Spirofused Tetrahydroisoquinoline-Oxindole Hybrids (Spiroquindolones) as Potential Multitarget Antimalarial Agents: Preliminary Hit Optimization and Efficacy Evaluation in Mice.

Previous studies suggest that 3',5'-dihydro-2'H-spiro[indoline-3,1'-isoquinolin]-2-ones (DSIIQs [spiroquindolones]) are multitarget antiplasmodial agents that combine the actions of spiroindolone and naphthylisoquinoline antimalarial agents. In this study, 12 analogues of compound (±)-5 (moxiquindole), the prototypical spiroquindolone, were synthesized and tested for antiplasmodial activity. Compound (±)-11 (a mixture of compounds 11a and 11b), the most potent analogue, displayed low-nanomolar activity against P. falciparum chloroquine-sensitive 3D7 strain (50% inhibitory concentration [IC50] for 3D7 = 21 ± 02 nM) and was active against all major erythrocytic stages of the parasite life cycle (ring, trophozoite, and schizont); it also inhibited hemoglobin metabolism and caused extensive vacuolation in parasites. In drug-resistant parasites, compound (±)-11 exhibited potent activity (IC50 for Dd2 = 58.34 ± 2.04 nM) against the P. falciparum multidrug-resistant Dd2 strain, and both compounds (±)-5 and (±)-11 displayed significant cross-resistance against the P. falciparum ATP4 mutant parasite Dd2 SJ733 but not against the Dd2 KAE609 strain. In mice, both compounds (±)-5 and (±)-11 displayed dose-dependent reduction of parasitemia with suppressive 50% effective dose (ED50) values of 0.44 and 0.11 mg/kg of body weight, respectively. The compounds were also found to be curative in vivo and are thus worthy of further investigation.

Bioactive natural products derived from the Central African flora against neglected tropical diseases and HIV.

This review discusses the medicinal potential of bioactive metabolites isolated from medicinal plants in Central Africa for the treatment of neglected tropical diseases and HIV. A correlation is established between the biological activities of the isolated compounds and the uses of the plants in traditional medicine. Insight is provided on how secondary metabolites from medicinal plants in Central Africa could be exploited for drug discovery.

Cameroonian medicinal plants: a bioactivity versus ethnobotanical survey and chemotaxonomic classification.

BACKGROUND: In Cameroon herbs are traditionally used to meet health care needs and plans are on the way to integrate traditional medicine in the health care system, even though the plans have not been put into action yet. The country however has a rich biodiversity, with ~8,620 plant species, some of which are commonly used in the treatment of several microbial infections and a range of diseases (malaria, trypanosomiasis, leishmaniasis, diabetes and tuberculosis). METHODS: Our survey consisted in collecting published data from the literature sources, mainly from PhD theses in Cameroonian university libraries and also using the author queries in major natural product and medicinal chemistry journals. The collected data includes plant sources, uses of plant material in traditional medicine, plant families, region of collection of plant material, isolated metabolites and type (e.g. flavonoid, terpenoid, etc.), measured biological activities of isolated compounds, and any comments on significance of isolated metabolites on the chemotaxonomic classification of the plant species. This data was compiled on a excel sheet and analysed. RESULTS: In this study, a literature survey led to the collection of data on 2,700 secondary metabolites, which have been previously isolated or derived from Cameroonian medicinal plants. This represents distinct phytochemicals derived from 312 plant species belonging to 67 plant families. The plant species are investigated in terms of chemical composition with respect to the various plant families. A correlation between the known biological activities of isolated compounds and the ethnobotanical uses of the plants is also attempted. Insight into future direction for natural product search within the Cameroonian forest and Savanna is provided. CONCLUSIONS: It can be verified that a phytochemical search of active secondary metabolites, which is inspired by knowledge from the ethnobotanical uses of medicinal plants could be very vital in a drug discovery program from plant-derived bioactive compounds.

CamMedNP: building the Cameroonian 3D structural natural products database for virtual screening.

BACKGROUND: Computer-aided drug design (CADD) often involves virtual screening (VS) of large compound datasets and the availability of such is vital for drug discovery protocols. We present CamMedNP - a new database beginning with more than 2,500 compounds of natural origin, along with some of their derivatives which were obtained through hemisynthesis. These are pure compounds which have been previously isolated and characterized using modern spectroscopic methods and published by several research teams spread across Cameroon. DESCRIPTION: In the present study, 224 distinct medicinal plant species belonging to 55 plant families from the Cameroonian flora have been considered. About 80 % of these have been previously published and/or referenced in internationally recognized journals. For each compound, the optimized 3D structure, drug-like properties, plant source, collection site and currently known biological activities are given, as well as literature references. We have evaluated the "drug-likeness" of this database using Lipinski's "Rule of Five". A diversity analysis has been carried out in comparison with the ChemBridge diverse database. CONCLUSION: CamMedNP could be highly useful for database screening and natural product lead generation programs.

Beyond Janzen's Hypothesis: How Amphibians That Climb Tropical Mountains Respond to Climate Variation.

Janzen's hypothesis (JH) posits that low thermal variation selects for narrow physiological tolerances, and thus small species distributional ranges and high species turnover along tropical elevational gradients. Although this hypothesis has been intensely revisited, it does not explain how many tropical species may exhibit broad distributions, encompassing altitudinal gradients. Moreover, the physiological responses of tropical species remain largely unknown, limiting our understanding on how they respond to climate variation. To fill these knowledge gaps, we tested a major component of JH, the climate variability hypothesis (CVH), which predicts broader thermal tolerance breadth (Tbr = CTmax - CTmin) with broader temperature variation. Specifically, we sampled populations of five amphibian species distributed in two mountain ranges in Brazil's Atlantic Forest to test how CTmin and CTmax vary along elevational gradients. Since both thermal and water balance traits are pivotal to the evolutionary history of amphibians, we also measured rates of dehydration and rehydration and their relations with thermal tolerances. We found that broader temperature variation with increasing altitude did not always lead to broader Tbr, since changes in CTmin and CTmax were species-specific. In addition, we found that water balance did not show consistent variation with altitude, also with low correlations between hydric and thermal traits. While we also found that highland populations are at lower risk of thermal stress than lowland counterparts, both are living far from their upper thermal limits. As a consequence of intraspecific variation in physiological traits and spatial variation in climate along altitude, responses to climate variation in tropical amphibian species were context-dependent and heterogeneous. Together with recent studies showing thermal tolerances of some tropical amphibians comparable to temperate taxa, our findings highlight that several responses to climate variation in tropical species may not conform to predictions made by either the CVH or other important hypotheses concerning physiological variation. This reinforces the need to overcome geographical bias in physiological data to improve predictions of climate change impacts on biodiversity. (Portuguese abstract) Resumo A Hipótese de Janzen (JH) postula que a baixa variação térmica seleciona tolerâncias fisiológicas estreitas e, portanto, amplitudes restritas de distribuição das espécies e alta substituição de espécies ao longo de gradientes altitudinais tropicais. Embora intensamente revisitada, essa hipótese não explica como espécies tropicais podem exibir amplas distribuições geográficas, abrangendo gradientes altitudinais. Além disso, as respostas fisiológicas das espécies tropicais permanecem amplamente desconhecidas, limitando nossa compreensão sobre como elas respondem à variação climática. Para preencher essas lacunas de conhecimento, testamos um componente importante da JH, a Hipótese de Variabilidade Climática (CVH), que prevê uma maior amplitude de tolerância térmica (Tbr = CTmax - CTmin) quando a variação da temperatura ambiental é mais ampla. Especificamente, amostramos populações de cinco espécies de anfíbios distribuídas em duas cadeias montanhosas na Mata Atlântica do Brasil para testar como CTmin e CTmax variam ao longo de gradientes de altitude. Dado que parâmetros térmicos e do balanço hídrico são fundamentais para a história evolutiva dos anfíbios, também medimos as taxas de desidratação e reidratação e suas relações com as tolerâncias térmicas. Encontramos que uma variação de temperatura ambiental mais ampla com o aumento da altitude nem sempre conduz a uma Tbr mais ampla, uma vez que as mudanças em CTmin e CTmax foram espécie-específicas. Além disso, encontramos que o balanço hídrico não apresentou variação consistente com a mudança de altitude, e que as correlações entre parâmetros hídricos e térmicos foram baixas. Embora populações das maiores altitudes apresentaram menor risco de estresse térmico do que populações da mesma espécie em altitudes menores, ambas estão vivendo longe de seus limites térmicos superiores. Em consequência da variação intraespecífica em parâmetros fisiológicos e variação espacial no clima ao longo da altitude, as respostas à variação climática em espécies de anfíbios tropicais foram contexto-dependentes e heterogêneas. Juntamente com estudos recentes indicando tolerâncias térmicas de alguns anfíbios tropicais comparáveis a de táxons temperados, nossas descobertas destacam que várias respostas à variação climática em espécies tropicais podem não estar de acordo com as previsões feitas pela CVH ou outras hipóteses importantes sobre a variação fisiológica. Isso reforça a necessidade de superar o viés geográfico em dados fisiológicos para aperfeiçoar previsões dos impactos das mudanças climáticas sobre a biodiversidade. (Spanish abstract) Resumen La hipótesis de Janzen (JH) postula que la baja variación térmica selecciona tolerancias fisiológicas estrechas y, por lo tanto, rangos de distribución de especies restringidos con alta rotación de especies a lo largo de gradientes de elevación tropicales. Aunque esta hipótesis ha sido intensamente discutida, no explica cómo várias especies tropicales pueden exhibir distribuciones amplias, abarcando gradientes altitudinales. Además, las respuestas fisiológicas de las especies tropicales siguen siendo bastante desconocidas, lo que limita la comprensión de cómo responden a la variación climática. Para llenar estos vacíos de conocimiento, examinamos un componente importante de JH, la Hipótesis de Variabilidad Climática (CVH), que predice mayor amplitud de tolerancia térmica (Tbr = CTmax - CTmin) cuando la variación de temperatura es más amplia. Específicamente, tomamos muestras de poblaciones de cinco especies de anfibios distribuidas en dos cadenas montañosas en el Bosque Atlántico de Brasil para verificar cómo CTmin y CTmax varían a lo largo de este gradiente de elevación. Dado que los rasgos de equilibrio térmico y hídrico son fundamentales para la historia evolutiva de los anfibios, también medimos las tasas de deshidratación y rehidratación y sus relaciones con las tolerancias térmicas. Encontramos que una variación de temperatura más amplia con el aumento de la altitud no siempre conduce a una Tbr más amplia, ya que los cambios en CTmin y CTmax son específicos de la especie. Además, encontramos que el balance hídrico no muestra variación consistente con la altitud, con bajas correlaciones también entre los rasgos hídricos y térmicos. Si bien las poblaciones de las tierras altas tienen un menor riesgo de estrés térmico que las contrapartes de las tierras bajas, ambas se encuentran lejos de sus límites térmicos superiores. Como consecuencia de la variación intraespecífica en los rasgos fisiológicos y la variación espacial en el clima a lo largo de la altitud, las respuestas a la variación climática en las especies de anfibios tropicales fueron dependientes del contexto y heterogéneas. Junto con estudios recientes que muestran tolerancias térmicas de algunos anfibios tropicales comparables a los taxones de zonas templadas, nuestros hallazgos resaltan que varias respuestas a la variación climática en especies tropicales pueden no ajustarse a las predicciones hechas por el CVH u otras hipótesis importantes sobre la variación fisiológica. Esto refuerza la necesidad de superar el sesgo geográfico en los datos fisiológicos para mejorar las predicciones de los impactos del cambio climático en la biodiversidad.

Bioassay-guided discovery of antibacterial agents: in vitro screening of Peperomia vulcanica, Peperomia fernandopoioana and Scleria striatinux.

BACKGROUND: The global burden of bacterial infections is high and has been further aggravated by increasing resistance to antibiotics. In the search for novel antibacterials, three medicinal plants: Peperomia vulcanica, Peperomia fernandopoioana (Piperaceae) and Scleria striatinux (Cyperaceae), were investigated for antibacterial activity and toxicity. METHODS: Crude extracts of these plants were tested by the disc diffusion method against six bacterial test organisms followed by bio-assay guided fractionation, isolation and testing of pure compounds. The minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations were measured by the microdilution method. The acute toxicity of the active extracts and cytotoxicity of the active compound were performed in mice and mammalian cells, respectively. RESULTS: The diameter of the zones of inhibition (DZI) of the extracts ranged from 7-13 mm on Escherichia coli and Staphylococcus aureus of which the methylene chloride:methanol [1:1] extract of Scleria striatinux recorded the highest activity (DZI = 13 mm). Twenty-nine pure compounds were screened and one, Okundoperoxide, isolated from S. striatinux, recorded a DZI ranging from 10-19 mm on S. aureus. The MICs and MBCs indicated that the Peperomias had broad-spectrum bacteriostatic activity. Toxicity tests showed that Okundoperoxide may have a low risk of toxicity with an LC50 of 46.88 µg/mL. CONCLUSIONS: The antibacterial activity of these plants supports their use in traditional medicine. The pure compound, Okundoperoxide, may yield new antibacterial lead compounds following medicinal chemistry exploration.

3',4'-Dihydro-2'H-spiro[indoline-3,1'-isoquinolin]-2-ones as potential anti-cancer agents: synthesis and preliminary screening.

Both tetrahydroisoquinolines (THIQs) and oxindoles (OXs) display a broad range of biological activities including anti-cancer activity, and are therefore recognized as two privileged scaffolds in drug discovery. In the present study, 24 3',4'-dihydro-2'H-spiro[indoline-3,1'-isoquinolin]-2-ones, designed as molecular hybrids of THIQ and OX, were synthesized and screened in vitro against 59 cell lines in the NCI-60 screen. Twenty compounds displayed weak to moderate inhibition of cell proliferation; among them, three compounds displayed at least 50% inhibition of cell proliferation. The compounds appeared to target primarily renal cell cancer lines; however, leukaemia, melanoma, non-small cell lung cancer, prostate, ovarian and even breast cancer cell lines were also affected. Therefore, this class of spirooxindoles may provide useful leads in the search for new anti-cancer agents.

Spirofused tetrahydroisoquinoline-oxindole hybrids as a novel class of fast acting antimalarial agents with multiple modes of action.

Molecular hybridization of privileged scaffolds may generate novel antiplasmodial chemotypes that display superior biological activity and delay drug resistance. In the present study, we describe the in vitro activities and mode of action of 3',4'-dihydro-2'H-spiro[indoline-3,1'-isoquinolin]-2-ones, a novel class of spirofused tetrahydroisoquinoline-oxindole hybrids, as novel antimalarial agents. Whole cell phenotypic screening of these compounds identified (14b), subsequently named (±)-moxiquindole, as the most potent compound in the current series with equipotent antiplasmodial activity against both chloroquine sensitive and multidrug resistant parasite strains with good selectivity. The compound was active against all asexual stages of the parasite including inhibition of merozoite egress. Additionally, (±)-moxiquindole exhibited significant inhibitory effects on hemoglobin degradation, and disrupted vacuolar lipid dynamics. Taken together, our data confirm the antiplasmodial activity of (±)-moxiquindole, and identify 3'4'-dihydro-2'H-spiro[indoline-3,1'-isoquinolin]-2-ones as a novel class of antimalarial agents with multiple modes of action.

The filamentous phage pIV multimer visualized by scanning transmission electron microscopy.

A family of homomultimeric outer-membrane proteins termed secretins mediates the secretion of large macromolecules such as enzymes and filamentous bacteriophages across bacterial outer membranes to the extracellular milieu. The secretin encoded by filamentous phage f1 was purified. Mass determination of individual molecules by scanning transmission electron microscopy revealed two forms, a unit multimer composed of about 14 subunits and a multimer dimer. The secretin is roughly cylindrical and has an internal diameter of about 80 angstroms, which is large enough to accommodate filamentous phage (diameter of 65 angstroms).

Okundoperoxide, a bicyclic cyclofarnesylsesquiterpene endoperoxide from Scleria striatinux with antiplasmodial activity.

Okundoperoxide (1) was isolated by bioassay-guided fractionation of extracts from Scleria striatinux (syn. S. striatonux). The compound contains a cyclic endoperoxide structural moiety and possesses moderate antimalarial activity.

Structurally simple synthetic 1, 4-disubstituted piperidines with high selectivity for resistant Plasmodium falciparum.

BACKGROUND: Emergence of resistance to artemisinins and some of their combinations in chemotherapy of clinical malaria has intensified the search for novel safe efficacious antimalarial molecules. Fourteen synthetic 1, 4-disubstituted piperidines with simple molecular structures were evaluated in this study. METHODS: Antiplasmodial activity were determined against cultured chloroquine-sensitive 3D7 and resistant Dd2 strains of P. falciparum by in vitro parasite growth inhibition. A primary screen was done to identify active compounds by fluorescence microscopy followed by a secondary screen to determine IC50 and IC90 values of active compounds by the parasite lactate dehydrogenase assay. Cytotoxicity of active compounds was assessed using the MTT/formazan assay and selectivity indices (SIs) determined. Optical densities were analysed to obtain experimental results. RESULTS: The compounds produced 56 to 93% inhibition of parasite growth at 40 µg/mL. Eight compounds (2 ketone, 5 alcohol and one amine analogues) showed high activity (IC50s between 1 and 5 µg/mL). Nine compounds were highly selective for the parasite (SIs = 15 to 182). Three promising (alcohol) analogues were identified: [1-(4-fluorobenzyl) piperidin-4-yl] [4-fluorophenyl] methanol, (7), [1-(3, 4-dichlorobenzyl) piperidin-4-yl] [4- fluorophenyl] methanol (8) and [1-(4-bromobenzyl) piperidin-4-yl] [4- fluorophenyl] methanol (11) which were more active on the resistant strain (IC50 values between 1.03 to 2.52 µg/mL), than the sensitive strain (IC50 values between 2.51 to 4.43 µg/mL). CONCLUSIONS: The alcohol analogues were the most active and most selective for the parasite with three promising hit molecules identified among them, suggesting the hydroxyl group at C-7' in these alcohol analogues is contributing greatly to their antiplasmodial activity. Further exploration of the core structure using chemistry approaches and biological screening including in vivo studies in an animal model of malaria may yield important antimalarial leads.

Site-directed photo-cross-linking of rRNA transcription initiation complexes.

Site-specific photo-cross-linking of the rRNA committed transcription complex was carried out by using 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP-derivatized promoter DNA. Putative TAFIs of 145, 99, 96, and 91 kDa, as well as TATA-binding protein (TBP), were found to specifically photo-cross-link to different positions along the promoter. These had been identified as potential subunits of the fundamental transcription initiation factor TIF-IB (also known as SL1, factor D, and TFID) from Acanthamoeba castellanii by purification to apparent homogeneity. No other polypeptides attributable to the rRNA architectural transcription factor UBF were identified, suggesting that this protein is not part of the committed complex. Scanning transmission electron microscopy of the complexes was used to estimate the mass of the complex and the contour length of the DNA in the complex. This showed that a single molecule of TIF-IB is in each committed complex and that the DNA is not looped around the protein, as would be expected if UBF were in the complex. A circular permutation analysis of DNA bending resulting from TIF-IB binding revealed a 45 +/- 3.1 degrees (n = 14) bend centered 23 bp upstream of the transcription initiation site. This degree of bending and the position of the bend relative to the site of TBP photo-cross-linking are consistent with earlier data showing that the TBP TATA box-binding domain is not utilized in the assembly of the rRNA committed complex (C. A. Radebaugh, J. L. Mathews, G. K. Geiss, F. Liu, J. Wong, E. Bateman, S. Camier, A. Sentenac, and M. R. Paule, Mol. Cell. Biol. 14:597-605, 1994).

In vitro anti-Helicobacter pylori activity of extracts of selected medicinal plants from North West Cameroon.

ETHNOPHARMACOLOGICAL RELEVANCE: Helicobacter pylori, a gram negative microaerophilic bacterium is a major etiological agent in duodenal, peptic and gastric ulcers. The growing problem of antibiotic resistance by the organism demands the search for novel compounds from plant based sources. AIM OF STUDY: The present study is aimed at evaluating the antimicrobial activity of some selected medicinal plants on clinical isolates of H. pylori circulating in Cameroon in a bid to identify potential sources of cheap starting materials for the synthesis of new drugs. MATERIALS AND METHODS: Gastric biopsy samples were obtained from patients presenting with gastroduodenal complications. H. pylori was isolated from the specimens following standard microbiology procedures. The disk diffusion method was used to determine the susceptibility of 15 isolates to ten methanol plant extracts (Ageratum conyzoides, Scleria striatinux, Lycopodium cernua, Acanthus montanus, Eryngium foetidium, Aulutandria kamerunensis, Tapeinachilus ananassae, Euphorbia hirta, Emilia coccinea and Scleria verrucosa). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for the most active plant extracts were also determined by the agar dilution method. Results were analyzed statistically by the Fisher's exact test. RESULTS: All the plants tested demonstrated antimicrobial activity with zone diameters of inhibition ranging from 0-30mm. Of these, A. conyzoides, S. striatinux and L. cernua showed very potent antibacterial activity on the isolates. The lowest MIC and MBC recorded were 0.032mg/mL and 0.098mg/mL respectively. However, the MIC of the extracts ranged from 0.032-1.0mg/mL for S. striatinux; 0.063-0.5mg/mL for L. cernua and 0.063-1.0mg/mL for A. conyzoides. The MBC of the extracts ranged from 0.098-15.0mg/mL for S. striatinux; 0.098-12.5mg/mL for A. conyzoides, and 0.195-12.5mg/mL for L. cernua. The extracts had a wide spectrum of activity. The three most potent extracts possessed significant (P<0.05) inhibitory activities. CONCLUSION: The plant extracts may contain compounds with therapeutic activity.

Antiparasitic Sesquiterpenes from the Cameroonian Spice Scleria striatinux and Preliminary In Vitro and In Silico DMPK Assessment.

The antiparasitic activity and preliminary in vitro and in silico drug metabolism and pharmacokinetic (DMPK) assessment of six isomeric sesquiterpenes (1-6), isolated from the Cameroonian spice Scleria striatinux De Wild (Cyperaceae) is reported. The study was prompted by the observation that two of the compounds (1 and 2) exhibited varying levels of antiparasitic activity on Plasmodium falciparum, Trypanosoma brucei rhodesiense, Trypanosoma cruzi and Leishmania donovani. The in silico method employed a total of 46 descriptors, calculated using Schrödinger QikProp software. 18 of these molecular descriptors that are often used to predict DMPK profiles of drug-like molecules have been selected for discussion. In vitro experimental assessment of metabolic stability made use of human liver microsomes, which was used to correlate theoretical predictions with experimental findings. Overall, the test compounds have been found to have acceptable physicochemical properties and fall within the ranges associated with "drug-like" molecules. Moreover, the compounds exhibited minimal degradation in incubations with human liver microsomes. Although some of these compounds have been reported previously (1, 2, 4 and 5), this is the first report on their antiparasitic activities, as well as assessment of their DMPK profiles. These results have therefore provided a window for further development of this novel class of sesquiterpene molecules as potential antiparasitic drugs.

Isolation and Characterization of Sclerienone C from Scleria striatinux.

Herein, we report the isolation and characterization of sclerienone C, a novel sesquiterpene isolated from the methylene chloride/methanol (1:1) extract of Scleria striatinux that we have deduced to have structure 1. This medicinal spice of Cameroon has been shown to display antimicrobial and antiplasmodial activities. The isolation and purification involved a combination of methods including silica gel column chromatography, Sephadex LH-20, and semi-prep HPLC separations. Structure elucidation was carried-out by means of spectroscopic analysis and comparison with previously isolated sesquiterpene derivatives from the plant.

4-aroylpiperidines and 4-(α-hydroxyphenyl)piperidines as selective sigma-1 receptor ligands: synthesis, preliminary pharmacological evaluation and computational studies.

BACKGROUND: Sigma (σ) receptors are membrane-bound proteins characterised by an unusual promiscuous ability to bind a wide variety of drugs and their high affinity for typical neuroleptic drugs, such as haloperidol, and their potential as alternative targets for antipsychotic agents. Sigma receptors display diverse biological activities and represent potential fruitful targets for therapeutic development in combating many human diseases. Therefore, they present an interesting avenue for further exploration. It was our goal to evaluate the potential of ring opened spipethiane (1) analogues as functional ligands (agonists) for σ receptors by chemical modification. RESULTS: Chemical modification of the core structure of the lead compound, (1), by replacement of the sulphur atom with a carbonyl group, hydroxyl group and 3-bromobenzylamine with the simultaneous presence of 4-fluorobenzoyl replacing the spirofusion afforded novel potent sigma-1 receptor ligands 7a-f, 8a-f and 9d-e. The sigma-1 receptor affinities of 7e, 8a and 8f were slightly lower than that of 1 and their selectivities for this receptor two to threefold greater than that of 1. CONCLUSIONS: It was found that these compounds have higher selectivities for sigma-1 receptors compared to 1. Quantitatitive structure-activity relationship studies revealed that sigma-1 binding is driven by hydrophobic interactions.Graphical abstractIdentified pharmacophore features for sigma binding.

A protein kinase from Dictyostelium discoideum with an unusual acidic repeat domain.

DdKinX codes for 1093 amino acids which are organized in four regions: the N-terminal catalytic domain, a region containing 30% acidic amino acids, tandem repeats of the motif VKVEEPVEE and the C-terminus. Identity with other protein kinases is 25 to 30%. Descendent trees show that DdKinX does not belong to any of the known kinase branches.

The molecular structure of agrobacterium VirE2-single stranded DNA complexes involved in nuclear import.

Nuclear import of DNA is a central event in genetic transformation of plant cells by Agrobacterium tumefaciens. Agrobacterium elicits tumors on plant hosts by transporting a single-stranded (ss) copy of the bacterial transferred DNA (T-DNA) from its Ti (tumor-inducing) plasmid into the plant cell nucleus. Presumably, the process of T-DNA nuclear import is mediated by two agrobacterium proteins, VirD2 and VirE2, which are thought to directly associate with the transported T-DNA. Both proteins have been shown to contain functional nuclear localizations signals (NLS). Recently, VirE2 alone has been shown to actively transport ssDNA into the plant cell nucleus. To understand the process of DNA nuclear import, it is important to know the structure of the transport intermediate. To this end, complexes of VirE2 and ssDNA were analyzed by scanning transmission electron microscopy (STEM). This analysis suggests that VirE2 packages ssDNA into semi-rigid, hollow cylindrical filaments with a telephone cord-like coiled structure. The outer diameter of these complexes is too large to enter the nucleus by diffusion but is within the size exclusion limits of the active nuclear import. Detailed mass analysis of VirE2-ssDNA filaments is presented and a structural model is proposed.

Stoichiometry and domainal organization of the long tail-fiber of bacteriophage T4: a hinged viral adhesin.

The long-tail fibers (LTFs) form part of bacteriophage T4's apparatus for host cell recognition and infection, being responsible for its initial attachment to susceptible bacteria. The LTF has two parts, each approximately 70 to 75 nm long; gp34 (140 kDa) forms the proximal half-fiber, while the distal half-fiber is composed of gp37 (109 kDa), gp36(23 kDa) and gp35 (30 kDa). LTFs have long been thought to be dimers of gp34, gp37 and gp36, with one copy of gp35. We have used mass mapping by scanning transmission electron microscopy (STEM), quantitative SDS-PAGE, and computational sequence analysis to study the structures of purified LTFs and half-fibers of both kinds. These data establish that the LTF is, in fact, trimeric, with a stoichiometry of gp34: gp37: gp36: gp35 = 3:3:3:1. Averaged images of stained and unstained molecules resolve the LTF into a linear stack of 17 domains. At the proximal end is a globular domain of approximately 145 kDa that becomes incorporated into the baseplate. It is followed by a rod-like shaft (33 x 4 mm; 151 kDa) which correlates with a cluster of seven quasi repeats, each 34 to 39 residues long. The proximal half-fiber terminates in three globular domains. The distal half-fiber consists of ten globular domains of variable size and spacing, preceding a needle-like end domain (15 x 2.5 nm; 31 kDa). The LTF is rigid apart from hinges between the two most proximal domains, and between the proximal and distal half-fibers. The latter hinge occurs at a site of local non-equivalence (the "kneecap") at which density, correlated with the presence of gp35, bulges asymmetrically out on one side. Several observations indicate that gp34 participates in the sharing of conserved structural modules among coliphage tail-fiber genes to which gp37 was previously noted to subscribe. Two adjacent globular domains in the proximal half-fiber match a pair of domains in the distal half-fiber, and the rod domain in the proximal half-fiber resembles a similar domain in the T4 short tail-fiber (gp12). Finally, possible structures are considered; combining our data with earlier observations, the most likely conformation for most of the LTF is a three-stranded beta-helix.

Filamentous hemagglutinin of Bordetella pertussis. A bacterial adhesin formed as a 50-nm monomeric rigid rod based on a 19-residue repeat motif rich in beta strands and turns.

The filamentous hemagglutinin (FHA) of Bordetella pertussis is an adhesin that binds the bacteria to cells of the respiratory epithelium in whooping-cough infections. Mature FHA is a 220 kDa secretory protein that is highly immunogenic and has been included in acellular vaccines. We have investigated its structure by combining electron microscopy and circular dichroism spectroscopy (CD) with computational analysis of its amino acid sequence. The FHA molecule is 50 nm in length and has the shape of a horseshoe nail: it has a globular head that appears to consist of two domains; a 35 nm-long shaft that averages 4 nm in width, but tapers slightly from the head end; and a small, flexible, tail. Mass measurements by scanning transmission electron microscopy establish that FHA is a monomer. Its sequence contains two regions of tandem 19-residue pseudo-repeats: the first, of 38 cycles, starts at residue 344; the second, of 13 cycles, starts at residue 1440. The repeat motifs are predicted to consist of short beta-strands separated by beta-turns, and secondary structure measurements by CD support this prediction. We propose a hairpin model for FHA in which the head is composed of the terminal domains; the shaft consists mainly of the repeat regions conformed as amphipathic, hyper-elongated beta-sheets, with their hydrophobic faces apposed; and the tail is composed of the intervening sequence. Further support for the model was obtained by immuno-labeling electron microscopy. The 19-residue repeats of FHA have features in common with the leucine-rich repeats (LRRs) that are present in many eukaryotic proteins, including some adhesion factors. The model is also compared with the two other classes of filamentous proteins that are rich in beta-structure, i.e. viral adhesins and two beta-helical secretory proteins. Our proposed structure implies how the functionally important adhesion sites and epitopes of FHA are distributed: its tripeptide (RGD) integrin-binding site is assigned to the tail; the putative hemagglutination site forms part of the head; and two classes of immunodominant epitopes are assigned to opposite ends of the molecule. Possible mechanisms are discussed for two modes of FHA-mediated adhesion.