BAK/BAX-deficient mice exhibited increased bleeding times and unstable thrombus formation. This phenotype was not caused by impaired PS exposure, but was associated with a defect in granule release from aged platelets. Strikingly, rejuvenation of BAK/BAX-deficient platelets in vivo completely rescued the observed hemostatic defects. Thus, apoptotic culling of old platelets from the bloodstream is essential to maintain a functional, hemostatically reactive platelet population. Inhibiting intrinsic apoptosis in blood banked platelets is unlikely to yield significant benefit.
Asunto(s)
Apoptosis , Plaquetas/metabolismo , Susceptibilidad a Enfermedades , Animales , Apoptosis/genética , Biomarcadores , Tiempo de Sangría , Recuento de Células Sanguíneas , Coagulación Sanguínea , Caspasas/metabolismo , Supervivencia Celular/genética , Femenino , Genotipo , Masculino , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Transducción de Señal , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
The use of arginine isosteres is a known strategy to overcome poor membrane permeability commonly associated with peptides or peptidomimetics that possess this highly polar amino acid. Here, we apply this strategy to peptidomimetics that are potent inhibitors of the malarial protease, plasmepsin V, with the aim of enhancing their activity against Plasmodium parasites, and exploring the structure-activity relationship of the P3 arginine within the S3 pocket of plasmepsin V. Of the arginine isosteres trialled in the P3 position, we discovered that canavanine was the ideal and that this peptidomimetic potently inhibits plasmepsin V, efficiently blocks protein export and inhibits parasite growth. Structure studies of the peptidomimetics bound to plasmepsin V provided insight into the structural basis for the enzyme activity observed in vitro and provides further evidence why plasmepsin V is highly sensitive to substrate modification.
Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Peptidomiméticos/química , Plasmodium vivax/enzimología , Animales , Espectroscopía de Resonancia Magnética , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Apoptotic caspases, including caspase-9, are thought to facilitate platelet shedding by megakaryocytes. They are known to be activated during platelet apoptosis, and have also been implicated in platelet hemostatic responses. However, the precise requirement for, and the regulation of, apoptotic caspases have never been defined in either megakaryocytes or platelets. To establish the role of caspases in platelet production and function, we generated mice lacking caspase-9 in their hematopoietic system. We demonstrate that both megakaryocytes and platelets possess a functional apoptotic caspase cascade downstream of Bcl-2 family-mediated mitochondrial damage. Caspase-9 is the initiator caspase, and its loss blocks effector caspase activation. Surprisingly, steady-state thrombopoiesis is unperturbed in the absence of caspase-9, indicating that the apoptotic caspase cascade is not required for platelet production. In platelets, loss of caspase-9 confers resistance to the BH3 mimetic ABT-737, blocking phosphatidylserine (PS) exposure and delaying ABT-737-induced thrombocytopenia in vivo. Despite this, steady-state platelet lifespan is normal. Casp9(-/-) platelets are fully capable of physiologic hemostatic responses and functional regulation of adhesive integrins in response to agonist. These studies demonstrate that the apoptotic caspase cascade is required for the efficient death of megakaryocytes and platelets, but is dispensable for their generation and function.
Asunto(s)
Apoptosis/fisiología , Plaquetas/citología , Caspasa 9/fisiología , Megacariocitos/citología , Trombopoyesis/fisiología , Animales , Compuestos de Bifenilo/farmacología , Compuestos de Bifenilo/toxicidad , Plaquetas/enzimología , Caspasa 9/deficiencia , Caspasa 9/genética , Linaje de la Célula , Hemostasis/efectos de los fármacos , Hemostasis/fisiología , Hirudinas/farmacología , Hígado/embriología , Trasplante de Hígado , Megacariocitos/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nitrofenoles/farmacología , Nitrofenoles/toxicidad , Piperazinas/farmacología , Piperazinas/toxicidad , Activación Plaquetaria/efectos de los fármacos , Activación Plaquetaria/fisiología , Quimera por Radiación , Sulfonamidas/farmacología , Sulfonamidas/toxicidad , Trombocitopenia/inducido químicamente , Proteína X Asociada a bcl-2/deficienciaRESUMEN
Emerging resistance to current antimalarials is reducing their effectiveness and therefore there is a need to develop new antimalarial therapies. Toward this goal, high throughput screens against the P. falciparum asexual parasite identified the pyrazolopyridine 4-carboxamide scaffold. Structure-activity relationship analysis of this chemotype defined that the N1-tert-butyl group and aliphatic foliage in the 3- and 6-positions were necessary for activity, while the inclusion of a 7'-aza-benzomorpholine on the 4-carboxamide motif resulted in potent anti-parasitic activity and increased aqueous solubility. A previous report that resistance to the pyrazolopyridine class is associated with the ABCI3 transporter was confirmed, with pyrazolopyridine 4-carboxamides showing an increase in potency against parasites when the ABCI3 transporter was knocked down. The low metabolic stability intrinsic to the pyrazolopyridine scaffold and the slow rate by which the compounds kill asexual parasites resulted in poor performance in a P. berghei asexual blood stage mouse model. Lowering the risk of resistance and mitigating the metabolic stability and cytochrome P450 inhibition will be challenges in the future development of the pyrazolopyrimidine antimalarial class.
Asunto(s)
Antimaláricos , Plasmodium falciparum , Pirazoles , Piridinas , Antimaláricos/farmacología , Antimaláricos/química , Antimaláricos/síntesis química , Plasmodium falciparum/efectos de los fármacos , Relación Estructura-Actividad , Pirazoles/química , Pirazoles/farmacología , Pirazoles/síntesis química , Animales , Piridinas/farmacología , Piridinas/química , Piridinas/síntesis química , Ratones , Pruebas de Sensibilidad Parasitaria , Estructura Molecular , Resistencia a Medicamentos/efectos de los fármacos , Relación Dosis-Respuesta a Droga , HumanosRESUMEN
Malaria is a devastating disease that causes significant morbidity worldwide. The development of new antimalarial chemotypes is urgently needed because of the emergence of resistance to frontline therapies. Independent phenotypic screening campaigns against the Plasmodium asexual parasite, including our own, identified the aryl amino acetamide hit scaffold. In a prior study, we identified the STAR-related lipid transfer protein (PfSTART1) as the molecular target of this antimalarial chemotype. In this study, we combined structural elements from the different aryl acetamide hit subtypes and explored the structure-activity relationship. It was shown that the inclusion of an endocyclic nitrogen, to generate the tool compound WJM-715, improved aqueous solubility and modestly improved metabolic stability in rat hepatocytes. Metabolic stability in human liver microsomes remains a challenge for future development of the aryl acetamide class, which was underscored by modest systemic exposure and a short half-life in mice. The optimized aryl acetamide analogs were cross resistant to parasites with mutations in PfSTART1, but not to other drug-resistant mutations, and showed potent binding to recombinant PfSTART1 by biophysical analysis, further supporting PfSTART1 as the likely molecular target. The optimized aryl acetamide analogue, WJM-715 will be a useful tool for further investigating the druggability of PfSTART1 across the lifecycle of the malaria parasite.
Asunto(s)
Antimaláricos , Proteínas Portadoras , Malaria Falciparum , Malaria , Ratas , Ratones , Humanos , Animales , Antimaláricos/química , Plasmodium falciparum , Malaria Falciparum/tratamiento farmacológico , Malaria/tratamiento farmacológico , Acetamidas/farmacología , LípidosRESUMEN
To contribute to the global effort to develop new antimalarial therapies, we previously disclosed initial findings on the optimization of the dihydroquinazolinone-3-carboxamide class that targets PfATP4. Here we report on refining the aqueous solubility and metabolic stability to improve the pharmacokinetic profile and consequently in vivo efficacy. We show that the incorporation of heterocycle systems in the 8-position of the scaffold was found to provide the greatest attainable balance between parasite activity, aqueous solubility, and metabolic stability. Optimized analogs, including the frontrunner compound S-WJM992, were shown to inhibit PfATP4-associated Na+-ATPase activity, gave rise to a metabolic signature consistent with PfATP4 inhibition, and displayed altered activities against parasites with mutations in PfATP4. Finally, S-WJM992 showed appreciable efficacy in a malaria mouse model and blocked gamete development preventing transmission to mosquitoes. Importantly, further optimization of the dihydroquinazolinone class is required to deliver a candidate with improved pharmacokinetic and risk of resistance profiles.
Asunto(s)
Antimaláricos , Plasmodium falciparum , Quinazolinonas , Antimaláricos/farmacología , Antimaláricos/química , Antimaláricos/farmacocinética , Animales , Plasmodium falciparum/efectos de los fármacos , Quinazolinonas/farmacología , Quinazolinonas/química , Quinazolinonas/farmacocinética , Ratones , Administración Oral , Relación Estructura-Actividad , Humanos , Malaria/tratamiento farmacológico , Femenino , SolubilidadRESUMEN
BH3 mimetics are a new class of proapo-ptotic anticancer agents that have shown considerable promise in preclinical animal models and early-stage human trials. These agents act by inhibiting the pro-survival function of one or more Bcl-2-related proteins. Agents that inhibit Bcl-x(L) induce rapid platelet death that leads to thrombocytopenia; however, their impact on the function of residual circulating platelets remains unclear. In this study, we demonstrate that the BH3 mimetics, ABT-737 or ABT-263, induce a time- and dose-dependent decrease in platelet adhesive function that correlates with ectodomain shedding of the major platelet adhesion receptors, glycoprotein Ibα and glycoprotein VI, and functional down-regulation of integrin α(IIb)ß(3). Analysis of platelets from mice treated with higher doses of BH3 mimetics revealed the presence of a subpopulation of circulating platelets undergoing cell death that have impaired activation responses to soluble agonists. Functional analysis of platelets by intravital microscopy revealed a time-dependent defect in platelet aggregation at sites of vascular injury that correlated with an increase in tail bleeding time. Overall, these studies demonstrate that Bcl-x(L)-inhibitory BH3 mimetics not only induce thrombocytopenia but also a transient thrombocytopathy that can undermine the hemostatic function of platelets.
Asunto(s)
Plaquetas/fisiología , Hemostasis/fisiología , Trombocitopenia/fisiopatología , Proteína bcl-X/metabolismo , Compuestos de Anilina/farmacología , Animales , Apoptosis/efectos de los fármacos , Compuestos de Bifenilo/farmacología , Plaquetas/metabolismo , Plaquetas/ultraestructura , Western Blotting , Colágeno/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Hemostasis/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Rastreo , Nitrofenoles/farmacología , Fosfatidilserinas/metabolismo , Piperazinas/farmacología , Adhesividad Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Recuento de Plaquetas , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Complejo GPIb-IX de Glicoproteína Plaquetaria , Glicoproteínas de Membrana Plaquetaria/metabolismo , Sulfonamidas/farmacología , Trombocitopenia/inducido químicamente , Factores de Tiempo , Proteína bcl-X/antagonistas & inhibidoresRESUMEN
There is an urgent need to populate the antimalarial clinical portfolio with new candidates because of resistance against frontline antimalarials. To discover new antimalarial chemotypes, we performed a high-throughput screen of the Janssen Jumpstarter library against the Plasmodium falciparum asexual blood-stage parasite and identified the 2,3-dihydroquinazolinone-3-carboxamide scaffold. We defined the SAR and found that 8-substitution on the tricyclic ring system and 3-substitution of the exocyclic arene produced analogues with potent activity against asexual parasites equivalent to clinically used antimalarials. Resistance selection and profiling against drug-resistant parasite strains revealed that this antimalarial chemotype targets PfATP4. Dihydroquinazolinone analogues were shown to disrupt parasite Na+ homeostasis and affect parasite pH, exhibited a fast-to-moderate rate of asexual kill, and blocked gametogenesis, consistent with the phenotype of clinically used PfATP4 inhibitors. Finally, we observed that optimized frontrunner analogue WJM-921 demonstrates oral efficacy in a mouse model of malaria.
Asunto(s)
Antimaláricos , Malaria Falciparum , Malaria , Animales , Ratones , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Plasmodium falciparum , Homeostasis , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitologíaRESUMEN
The development of new antimalarials is required because of the threat of resistance to current antimalarial therapies. To discover new antimalarial chemotypes, we screened the Janssen Jumpstarter library against the P. falciparum asexual parasite and identified the 7-N-substituted-3-oxadiazole quinolone hit class. We established the structure-activity relationship and optimized the antimalarial potency. The optimized analog WJM228 (17) showed robust metabolic stability in vitro, although the aqueous solubility was limited. Forward genetic resistance studies uncovered that WJM228 targets the Qo site of cytochrome b (cyt b), an important component of the mitochondrial electron transport chain (ETC) that is essential for pyrimidine biosynthesis and an established antimalarial target. Profiling against drug-resistant parasites confirmed that WJM228 confers resistance to the Qo site but not Qi site mutations, and in a biosensor assay, it was shown to impact the ETC via inhibition of cyt b. Consistent with other cyt b targeted antimalarials, WJM228 prevented pre-erythrocytic parasite and male gamete development and reduced asexual parasitemia in a P. berghei mouse model of malaria. Correcting the limited aqueous solubility and the high susceptibility to cyt b Qo site resistant parasites found in the clinic will be major obstacles in the future development of the 3-oxadiazole quinolone antimalarial class.
Asunto(s)
Antimaláricos , Antagonistas del Ácido Fólico , Malaria Falciparum , Quinolonas , Animales , Ratones , Antimaláricos/farmacología , Citocromos b , Antagonistas del Ácido Fólico/metabolismo , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Plasmodium falciparum , Quinolonas/farmacologíaRESUMEN
Plasmepsin X (PMX) is an aspartyl protease that processes proteins essential for Plasmodium parasites to invade and egress from host erythrocytes during the symptomatic asexual stage of malaria. PMX substrates possess a conserved cleavage region denoted by the consensus motif, SFhE (h=hydrophobic amino acid). Peptidomimetics reflecting the P3 -P1 positions of the consensus motif were designed and showed potent and selective inhibition of PMX. It was established that PMX prefers Phe in the P1 position, di-substitution at the ß-carbon of the P2 moiety and a hydrophobic P3 group which was supported by modelling of the peptidomimetics in complex with PMX. The peptidomimetics were shown to arrest asexual P. falciparum parasites at the schizont stage by impairing PMX substrate processing. Overall, the peptidomimetics described will assist in further understanding PMX substrate specificity and have the potential to act as a template for future antimalarial design.
Asunto(s)
Antimaláricos , Antagonistas del Ácido Fólico , Malaria Falciparum , Peptidomiméticos , Aminoácidos , Antimaláricos/química , Antimaláricos/farmacología , Ácido Aspártico Endopeptidasas , Carbono , Humanos , Malaria Falciparum/tratamiento farmacológico , Peptidomiméticos/química , Peptidomiméticos/farmacología , Plasmodium falciparum/metabolismo , Inhibidores de Proteasas/química , Proteínas ProtozoariasRESUMEN
Drug resistance to first-line antimalarials-including artemisinin-is increasing, resulting in a critical need for the discovery of new agents with novel mechanisms of action. In collaboration with the Walter and Eliza Hall Institute and with funding from the Wellcome Trust, a phenotypic screen of Merck's aspartyl protease inhibitor library identified a series of plasmepsin X (PMX) hits that were more potent than chloroquine. Inspired by a PMX homology model, efforts to optimize the potency resulted in the discovery of leads that, in addition to potently inhibiting PMX, also inhibit another essential aspartic protease, plasmepsin IX (PMIX). Further potency and pharmacokinetic profile optimization efforts culminated in the discovery of WM382, a very potent dual PMIX/X inhibitor with robust in vivo efficacy at multiple stages of the malaria parasite life cycle and an excellent resistance profile.
RESUMEN
The COVID-19 pandemic continues unabated, emphasizing the need for additional antiviral treatment options to prevent hospitalization and death of patients infected with SARS-CoV-2. The papain-like protease (PLpro) domain is part of the SARS-CoV-2 non-structural protein (nsp)-3, and represents an essential protease and validated drug target for preventing viral replication. PLpro moonlights as a deubiquitinating (DUB) and deISGylating enzyme, enabling adaptation of a DUB high throughput (HTS) screen to identify PLpro inhibitors. Drug repurposing has been a major focus through the COVID-19 pandemic as it may provide a fast and efficient route for identifying clinic-ready, safe-in-human antivirals. We here report our effort to identify PLpro inhibitors by screening the ReFRAME library of 11,804 compounds, showing that none inhibit PLpro with any reasonable activity or specificity to justify further progression towards the clinic. We also report our latest efforts to improve piperidine-scaffold inhibitors, 5c and 3k, originally developed for SARS-CoV PLpro. We report molecular details of binding and selectivity, as well as in vitro absorption, distribution, metabolism and excretion (ADME) studies of this scaffold. A co-crystal structure of SARS-CoV-2 PLpro bound to inhibitor 3k guides medicinal chemistry efforts to improve binding and ADME characteristics. We arrive at compounds with improved and favorable solubility and stability characteristics that are tested for inhibiting viral replication. Whilst still requiring significant improvement, our optimized small molecule inhibitors of PLpro display decent antiviral activity in an in vitro SARS-CoV-2 infection model, justifying further optimization.
RESUMEN
Phosphoinositide (PI) 3-kinase (PI3K) signaling processes play an important role in regulating the adhesive function of integrin alpha(IIb)beta(3), necessary for platelet spreading and sustained platelet aggregation. PI3K inhibitors are effective at reducing platelet aggregation and thrombus formation in vivo and as a consequence are currently being evaluated as novel antithrombotic agents. PI3K regulation of integrin alpha(IIb)beta(3) activation (affinity modulation) primarily occurs downstream of G(i)-coupled and tyrosine kinase-linked receptors linked to the activation of Rap1b, AKT, and phospholipase C. In the present study, we demonstrate an important role for PI3Ks in regulating the avidity (strength of adhesion) of high affinity integrin alpha(IIb)beta(3) bonds, necessary for the cellular transmission of contractile forces. Using knock-out mouse models and isoform-selective PI3K inhibitors, we demonstrate that the Type Ia p110 beta isoform plays a major role in regulating thrombin-stimulated fibrin clot retraction in vitro. Reduced clot retraction induced by PI3K inhibitors was not associated with defects in integrin alpha(IIb)beta(3) activation, actin polymerization, or actomyosin contractility but was associated with a defect in integrin alpha(IIb)beta(3) association with the contractile cytoskeleton. Analysis of integrin alpha(IIb)beta(3) adhesion contacts using total internal reflection fluorescence microscopy revealed an important role for PI3Ks in regulating the stability of high affinity integrin alpha(IIb)beta(3) bonds. These studies demonstrate an important role for PI3K p110 beta in regulating the avidity of high affinity integrin alpha(IIb)beta(3) receptors, necessary for the cellular transmission of contractile forces. These findings may provide new insight into the potential antithrombotic properties of PI3K p110 beta inhibitors.
Asunto(s)
Plaquetas/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Adhesividad Plaquetaria/fisiología , Agregación Plaquetaria/fisiología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Adenosina Difosfato/metabolismo , Animales , Plaquetas/efectos de los fármacos , Fosfatidilinositol 3-Quinasa Clase I , Retracción del Coagulo/efectos de los fármacos , Retracción del Coagulo/fisiología , Citoesqueleto/fisiología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/genética , Estrés Mecánico , Trombina/farmacología , Tromboxano A2/metabolismoRESUMEN
SK1 (sphingosine kinase 1) plays an important role in many aspects of cellular regulation. Most notably, elevated cellular SK1 activity leads to increased cell proliferation, protection from apoptosis, and induction of neoplastic transformation. We have previously shown that translocation of SK1 from the cytoplasm to the plasma membrane is integral for oncogenesis mediated by this enzyme. The molecular mechanism mediating this translocation of SK1 has remained undefined. Here, we demonstrate a direct role for CIB1 (calcium and integrin-binding protein 1) in this process. We show that CIB1 interacts with SK1 in a Ca(2+)-dependent manner at the previously identified "calmodulin-binding site" of SK1. We also demonstrate that CIB1 functions as a Ca(2+)-myristoyl switch, providing a mechanism whereby it translocates SK1 to the plasma membrane. Both small interfering RNA knockdown of CIB1 and the use of a dominant-negative CIB1 we have generated prevent the agonist-dependent translocation of SK1. Furthermore, we demonstrate the requirement of CIB1-mediated translocation of SK1 in controlling cellular sphingosine 1-phosphate generation and associated anti-apoptotic signaling.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Membrana Celular/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Apoptosis/efectos de los fármacos , Sitios de Unión , Calcio/metabolismo , Calmodulina/metabolismo , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Genes Dominantes/genética , Humanos , Ácido Mirístico/metabolismo , FN-kappa B/metabolismo , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
AIM: Refugee arrivals to Europe have numbered more than one million since 2015 with the majority arriving through Greece. The healthcare needs of refugees have placed strains on Greece's healthcare system which has already been affected by its ongoing economic crisis. At the peak of arrivals during 2016, primary healthcare was primarily provided by humanitarian organizations with specialist referrals into the Greek healthcare system. There is little published literature on the type and impacts of specialist referrals for refugees in Greece. The aim of this retrospective review is to identify the type and impacts of specialist referrals for refugees into Greece's health system. METHODS: This retrospective study reviewed the number and type of specialty referrals from one humanitarian organization providing primary healthcare for refugees in Greece. All consultations during an 8-month period (December 1, 2016-July 31, 2017) were reviewed. RESULTS: Of 4168 consultations, 42% were patients aged 17 years or younger, 52% were male, and 90% were Syrian. Two hundred and thirty-three patients (11%) required a specialist referral; 25% were for dental (provided by another humanitarian organization), 10% each for obstetrics and gynecology and pediatrics, and 8% for ophthalmology. Respiratory complaints were most frequently seen, and these were more predominant in the winter months. Pediatric consultations varied according to month, likely due to population movements. CONCLUSION: Dentistry was noted to be a gap in humanitarian response programming and accounted for the greatest need for specialist input with referrals for women and children accounting for a large proportion of referrals.
RESUMEN
Limited therapeutic options are available for the treatment of human schistosomiasis caused by the parasitic Schistosoma flatworm. The B cell lymphoma-2 (BCL-2)-regulated apoptotic cell death pathway in schistosomes was recently characterized and shown to share similarities with the intrinsic apoptosis pathway in humans. Here, we exploit structural differences in the human and schistosome BCL-2 (sBCL-2) pro-survival proteins toward a novel treatment strategy for schistosomiasis. The benzothiazole hydrazone scaffold previously employed to target human BCL-XL was repurposed as a starting point to target sBCL-2. We utilized X-ray structural data to inform optimization and then applied a scaffold-hop strategy to identify the 5-carboxamide thiazole hydrazone scaffold (43) with potent sBCL-2 activity (IC50 30 nM). Human BCL-XL potency (IC50 13 nM) was inadvertently preserved during the optimization process. The lead analogues from this study exhibit on-target activity in model fibroblast cell lines dependent on either sBCL-2 or human BCL-XL for survival. Further optimization of the thiazole hydrazone class is required to exhibit activity in schistosomes and enhance the potential of this strategy for treating schistosomiasis.
Asunto(s)
Hidrazonas , Schistosoma , Animales , Apoptosis , Benzotiazoles , Humanos , Hidrazonas/farmacología , Proteína bcl-X/genéticaRESUMEN
Introduction: As of January 2020, 115,600 refugees remain in Greece; most are Afghani, Iraqi or Syrian nationals. This qualitative research study explores the views of key stakeholders providing healthcare for refugees in Greece between 2015 and 2018. The focus was on identifying key barriers and facilitators to healthcare access for refugees in Greece. Methods: 16 interviewees from humanitarian and international organisations operating in Greece were identified through purposive and snowball sampling. Semi-structured interviews were conducted between March and April 2018. Data were analysed using the Framework Method. Results: Key themes affecting healthcare access included the influence of socio-cultural factors (healthcare expectations, language, gender) and the ability of the Greek health system to respond to existing and evolving demands; these included Greece's ongoing economic crisis, human resource shortages, weak primary healthcare system, legal barriers and logistics. The evolution of the humanitarian response from emergency to sustained changes to EU funding, coordination and comprehensiveness of services affected healthcare access for refugees. Conclusion: The most noted barriers cited by humanitarian stakeholders to healthcare access for refugees in Greece were socio-cultural and language differences between refugees and healthcare providers and poor coordination among stakeholders. Policies and interventions which address these could improve healthcare access for refugees in Greece with coordination led by the EU.
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Personal de Salud/psicología , Accesibilidad a los Servicios de Salud/estadística & datos numéricos , Disparidades en Atención de Salud , Refugiados/psicología , Altruismo , Femenino , Grecia , Humanos , Entrevistas como Asunto , Masculino , Organizaciones , Investigación Cualitativa , Refugiados/estadística & datos numéricos , Participación de los Interesados , Siria/etnologíaRESUMEN
The P. falciparum parasite, responsible for the disease in humans known as malaria, must invade erythrocytes to provide an environment for self-replication and survival. For invasion to occur, the parasite must engage several ligands on the host erythrocyte surface to enable adhesion, tight junction formation and entry. Critical interactions include binding of erythrocyte binding-like ligands and reticulocyte binding-like homologues (Rhs) to the surface of the host erythrocyte. The reticulocyte binding-like homologue 5 (Rh5) is the only member of this family that is essential for invasion and it binds to the basigin host receptor. The essential nature of Rh5 makes it an important vaccine target, however to date, Rh5 has not been targeted by small molecule intervention. Here, we describe the development of a high-throughput screening assay to identify small molecules which interfere with the Rh5-basigin interaction. To validate the utility of this assay we screened a known drug library and the Medicines for Malaria Box and demonstrated the reproducibility and robustness of the assay for high-throughput screening purposes. The screen of the known drug library identified the known leukotriene antagonist, pranlukast. We used pranlukast as a model inhibitor in a post screening evaluation cascade. We procured and synthesised analogues of pranlukast to assist in the hit confirmation process and show which structural moieties of pranlukast attenuate the Rh5 - basigin interaction. Evaluation of pranlukast analogues against P. falciparum in a viability assay and a schizont rupture assay show the parasite activity was not consistent with the biochemical inhibition of Rh5, questioning the developability of pranlukast as an antimalarial. The high-throughput assay developed from this work has the capacity to screen large collections of small molecules to discover inhibitors of P. falciparum Rh5 for future development of invasion inhibitory antimalarials.