Repurposing ciclopirox as a pharmacological chaperone in a model of congenital erythropoietic porphyria.

Congenital erythropoietic porphyria is a rare autosomal recessive disease produced by deficient activity of uroporphyrinogen III synthase, the fourth enzyme in the heme biosynthetic pathway. The disease affects many organs, can be life-threatening, and currently lacks curative treatments. Inherited mutations most commonly reduce the enzyme's stability, altering its homeostasis and ultimately blunting intracellular heme production. This results in uroporphyrin by-product accumulation in the body, aggravating associated pathological symptoms such as skin photosensitivity and disfiguring phototoxic cutaneous lesions. We demonstrated that the synthetic marketed antifungal ciclopirox binds to the enzyme, stabilizing it. Ciclopirox targeted the enzyme at an allosteric site distant from the active center and did not affect the enzyme's catalytic role. The drug restored enzymatic activity in vitro and ex vivo and was able to alleviate most clinical symptoms of congenital erythropoietic porphyria in a genetic mouse model of the disease at subtoxic concentrations. Our findings establish a possible line of therapeutic intervention against congenital erythropoietic porphyria, which is potentially applicable to most of deleterious missense mutations causing this devastating disease.

Discovery of Enhancers of the Secretion of Leukemia Inhibitory Factor for the Treatment of Multiple Sclerosis.

Multiple sclerosis (MS) is an autoimmune neurodegenerative disease that involves activation of T cells, microglia, and astrocytes. There is a clear unmet medical need for MS, as current therapies reduce the relapse rate, but are unable to prevent the neurological deterioration. Leukemia inhibitory factor (LIF) is a proinflammatory cytokine that can also positively modulate the immune response, by inducing the inhibition of myelin-reactive TH17 differentiation, and by promoting oligodendrocyte-mediated myelination. The aim of this project was to find central nervous system (CNS)-permeable and orally available small molecules that upregulate production of endogenous LIF. We describe here the development of a phenotypic assay and screening of 1.7 million compounds to identify LIF enhancers using U87 MG cells. Five chemically tractable series of compounds and a few singletons were selected for further progression. Some of them were also active in a different LIF-expressing cell line and in primary rat astrocytes. Although further studies would be required to deconvolute the targets involved in LIF induction and to confirm activity of hits in more disease-relevant assays, our results have demonstrated the potential of the phenotypic approach to identify specific and chemically tractable small molecules that trigger the production of LIF in relevant cell lines.

La enfermedad vascular carotídea como marcador de isquemia inducible en pacientes asintomáticos con múltiples factores de riesgo / Carotid vascular disease as a marker of inducible ischemia in asymptomatic patients with multiple risk factors

Antecedentes: está bien establecido el valor como marcador de riesgo para eventos cardiovasculares a largo plazo del engrosamiento de íntima media carotídeo (EIM) y la correlación entre las placas carotídeas (PC) y la anatomía coronaria (SYNTAX score). Existe escasa evidencia sobre la incidencia de isquemia miocárdica en función de los diferentes grados de severidad de la enfermedad vascular carotídea (EVC). Objetivo: evaluar la incidencia y la severidad de la isquemia miocárdica inducible en pacientes con diferentes grados de EVC, utilizando los datos cuantificados del eco Doppler carotídeo (EDC) y del estudio funcional de perfusión miocárdica con radioisótopos (SPECT). Material y método: se incluyeron sucesivamente 397 pacientes, asintomáticos, 251 varones, con edad media de 65 ± 9 años, con factores de riesgo vascular, y score de Framingham ³ 15. Los pacientes fueron estudiados mediante EDC y SPECT y divididos en cinco grupos (G), según el grado de la EVC, cuantificada mediante el score de placa (SP). G1 (control, n: 50): sin alteraciones carotídeas: íntima media carotídea (IMC): <1,1 mm; G2 (n: 150): con EIM: ³1,1-<1,5 mm; G3 (n: 88): SP: ³1,5-<6 mm; G4 (n: 62): SP: ³6-<12 mm; G5 (n: 47): SP: ³12 mm, determinando la incidencia de isquemia en cada grupo, correlación (r) entre el SP con el score diferencial de suma (SDS) por SPECT, y curva ROC. Resultado: del total de los 397 pacientes, desarrollaron isquemia con SPECT: 169 (42%). Para cada grupo, G1: 14 (28%); G2: 41 (26%); G3: 30 (34%); G4: 45 (72%)*; G5: 39 (83%)*. Correlación: r = SP/SDS. G1: 0,13; G2: 0,23; G3: 0,25; G4: 0,47*; G5: 0,65* (* = p: <0,01). Curva ROC: 0,72 ± 0,04 (línea de corte de SP: 6 mm). Conclusión: la cuantía de la enfermedad vascular carotídea se correlacionó con una elevada incidencia y severidad de isquemia miocárdica inducible. Este hallazgo reafirma su valor clínico como marcador de riesgo adicional a los scores disponibles en la actualidad.

Background: the value of the carotid intima-media thickness (IMT) as a long-term risk marker for cardiovascular events and the correlation between carotid plaques (CP) and coronary anatomy (SYNTAX score) have been well demonstrated. There is little evidence about the incidence of myocardial ischemia depending on the different degrees of severity of the carotid artery disease (CAD). Objective: to assess the incidence and severity of inducible myocardial ischemia in patients (P) with different degrees of CAD, using quantitative data of Carotid Doppler (CD) and of the functional study of radioisotope myocardial perfusion (SPECT). Materials and methods: they were included 397 consecutive asymptomatic patients were followed up, 251 males, average age of 65 ± 9 years old, with cardiovascular risk factors and Framingham score ³ 15, patients were studied by CD and SPECT and divided into 5 groups (G) according to plaque score (PS). G1 (control, n: 50): with no carotid alterations: carotid intima media: <1,1 mm; G2 (n: 150): with thickening of IMT: PS: ³1,1-<1,5 mm; G3 (n: 88): PS: ³1,5-<6 mm, G4 (n: 62): PS: ³6-<12 mm; G5 (n: 47): PS: ³12 mm. Determination of ischemic incidence in each group, correlation (r) between PS by means of CDE with summed difference score (SDS) by means of SPECT and ROC curve. Results: from the total of 397 P, 169 (42%) developed ischemia under SPECT. For each group: G1:14 (28%), G2:41 (26%), G3:30 (34%), G4:45 (72%) *, G5:39 (83%) *. Correlation: r=PS/SDS: G1:0.13. G2:0.23. G3:0.25. G4:0.47 *. G5:0.65 *. (* = p: <0.01). ROC curve: 0.72 ± 0.04 (PS cutting line: 6 mm). Conclusion: the amount of carotid vascular disease correlated with a high incidence and severity of inducible myocardial ischemia. This finding reinforces their clinical marker value to additional risk scores available today.

La aplicación de planificación estratégica y de herramientas genéricas ISO 9001: 2008 en el control integral de los factores de riesgo para enfermedad cardiovascular determinan una mayor tasa de éxito a largo plazo / The application of strategic planning and generic tools ISO 9001: 2008 in the comprehensive control of risk factors for cardiovascular disease determine a higher long-term success rate

Antecedentes: una adecuada planificación estratégica (PE) y la utilización de herramientas genéricas de las normas ISO 9001:2008, aplicadas en medicina, ha demostrado mejorar la eficacia y la eficiencia de los procesos tanto en áreas administrativas como asistenciales. Objetivo: alcanzar y sostener en el tiempo el control integral de los factores de riesgo para enfermedad cardiovascular (FRCV), implementando y aplicado un seguimiento por procesos (SPP) y evaluando su respuesta por el lapso de tres años en pacientes de alto riesgo clínico, comparándolo con el manejo habitual conocido hasta ahora. Método: 310 pacientes con múltiples FRCV iniciaron el programa de seguimiento (192 varones y 118 mujeres, con edad de 59±11 años, 206 en prevención primaria y 104 en secundaria), divididos aleatoriamente en dos grupos, según el sistema de seguimiento: grupo 1 (n:160: SPP) y grupo 2 (n:150, seguimiento según criterios de sus profesionales asistentes). La PE en cuanto a tiempo y plazos se objetivó previamente mediante gráfica de Gantt, el primer año en cuatro trimestres, segundo y tercer año en dos semestres cada uno, y su implementación mediante herramienta de suceso real PERT (evaluación de programa y técnica de revisión); se evaluaron los resultados comparativos entre ambos grupos, teniendo, como objetivo de calidad, el control integral y sostenido de los FRCV. Test de t para datos apareados, con nivel de rechazo de hipótesis nula: p<0,05. Resultado: 294 pacientes (95%) completaron el seguimiento a tres años, lograron alcanzar y mantener las metas de control previstas, para el grupo 1: 58%, 56% y 55%, y para el grupo 2: 32%, 28% y 25%, para cada año respectivamente (p<0,01). Conclusión: el cumplimiento y sostenimiento de objetivos de calidad mediante planificación estratégica y utilización de herramientas de gestión genéricas ISO 9001:2008 logra a largo plazo una tasa de éxito elevada en el control integral de los FRCV.

The application of strategic planning and generic tools ISO 9001:2008 in the integral control of risk factors for cardiovascular disease, improve efficiency and efficacy of administrative and care processes. Background: adequate strategic planning (SP) and the use of generic tools of ISO 9001:2008, applied in medicine has been shown to improve the effectiveness and efficiency of processes, both in administrative areas such as healthcare. Objective: to achieve and sustain over time the integral control of risk factors for cardiovascular disease (CVRF), implementing and applying a tracking process (TPP) and evaluating their response for a period of 3 years in high clinical risk patients, compared with standard management known so far. Methods: 310 patients with multiple CVRF began the monitoring program (192 men and 118 women, aged 59 ± 11 years , 206 in primary prevention and 104 in secondary), were randomly divided into two groups according to the tracking system Group 1 (n = 160 : TPP) and Group 2 (n = 150, follow criteria of professional assistants). The SP in terms of time and deadlines previously studied using Gantt chart, the first year in 4 quarters, second and third year of 2 semesters each, and deployment tool real event PERT (program evaluation and technique review) , the comparative results between the two groups were evaluated, taking aim quality, integrated control and sustained CVRF. T test for paired data, with the level of rejection of null hypothesis: p: < 0.05. Results: 294 patients (95%) completed follow-up to 3 years, managed to achieve and maintain control targets planned: for Group 1: 58%, 56% and 55%, for Group 2 : 32%, 28% and 25% for each year respectively (p: <0.01). Conclusion: the implementation and maintenance of quality objectives through strategic planning and use of generic management tools ISO 9001:2008 , achieved long-term success rate in the comprehensive control of CVRF.

Mining Natural-Products Screening Data for Target-Class Chemical Motifs.

In this article, we describe two complementary data-mining approaches used to characterize the GlaxoSmithKline (GSK) natural-products set (NPS) based on information from the high-throughput screening (HTS) databases. Both methods rely on the aggregation and analysis of a large set of single-shot screening data for a number of biological assays, with the goal to reveal natural-product chemical motifs. One of them is an established method based on the data-driven clustering of compounds using a wide range of descriptors,(1)whereas the other method partitions and hierarchically clusters the data to identify chemical cores.(2,3)Both methods successfully find structural scaffolds that significantly hit different groups of discrete drug targets, compared with their relative frequency of demonstrating inhibitory activity in a large number of screens. We describe how these methods can be applied to unveil hidden information in large single-shot HTS data sets. Applied prospectively, this type of information could contribute to the design of new chemical templates for drug-target classes and guide synthetic efforts for lead optimization of tractable hits that are based on natural-product chemical motifs. Relevant findings for 7TM receptors (7TMRs), ion channels, class-7 transferases (protein kinases), hydrolases, and oxidoreductases will be discussed.

Screening technologies for G protein-coupled receptors: from HTS to uHTS.

The discovery of drugs for G protein-coupled receptors (GPCRs) has traditionally been very successful, even before the structural nature of these molecular targets was elucidated. Over the years, this family of proteins has become more important in the understanding and treatment of different human pathologies, representing today close to 30% of the molecular targets of all marketed drugs. The sequencing of the human genome unveiled the existence of many new GPCRs and this has increased even more the interest of this family of proteins as potential drug targets. Today the search for compounds that interfere or modulate the function of GPCRs is one of the major focuses of pharmaceutical companies. The understanding of the molecular events that take place upon receptor activation, together with the need of testing large chemical libraries, has resulted in the development of a variety of methods and technologies to measure the activity of these receptors. In this chapter we will review most of the assay technologies currently in use for "in vitro" pharmacological screening, their evolution, their capabilities, and their limitations.

Process validation and screen reproducibility in high-throughput screening.

The use of large-scale compound screening has become a key component of drug discovery projects in both the pharmaceutical and the biotechnological industries. More recently, these activities have also been embraced by the academic community as a major tool for chemical genomic activities. High-throughput screening (HTS) activities constitute a major step in the initial drug discovery efforts and involve the use of large quantities of biological reagents, hundreds of thousands to millions of compounds, and the utilization of expensive equipment. All these factors make it very important to evaluate in advance of the HTS campaign any potential issues related to reproducibility of the experimentation and the quality of the results obtained at the end of these very costly activities. In this article, the authors describe how GlaxoSmithKline (GSK) has addressed the need of a true validation of the HTS process before embarking in full HTS campaigns. They present 2 different aspects of the so-called validation process: (1) optimization of the HTS workflow and its validation as a quality process and (2) the statistical evaluation of the HTS, focusing on the reproducibility of results and the ability to distinguish active from nonactive compounds in a vast collection of samples. The authors describe a variety of reproducibility indexes that are either innovative or have been adapted from generic medical diagnostic screening strategies. In addition, they exemplify how these validation tools have been implemented in a number of case studies at GSK.

A new destruxin as inhibitor of vacuolar-type H+-ATPase of Saccharomyces cerevisiae.

In the course of our screening efforts to discover small molecules as selective inhibitors of vacuolar-type H+-ATPase of Saccharomyces cerevisiae, we have identified eight active destruxins, 1-8, from the fungus Metarhizium anisopliae. The structures were elucidated by extensive 1D- and 2D-NMR spectroscopy, and MS spectrometry. One of these compounds, 8, a regioisomer of chlorohydrin destruxin E (7), is a new destruxin.

L-cystine inhibits aspartate-beta-semialdehyde dehydrogenase by covalently binding to the essential 135Cys of the enzyme.

Aspartate-beta-semialdehyde dehydrogenase (ASADH) from Escherichia coli is inhibited by L- and D-cystine, and by other cystine derivatives. Enzyme inhibition is quantitatively reversed by addition of dithiothreitol (DTT), dithioerythrytol, beta-mercaptoethanol, di-mercaptopropanol or glutathione to the cystine-inactivated enzyme. Cystine labeling of the enzyme is a pH dependent process and is optimal at pH values ranging from 7.0 to 7.5. Both the cysteine incorporation profile and the inactivation curve of the enzyme as a function of pH suggest that a group(s) with pK(a) of 8.5 could be involved in cystine binding. Stoichiometry of the inactivation reaction indicates that one cysteine residue from the enzyme subunit is reactive against cystine, as found by direct incorporation of radioactive cystine into the enzyme and by free-thiol titration of the enzyme with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) before and after the cystine treatment. One mole of cysteine is released from each mol of cystine after reaction with the enzyme. ASA, NADP and NADPH did not prevent cystine inhibition. The [35S]cysteine-labelled enzyme can be visualized after electrophoresis in polyacrylamide gels and further detection by autoradiography. After pepsin treatment of the [35S]cysteine-inactivated enzyme, a main radioactive peptide was isolated by HPLC. The amino acid sequence of this peptide was determined as FVGGN(Cys)(2)TVSL, thus demonstrating that the essential 135Cys is the amino acid residue modified by the treatment with cystine.

A novel ergot alkaloid as a 5-HT(1A) inhibitor produced by Dicyma sp.

In the course of a search for small-molecule inhibitors of 5-hydroxytryptamine receptors we have identified a novel ergoline derivative (1) from the fungal culture of Dicyma sp. This compound has a pK(i) of 10.2 versus the 5-hydroxytryptamine(1A) receptor subtype. The structure was elucidated by extensive NMR spectroscopy and mass spectrometry.

Identification of a series of tricyclic natural products as potent broad-spectrum inhibitors of metallo-beta-lactamases.

This work describes the discovery and characterization of a novel series of tricyclic natural product-derived metallo-beta-lactamase inhibitors. Natural product screening of the Bacillus cereus II enzyme identified an extract from a strain of Chaetomium funicola with inhibitory activity against metallo-beta-lactamases. SB236050, SB238569, and SB236049 were successfully extracted and purified from this extract. The most active of these compounds was SB238569, which possessed K(i) values of 79, 17, and 3.4 microM for the Bacillus cereus II, Pseudomonas aeruginosa IMP-1, and Bacteroides fragilis CfiA metallo-beta-lactamases, respectively, yet none of the compounds exhibited any inhibitory activity against the Stenotrophomonas maltophilia L-1 metallo-beta-lactamase (50% inhibitory concentration > 1,000 microM). The lack of activity against angiotensin-converting enzyme and serine beta-lactamases demonstrated the selective nature of these compounds. The crystal structure of SB236050 complexed in the active site of CfiA has been obtained to a resolution of 2.5 A. SB236050 exhibits key polar interactions with Lys184, Asn193, and His162 and a stacking interaction with the indole ring of Trp49 in the flap, which is in the closed conformation over the active site groove. SB236050 and SB238569 also demonstrate good antibacterial synergy with meropenem. Eight micrograms of SB236050 per ml gave rise to an eightfold drop in the MIC of meropenem for two clinical isolates of B. fragilis producing CfiA, making these strains sensitive to meropenem (MIC < or = 4 microg/ml). Consequently, this series of metallo-beta-lactamase inhibitors exhibit the most promising antibacterial synergy activity so far observed against organisms producing metallo-beta-lactamases.