Discovery of new organoselenium compounds as antileishmanial agents

We report new organoselenium compounds bearing the sulfonamide moiety as effective inhibitors of the β-isoform of Carbonic Anhydrase from the unicellular parasitic protozoan L. donovani chagasi. All derivatives were evaluated in vitro for their leishmanicidal activities against Leishmania infantum amastigotes along with their cytotoxicities in human THP-1 cells. Compounds 3e-g showed their activity in the low micromolar range with IC₅₀ values spanning from 0.72 to 0.81 µM and selectivity indexes (SI) > 8 (for 3g SI > 30), thus much higher than those observed for the reference drugs miltefosine and edelfosine. This is the first study which reports new selenoderivatives with promising leishmanicidal properties and acting as Carbonic Anhydrase inhibitors too thus paving the way to the development of innovative agents for the treatment of neglected diseases such as leishmaniasis.     

Studies on the antileishmanial properties of the antimicrobial peptides temporin A,B and 1Sa

Given the paucity and toxicity of available drugs for leishmaniasis, coupled with the advent of drug resistance, the discovery of new therapies for this neglected tropical disease is recognised as being of the utmost urgency. As such antimicrobial peptides (AMPs) have been proposed as promising compounds against the causative Leishmania species, insect vector‐borne protozoan parasites. Here the AMP temporins A, B and 1Sa have been synthesised and screened for activity against Leishmania mexicana insect stage promastigotes and mammalian stage amastigotes, a significant cause of human cutaneous disease. In contrast to previous studies with other species the activity of these AMPs against L. mexicana amastigotes was low. This suggests that amastigotes from different Leishmania species display varying susceptibility to peptides from the temporin family, perhaps indicating differences in their surface structure, the proposed target of these AMPs. In contrast, insect stage L. mexicana promastigotes were sensitive to two of the screened temporins which clearly demonstrates the importance of screening AMPs against both forms of the parasite. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

High Content Analysis of Primary Macrophages Hosting Proliferating Leishmania Amastigotes: Application to Anti-leishmanial Drug Discovery

Background/Objectives

Human leishmaniases are parasitic diseases causing severe morbidity and mortality. No vaccine is available and numerous factors limit the use of current therapies. There is thus an urgent need for innovative initiatives to identify new chemotypes displaying selective activity against intracellular Leishmania amastigotes that develop and proliferate inside macrophages, thereby causing the pathology of leishmaniasis.

Methodology/Principal Findings

We have developed a biologically sound High Content Analysis assay, based on the use of homogeneous populations of primary mouse macrophages hosting Leishmania amazonensis amastigotes. In contrast to classical promastigote-based screens, our assay more closely mimics the environment where intracellular amastigotes are growing within acidic parasitophorous vacuoles of their host cells. This multi-parametric assay provides quantitative data that accurately monitors the parasitic load of amastigotes-hosting macrophage cultures for the discovery of leishmanicidal compounds, but also their potential toxic effect on host macrophages. We validated our approach by using a small set of compounds of leishmanicidal drugs and recently published chemical entities. Based on their intramacrophagic leishmanicidal activity and their toxicity against host cells, compounds were classified as irrelevant or relevant for entering the next step in the drug discovery pipeline.

Conclusions/Significance

Our assay represents a new screening platform that overcomes several limitations in anti-leishmanial drug discovery. First, the ability to detect toxicity on primary macrophages allows for discovery of compounds able to cross the membranes of macrophage, vacuole and amastigote, thereby accelerating the hit to lead development process for compounds selectively targeting intracellular parasites. Second, our assay allows discovery of anti-leishmanials that interfere with biological functions of the macrophage required for parasite development and growth, such as organelle trafficking/acidification or production of microbicidal effectors. These data thus validate a novel phenotypic screening assay using virulent Leishmania amastigotes growing inside primary macrophage to identify new chemical entities with bona fide drug potential.     

Gold complexes with benzimidazole derivatives: synthesis,characterization and biological studies

Synthesis, characterization, DFT studies and biological assays of new gold(I) and gold(III) complexes of benzimidazole are reported. Molecular and structural characterizations of the compounds were based on elemental (C, H and N) and thermal (TG–DTA) analyses, and FT-IR and UV–Visible spectroscopic measurements. The structures of complexes were proposed based DFT calculations. The benzimidazole compounds (Lig1 and Lig2) and the gold complexes were tested against three Leishmania species related to cutaneous manifestations of leishmaniasis. The free benzimidazole compounds showed no leishmanicidal activity. On the other hand, the gold(I and III) complexes have shown to possess significant activity against Leishmania in both stages of parasite, and the gold(III) complex with Lig2 exhibited expressive leishmanicidal activity with IC₅₀ values below 5.7 μM. Also, the gold complexes showed high leishmania selectivity. The gold(I) complex with Lig1, for example, is almost 50 times more toxic for the parasite than for macrophages. Besides the leishmanicidal activity, all complexes exhibited toxic effect against SK-Mel 103 and Balb/c 3T3, cancer cells.     

Trypanosoma brucei glycogen synthase kinase-3, a target for anti-trypanosomal drug development: a public-private partnership to identify novel leads

Background

Trypanosoma brucei, the causative agent of Human African Trypanosomiasis (HAT), expresses two proteins with homology to human glycogen synthase kinase 3β (HsGSK-3) designated TbruGSK-3 short and TbruGSK-3 long. TbruGSK-3 short has previously been validated as a potential drug target and since this enzyme has also been pursued as a human drug target, a large number of inhibitors are available for screening against the parasite enzyme. A collaborative industrial/academic partnership facilitated by the World Health Organisation Tropical Diseases Research division (WHO TDR) was initiated to stimulate research aimed at identifying new drugs for treating HAT.

Methodology/Principal Findings

A subset of over 16,000 inhibitors of HsGSK-3 β from the Pfizer compound collection was screened against the shorter of two orthologues of TbruGSK-3. The resulting active compounds were tested for selectivity versus HsGSK-3β and a panel of human kinases, as well as in vitro anti-trypanosomal activity. Structural analysis of the human and trypanosomal enzymes was also performed.

Conclusions/Significance

We identified potent and selective compounds representing potential attractive starting points for a drug discovery program. Structural analysis of the human and trypanosomal enzymes also revealed hypotheses for further improving selectivity of the compounds.     

High throughput screens yield small molecule inhibitors of Leishmania CRK3:CYC6 cyclin-dependent kinase

Background

Leishmania species are parasitic protozoa that have a tightly controlled cell cycle, regulated by cyclin-dependent kinases (CDKs). Cdc2-related kinase 3 (CRK3), an essential CDK in Leishmania and functional orthologue of human CDK1, can form an active protein kinase complex with Leishmania cyclins CYCA and CYC6. Here we describe the identification and synthesis of specific small molecule inhibitors of bacterially expressed Leishmania CRK3:CYC6 using a high throughput screening assay and iterative chemistry. We also describe the biological activity of the molecules against Leishmania parasites.

Methodology/Principal Findings

In order to obtain an active Leishmania CRK3:CYC6 protein kinase complex, we developed a co-expression and co-purification system for Leishmania CRK3 and CYC6 proteins. This active enzyme was used in a high throughput screening (HTS) platform, utilising an IMAP fluorescence polarisation assay. We carried out two chemical library screens and identified specific inhibitors of CRK3:CYC6 that were inactive against the human cyclin-dependent kinase CDK2:CycA. Subsequently, the best inhibitors were tested against 11 other mammalian protein kinases. Twelve of the most potent hits had an azapurine core with structure activity relationship (SAR) analysis identifying the functional groups on the 2 and 9 positions as essential for CRK3:CYC6 inhibition and specificity against CDK2:CycA. Iterative chemistry allowed synthesis of a number of azapurine derivatives with one, compound 17, demonstrating anti-parasitic activity against both promastigote and amastigote forms of L. major. Following the second HTS, 11 compounds with a thiazole core (active towards CRK3:CYC6 and inactive against CDK2:CycA) were tested. Ten of these hits demonstrated anti-parasitic activity against promastigote L. major.

Conclusions/Significance

The pharmacophores identified from the high throughput screens, and the derivatives synthesised, selectively target the parasite enzyme and represent compounds for future hit-to-lead synthesis programs to develop therapeutics against Leishmania species. Challenges remain in identifying specific CDK inhibitors with both target selectivity and potency against the parasite.     

Discovery of new GSK-3β inhibitors through structure-based virtual screening

Glycogen synthase kinase-3β (GSK-3β) is an attractive therapeutic target for human diseases, such as diabetes, cancer, neurodegenerative diseases, and inflammation. Thus, structure-based virtual screening was performed to identify novel scaffolds of GSK-3β inhibitors, and we observed that conserved water molecules of GSK-3β were suitable for virtual screening. We found 14 hits and D1 (IC₅₀ of 0.71?μM) were identified. Furthermore, the neuroprotection activity of D1–D3 was validated on a cellular level. 2D similarity searches were used to find derivatives of high inhibitory compounds and an enriched structure–activity relationship suggested that these skeletons were worthy of study as potent GSK-3β inhibitors.     

Antimicrobial peptides isolated from Phyllomedusa nordestina (Amphibia) alter the permeability of plasma membrane of Leishmania and Trypanosoma cruzi

Nature has provided inspiration for Drug Discovery studies and amphibian secretions have been used as a promising source of effective peptides which could be explored as novel drug prototypes for neglected parasitic diseases as Leishmaniasis and Chagas disease. In this study, we isolated four antimicrobial peptides (AMPs) from Phyllomedusa nordestina secretion, and studied their effectiveness against Leishmania (L.) infantum and Trypanosoma cruzi. The antiparasitic fractions were characterized by mass spectrometry and Edman degradation, leading to the identification of dermaseptins 1 and 4 and phylloseptins 7 and 8. T. cruzi trypomastigotes were susceptible to peptides, showing IC₅₀ values in the range concentration of 0.25–0.68 μM. Leishmania (L.) infantum showed susceptibility to phylloseptin 7, presenting an IC₅₀ value of 10 μM. Except for phylloseptin 7 which moderate showed cytotoxicity (IC₅₀ = 34 μM), the peptides induced no cellular damage to mammalian cells. The lack of mitochondrial oxidative activity of parasites detected by the MTT assay, suggested that peptides were leishmanicidal and trypanocidal. By using the fluorescent probe SYTOX® Green, dermaseptins 1 and 4 and phylloseptins 7 and 8 showed time-dependent plasma membrane permeabilization of T. cruzi; phylloseptin 7 also showed a similar effect in Leishmania parasites. The present study demonstrates for the first time that AMPs target the plasma membrane of Leishmania and T. cruzi, leading to cellular death. Considering the potential of amphibian peptides against protozoan parasites and the reduced mammalian toxicity, they may contribute as scaffolds for drug design studies.     

Inhibition of Leishmania infantum trypanothione reductase by diaryl sulfide derivatives

The study presented here aimed at identifying a new class of compounds acting against Leishmania parasites, the causative agent of Leishmaniasis. For this purpose, the thioether derivatives of our in-house library have been evaluated in whole-cell screening assays in order to determine their in vitro activity against Leishmania protozoan. Among them, promising results have been achieved with compound RDS 777 (6-(sec-butoxy)-2-((3-chlorophenyl)thio)pyrimidin-4-amine) (IC_50?=_?29.43?µM), which is able to impair the mechanism of the parasite defence against the reactive oxygen species by inhibiting the trypanothione reductase (TR) with high efficiency (K_i 0.25?±?0.18?µM). The X-ray structure of L. infantum TR in complex with RDS 777 disclosed the mechanism of action of this compound that binds to the catalytic site and engages in hydrogen bonds the residues more involved in the catalysis, namely Glu466', Cys57 and Cys52, thereby inhibiting the trypanothione binding and avoiding its reduction.     

Plant derived therapeutics for the treatment of Leishmaniasis

Diseases caused by insect borne trypanosomatid parasites are significant, yet remain a neglected public health problem. Leishmania, a unicellular protozoan parasite is the causative organism of Leishmaniasis and is transmitted by female phlebotamine sandflies affecting millions of people worldwide. In the wake of resistance to pentavalent antimonial drugs, new therapeutic alternatives are desirable. The plant kingdom has in the past provided several affordable compounds and this review aims to provide an overview of the current status of available leishmanicidal plant derived compounds that are effective singly or in combination with conventional anti-leishmanial drugs, yet are non toxic to mammalian host cells. Furthermore, delineation of the contributory biochemical mechanisms involved in mediating their effect would help develop new chemotherapeutic approaches.     

Potential of 2-Hydroxy-3-Phenylsulfanylmethyl-[1,4]-Naphthoquinones against Leishmania (L.) infantum: Biological Activity and Structure-Activity Relationships

Naphtoquinones have been used as promising scaffolds for drug design studies against protozoan parasites. Considering the highly toxic and limited therapeutic arsenal, the global negligence with tropical diseases and the elevated prevalence of co-morbidities especially in developing countries, the parasitic diseases caused by various Leishmania species (leishmaniasis) became a significant public health threat in 98 countries. The aim of this work was the evaluation of antileishmanial in vitro potential of thirty-six 2-hydroxy-3-phenylsulfanylmethyl-[1,4]-naphthoquinones obtained by a three component reaction of lawsone, the appropriate aldehyde and thiols adequately substituted, exploiting the in situ generation of o-quinonemethides (o-QM) via the Knoevenagel condensation. The antileishmanial activity of the naphthoquinone derivatives was evaluated against promastigotes and intracellular amastigotes of Leishmania (Leishmania) infantum and their cytotoxicity was verified in mammalian cells. Among the thirty-six compounds, twenty-seven were effective against promastigotes, with IC₅₀ values ranging from 8 to 189 µM; fourteen compounds eliminated the intracellular amastigotes, with IC₅₀ values ranging from 12 to 65 µM. The compounds containing the phenyl groups at R₁ and R₂ and with the fluorine substituent at the phenyl ring at R₂, rendered the most promising activity, demonstrating a selectivity index higher than 15 against amastigotes. A QSAR (quantitative structure activity relationship) analysis yielded insights into general structural requirements for activity of most compounds in the series. Considering the in vitro antileishmanial potential of 2-hydroxy-3-phenylsulfanylmethyl-[1,4]-naphthoquinones and their structure-activity relationships, novel lead candidates could be exploited in future drug design studies for leishmaniasis.     

Leishmania major: activity of tamoxifen against experimental cutaneous leishmaniasis

Leishmaniasis is a family of diseases caused by protozoan parasites of the genus Leishmania. Various Leishmania species can cause human infection, producing a spectrum of clinical manifestations. The current treatments are unsatisfactory, and in absence of a vaccine, there is an urgent need for effective drugs to replace/supplement those currently in use. Recent studies have shown that the antineoplastic drug, tamoxifen, had direct leishmanicidal effect on several Leishmania species in vitro. Moreover, in vivo testing was carried out on some of the species and showed promising results. The authors have carried out the present work to complement previous published studies by investigating in vivo activity of tamoxifen in an experimental model of cutaneous leishmaniasis (CL) caused by Leishmania major. Groups of infected mice were given tamoxifen, orally, at a dose of 20 mg/kg/day for 15 days. Efficacy was assessed clinically, parasitologically, histopathologically by light and transmission electron microscope (TEM). Results showed that untreated infected mice suffered from autoamputation of the inoculated foot pad. However, those which received tamoxifen showed marked improvement of the cutaneous lesions and reduction of parasite burden. TEM of the cutaneous lesions from infected mice revealed the fine structure of normal Leishmania amastigotes, whereas those from infected mice treated with tamoxifen showed considerable changes. All male mice that received tamoxifen showed scrotal swelling with evident histopathological changes in the testes that could seriously compromise fertility of male mice. In conclusion, although tamoxifen causes significant side effects to the male reproductive system in the mouse model, it could provide an alternative to current agents. Results of this study demonstrated in vivo activity of tamoxifen against Leishmania major, thus, suggesting that tamoxifen is a suitable lead for the synthesis of more effective and less toxic antileishmanial derivatives.     

In vitro Leishmanicidal and Cytotoxic Activities of the Glycoalkaloids from Solanum lycocarpum (Solanaceae) Fruits

Leishmaniasis is an infection caused by a protozoan parasite of the genus Leishmania and is the second most prevalent parasitic protozoal disease after malaria in the world. We report the in vitro leishmanicidal activity on promastigote forms of Leishmania amazonensis and cytotoxicity, using LLCMK₂ cells, of the glycoalkaloids from the fruits of Solanum lycocarpum, determined by colorimetric methods. The alkaloidic extract was obtained by acid‐base extraction; solamargine and solasonine were isolated by silica‐gel chromatography, followed by reversed‐phase HPLC final purification. The alkaloidic extract, solamargine, solasonine, as well as the equimolar mixture of the glycoalkaloids solamargine and solasonine displayed leishmanicidal activity against promastigote forms of L. amazonensis, whereas the aglycone solasodine was inactive. After 24 and 72 h of incubation, most of the samples showed lower cytotoxicities (IC₅₀ 6.5 to 124 μM ) as compared to leishmanicidal activity (IC₅₀ 1.1 to 23.6 μM ). The equimolar mixture solamargine/solasonine was the most active with an IC₅₀ value of 1.1 μM , after 72 h. Likewise, solamargine was the most active after 24 h with an IC₅₀ value of 14.4 μM , both in comparison with the positive control amphotericin B.     

Enhanced leishmanicidal activity of cryptopeptide chimeras from the active N1 domain of bovine lactoferrin

Two antimicrobial cryptopeptides from the N1 domain of bovine lactoferrin, lactoferricin (LFcin17–30) and lactoferrampin (LFampin265–284), together with a hybrid version (LFchimera), were tested against the protozoan parasite Leishmania. All peptides were leishmanicidal against Leishmania donovani promastigotes, and LFchimera showed a significantly higher activity over its two composing moieties. Besides, it was the only peptide active on Leishmania pifanoi axenic amastigotes, already showing activity below 10?μM. To investigate their leishmanicidal mechanism, promastigote membrane permeabilization was assessed by decrease of free ATP levels in living parasites, entrance of the vital dye SYTOX Green (MW?=?600?Da) and confocal and transmission electron microscopy. The peptides induced plasma membrane permeabilization and bioenergetic collapse of the parasites. To further clarify the structural traits underlying the increased leishmanicidal activity of LFchimera, the activity of several analogues was assessed. Results revealed that the high activity of these hybrid peptides seems to be related to the order and sequence orientation of the two cryptopeptide moieties, rather than to their particular linkage through an additional lysine, as in the initial LFchimera. The incorporation of both antimicrobial cryptopeptide motifs into a single linear sequence facilitates chemical synthesis and should help in the potential clinical application of these optimized analogues.     

Computer-aided molecular design of pyrazolotriazines targeting glycogen synthase kinase 3

Numerous studies have highlighted the implications of the glycogen synthase kinase 3 (GSK-3) in several processes associated with Alzheimer’s disease (AD). Therefore, GSK-3 has become a crucial therapeutic target for the treatment of this neurodegenerative disorder. Hereby, we report the design and multistep synthesis of ethyl 4-oxo-pyrazolo[4,3-d][1–3]triazine-7-carboxylates and their biological evaluation as GSK-3 inhibitors. Molecular modelling studies allow us to develop this new scaffold optimising the chemical structure. Potential binding mode determination in the enzyme and the analysis of the key features in the catalytic site are also described. Furthermore, the ability of pyrazolotriazinones to cross the blood–brain barrier (BBB) was evaluated by passive diffusion and those who showed great GSK-3 inhibition and permeation to the central nervous system (CNS) showed neuroprotective properties against tau hyperphosphorylation in a cell-based model. These new brain permeable pyrazolotriazinones may be used for key in vivo studies and may be considered as new leads for further optimisation for the treatment of AD.     

Protease Inhibitors. Part 2. Weakly Basic Thrombin Inhibitors Incorporating Sulfonyl-Aminoguanidine Moieties as S1 Anchoring Groups: Synthesis and Structure-Activity Correlations

Abstract

Two series of derivatives have been prepared and assayed as inhibitors of two physiologically relevant serine proteases, human thrombin and human trypsin. The first series includes alkyl-/ aralkyl-/aryl- and hetarylsulfonyl-aminoguanidines. It was thus observed that sulfanilyl-aminoguanidine possesses moderate but intrinsically selective thrombin inhibitory properties, with K_i values around 90 and 1400 nM against thrombin and trypsin respectively. Further elaboration of this molecule afforded compounds that inhibited thrombin with K_i values in the range 10–50 nM, whereas affinity for trypsin remained relatively low. Such compounds were obtained either by attaching benzyloxycarbonyl- or 4-toluenesulfonylureido-protected amino acids (such as D-Phe, L-Pro) or dipeptides (such as Phe-Pro, Gly-His, β-Ala-His or Pro-Gly) to the N-4 atom of the lead molecule, sulfanilyl-aminoguanidine, or by attaching substituted-pyridinium-propylcarboxamido moieties to this lead. Thus, this study brings novel insights regarding a novel non-basic S1 anchoring moiety (i, e., SO₂NHNHC(=NH)NH₂), and new types of peptidomimetic scaffolds obtained by incorporating tosylureido-amino acids/pyridinium-substituted-GABA moieties in the hydrophobic binding site(s). Structure-activity correlations of the new serine protease inhibitors are also discussed based on a QSAR model described previously for a large series of structurally-related derivatives (Supuran et al. (1999) J. Med. Chem., in press).     

Design,synthesis, and antiprotozoal evaluation of new 2,4-bis[(substituted-aminomethyl)phenyl]quinoline, 1,3-bis[(substituted-aminomethyl)phenyl]isoquinoline and 2,4-bis[(substituted-aminomethyl)phenyl]quinazoline derivatives

Abstract

A series of new 2,4-bis[(substituted-aminomethyl)phenyl]quinoline, 1,3-bis[(substituted-aminomethyl)phenyl]isoquinoline, and 2,4-bis[(substituted-aminomethyl)phenyl]quinazoline derivatives was designed, synthesised, and evaluated in?vitro against three protozoan parasites (Plasmodium falciparum, Leishmania donovani, and Trypanosoma brucei brucei). Biological results showed antiprotozoal activity with IC₅₀ values in the µM range. In addition, the in?vitro cytotoxicity of these original molecules was assessed with human HepG2 cells. The quinoline 1c was identified as the most potent antimalarial candidate with a ratio of cytotoxic to antiparasitic activities of 97 against the P. falciparum CQ-sensitive strain 3D7. The quinazoline 3h was also identified as the most potent trypanosomal candidate with a selectivity index (SI) of 43 on T. brucei brucei strain. Moreover, as the telomeres of the parasites P. falciparum and Trypanosoma are possible targets of this kind of nitrogen heterocyclic compounds, we have also investigated stabilisation of the Plasmodium and Trypanosoma telomeric G-quadruplexes by our best compounds through FRET melting assays.     

Synthesis of benzimidazole-linked-1,3,4-oxadiazole carboxamides as GSK-3β inhibitors with in vivo antidepressant activity

Recent findings of potential implications of glycogen synthase kinase-3β (GSK-3β) dysfunction in psychiatric disorders like depression, have increased focus for development of GSK-3β inhibitors with possible anti-depressant activity. Keeping this in view, we synthesized a series of benzimidazole-linked-1,3,4-oxadiazole carboxamides and evaluated them for in vitro GSK-3β inhibition. Active compounds were investigated for in vivo antidepressant activity in Wistar rats. Docking studies of active compounds have also been performed. Among nineteen compounds synthesized, compounds 7a, 7r, 7j, and 7d exhibited significant potency against GSK-3β in sub-micromolar range with IC₅₀ values of 0.13 μM, 0.14 μM, 0.20 μM, 0.22 μM respectively and significantly reduced immobility time (antidepressant-like activity) in rats compared to control group. Docking study showed key interactions of these compounds with GSK-3β. These compounds may thus serve as valuable candidates for subsequent development of effective drugs against depression and related disorders.     

Drosophila melanogaster: a model organism for controlling Dipteran vectors and pests

Abstract

Beta-carbonic anhydrases (β-CAs) have been recently reported to be present in many protozoan and metazoan species, whereas it is absent in mammals. In this review, we introduce β-CA from Drosophila melanogaster as a model enzyme for pesticide development. These enzymes can be targeted with various enzyme inhibitors, which can have deleterious effects on pathogenic and other harmful organisms. Therefore, β-CAs represent a new potential target to fight against Dipteran vectors and pests relevant to medicine, veterinary medicine, and agriculture.