Structure- and ligand-based drug design approaches for neglected tropical diseases

Drug discovery has moved toward more rational strategies based on our increasing understanding of the fundamental principles of protein-ligand interactions. Structure( SBDD) and ligand-based drug design (LBDD) approaches bring together the most powerful concepts in modern chemistry and biology, linking medicinal chemistry with structural biology. The definition and assessment of both chemical and biological space have revitalized the importance of exploring the intrinsic complementary nature of experimental and computational methods in drug design. Major challenges in this field include the identification of promising hits and the development of high-quality leads for further development into clinical candidates. It becomes particularly important in the case of neglected tropical diseases (NTDs) that affect disproportionately poor people living in rural and remote regions worldwide, and for which there is an insufficient number of new chemical entities being evaluated owing to the lack of innovation and R&D investment by the pharmaceutical industry. This perspective paper outlines the utility and applications of SBDD and LBDD approaches for the identification and design of new small-molecule agents for NTDs.

Evaluation of HIV-1 resistance to antiretroviral drugs among 150 patients after six months of therapeutic interruption

Most of the antiretroviral (ARV) studies in Brazil have been reported in treatment-experienced and naive patients rather than in the setting of treatment interruption (TI). In this study, we analysed reasons given for TI and resistance mutations occurring in 150 HIV-1-infected patients who underwent TI. Of the patients analysed, 110 (73.3%) experienced TI following medical advice, while the remaining patients stopped antiretroviral therapy (ART) of their own accord. The main justifications for TI were: ARV-related toxicities (38.7%), good laboratory parameters (30%) and poor adherence (20%). DNA sequencing of the partial pol gene was successful in 137 (91.3%) patients, of whom 38 (27.7%) presented mutations conferring ARV resistance. A higher viral load prior to TI correlated with drug resistance (P < 0.05). Our results demonstrate that there are diverse rationales for TI and that detection of resistant strains during TI most likely indicates a fitter virus than the wild type. High viral loads coupled with unprotected sex in this group could increase the likelihood of transmission of drug-resistant virus. Thus, treating physicians should be alerted to this problem when the use of ARVs is interrupted.

Evaluation of inadequate anti-retroviral treatment in patients with HIV/AIDS

Introduction: Since the emergence of antiretroviral therapy, the survival of patients infected with human immunodeficiency virus has increased. Non-adherence to this therapy is directly related to treatment failure, which allows the emergence of resistant viral strains. Methods: A retrospective descriptive study of the antiretroviral dispensing records of 229 patients from the Center for Health Care, University Hospital, Federal University of Juiz de Fora, Brazil, was conducted between January and December 2009. Results: The study aimed to evaluate patient compliance and determine if there was an association between non-adherence and the therapy. Among these patients, 63.8% were men with an average age of 44.0 +/- 9.9 years. The most used treatment was a combination of 2 nucleoside reverse transcriptase inhibitors with 1 non-nucleoside reverse transcriptase inhibitor (55.5%) or with 2 protease inhibitors (28.8%). It was found that patients taking lopinavir/ritonavir with zidovudine and lamivudine had a greater frequency of inadequate treatment than those taking atazanavir with zidovudine and lamivudine (85% and 83.3%, respectively). Moreover, when the combination of zidovudine/lamivudine was used, the patients were less compliant (chi(2) = 4.468, 1 degree of freedom, p = 0.035). Conclusions: The majority of patients failed to correctly adhere to their treatment; therefore, it is necessary to implement strategies that lead to improved compliance, thus ensuring therapeutic efficacy and increased patient survival.

Structure of the haptoglobin-haemoglobin complex

Red cell haemoglobin is the fundamental oxygen-transporting molecule in blood, but also a potentially tissue-damaging compound owing to its highly reactive haem groups. During intravascular haemolysis, such as in malaria and haemoglobinopathies(1), haemoglobin is released into the plasma, where it is captured by the protective acute-phase protein haptoglobin. This leads to formation of the haptoglobin-haemoglobin complex, which represents a virtually irreversible non-covalent protein-protein interaction(2). Here we present the crystal structure of the dimeric porcine haptoglobin-haemoglobin complex determined at 2.9 angstrom resolution. This structure reveals that haptoglobin molecules dimerize through an unexpected beta-strand swap between two complement control protein (CCP) domains, defining a new fusion CCP domain structure. The haptoglobin serine protease domain forms extensive interactions with both the alpha- and beta-subunits of haemoglobin, explaining the tight binding between haptoglobin and haemoglobin. The haemoglobin-interacting region in the alpha beta dimer is highly overlapping with the interface between the two alpha beta dimers that constitute the native haemoglobin tetramer. Several haemoglobin residues prone to oxidative modification after exposure to haem-induced reactive oxygen species are buried in the haptoglobin-haemoglobin interface, thus showing a direct protective role of haptoglobin. The haptoglobin loop previously shown to be essential for binding of haptoglobin-haemoglobin to the macrophage scavenger receptor CD163 (ref. 3) protrudes from the surface of the distal end of the complex, adjacent to the associated haemoglobin alpha-subunit. Small-angle X-ray scattering measurements of human haptoglobin-haemoglobin bound to the ligand-binding fragment of CD163 confirm receptor binding in this area, and show that the rigid dimeric complex can bind two receptors. Such receptor cross-linkage may facilitate scavenging and explain the increased functional affinity of multimeric haptoglobin-haemoglobin for CD163 (ref. 4).

Body changes: antiretroviral therapy and lipodystrophy syndrome in people living with HIV/aids

Objective: to identify the different types of morphological alterations from lipodystrophy syndrome (LS) in outpatients and relate them to the therapeutic regimen used. Method: a cross-sectional study which recruited 60 patients with HIV and LS and 79 without LS, who consented to interview and data collection from their medical notes. Results: the region most affected by lipoatrophy was the face; by lipohypertrophy, the abdomen, and by the mixed form, the alterations to the abdomen, face, and upper and lower limbs. Conclusion: among the therapeutic regimens, that comprised of zidovudine, lamivudine and efavirenz seemed to protect against LS. Nursing can act in the early identification of the changes, as well as providing guidance and support for patients affected by the changes in their body image.

Analysis of Hepatitis C Virus Intrahost Diversity across the Coding Region by Ultradeep Pyrosequencing

Hepatitis C virus (HCV) is the leading cause of liver disease worldwide. In this study, we analyzed four treatment-naive patients infected with subtype 1a and performed Roche/454 pyrosequencing across the coding region. We report the presence of low-level drug resistance mutations that would most likely have been missed using conventional sequencing methods. The approach described here is broadly applicable to studies of viral diversity and could help to improve the efficacy of direct-acting antiviral agents (DAA) in the treatment of HCV-infected patients.

A tellurium-based cathepsin B inhibitor: Molecular structure, modelling, molecular docking and biological evaluation

The crystallographically determined structure of biologically active 4,4-dichloro-1,3-diphenyl-4-telluraoct-2-en-1-one, 3, shows the coordination geometry for Te to be distorted psi-pentagonal bipyramidal based on a C2OCl3(lone pair) donor set. Notable is the presence of an intramolecular axial Te center dot center dot center dot O (carbonyl) interaction, a design element included to reduce hydrolysis. Raman and molecular modelling studies indicate the persistence of the Te center dot center dot center dot O(carbonyl) interaction in the solution (CHCl3) and gasphases, respectively. Docking studies of 3' (i.e. original 3 less one chloride) with Cathepsin B reveals a change in the configuration about the vinyl C = C bond. i.e. to E from Z (crystal structure). This isomerism allows the optimisation of interactions in the complex which features a covalent Te-SGCys29 bond. Crucially, the E configuration observed for 3' allows for the formation of a hypervalent Te center dot center dot center dot O interaction as well as an O center dot center dot center dot H-O hydrogen bond with the Gly27 and Glu122 residues, respectively. Additional stabilisation is afforded by a combination of interactions spanning the S1, S2, S1' and S2' sub-sites of Cathepsin B. The greater experimental inhibitory activity of 3 compared with analogues is rationalised by the additional interactions formed between 3' and the His110 and His111 residues in the occluding loop, which serve to hinder the entrance to the active site. (C) 2012 Elsevier B.V. All rights reserved.

Design and synthesis of new (E)-cinnamic N-acylhydrazones as potent antitrypanosomal agents

We report herein the synthesis and trypanocidal profile of new (E)-cinnamic N-acylhydrazones (NAHs) designed by exploiting molecular hybridization between the potent cruzain inhibitors (E)-1-(benzo[d] 11,3)dioxol-5-yl)-3-(4-bromophenyl)prop-2-en-1-one and (E)-3-hydroxy-N'-((2-hydroxynaphthalen-1-yl)methylene)-7-methoxy-2-naphthohydrazide. These derivatives were evaluated against both amastigote and trypomastigote forms of Trypanosoma cruzi and lead us to identify two compounds that were approximately two times more active than the reference drug, benznidazole, and with good cytotoxic index. Although designed as cruzain inhibitors, the weak potency displayed by the best cinnamyl NAH derivatives indicated that another mechanism of action was likely responsible for their trypanocide action. (C) 2012 Elsevier Masson SAS. All rights reserved.

Structural Investigation of Anti-Trypanosoma cruzi 2-Iminothiazolidin-4-ones Allows the Identification of Agents with Efficacy in Infected Mice

We modified the thiazolidinic ring at positions N3, C4, and C5, yielding compounds 6-24. Compounds with a phenyl at position N3, 15-19, 22-24, exhibited better inhibitory properties for cruzain and against the parasite than 2-iminothiazolidin-4-one S. We were able to identify one high-efficacy trypanocidal compound, 2-minothiazolidin-4-one 18, which inhibited the activity of cruzain and the proliferation of epirnastigotes and was cidal for trypomastigotes but was not toxic for splenocytes. Having located some of the structural determinants of the trypanocidal properties, we subsequently wished to determine if the exchange of the thiazolidine for a thiazole ring leaves the functional properties unaffected. We therefore tested thiazoles 26-45 and observed that they did not inhibit cruzain, but they exhibited trypanocidal effects. Parasite development was severely impaired when treated with 18, thus reinforcing the notion that this class of heterocycles can lead to useful cidal agents for Chagas disease.

Recombinant expression, localization and in vitro inhibition of midgut cysteine peptidase (Sl-CathL) from sugarcane weevil, Sphenophorus levis

A cDNA coding for a digestive cathepsin L, denominated Sl-CathL, was isolated from a cDNA library of Sphenophorus levis larvae, representing the most abundant EST (10.49%) responsible for proteolysis in the midgut. The open reading frame of 972 bp encodes a preproenzyme similar to midgut cathepsin L-like enzymes in other coleopterans. Recombinant Sl-CathL was expressed in Pichia pastoris, with molecular mass of about 42 kDa. The recombinant protein was catalytically activated at low pH and the mature enzyme of 39 kDa displayed thermal instability and maximal activity at 37 degrees C and pH 6.0. Immunocytochemical analysis revealed Sl-CathL production in the midgut epithelium and secretion from vesicles containing the enzyme into the gut lumen, confirming an important role for this enzyme in the digestion of the insect larvae. The expression profile identified by RT-PCR through the biological cycle indicates that Sl-CathL is mainly produced in larval stages, with peak expression in 30-day-old larvae. At this stage, the enzyme is 1250-fold more expressed than in the pupal fase, in which the lowest expression level is detected. This enzyme is also produced in the adult stage, albeit in lesser abundance, assuming the presence of a different array of enzymes in the digestive system of adults. Tissue-specific analysis revealed that Sl-CathL mRNA synthesis occurs fundamentally in the larval midgut, thereby confirming its function as a digestive enzyme, as detected in immunolocalization assays. The catalytic efficiency of the purified recombinant enzyme was calculated using different substrates (Z-Leu-Arg-AMC, Z-Arg-Arg-AMC and Z-Phe-Arg-AMC) and rSl-CathL exhibited hydrolysis preference for Z-Leu-Arg-AMC (k(cat)/K-m = 37.53 mM S-1), which is similar to other insect cathepsin L-like enzymes. rSl-CathL activity inhibition assays were performed using four recombinant sugarcane cystatins. rSl-CathL was strongly inhibited by recombinant cystatin CaneCPI-4 (K-i = 0.196 nM), indicating that this protease is a potential target for pest control. (C) 2011 Elsevier Ltd. All rights reserved.

Molecular cloning and insecticidal effect of Inga laurina trypsin inhibitor on Diatraea saccharalis and Heliothis virescens

Native Inga laurina (Fabaceae) trypsin inhibitor (ILTI) was tested for anti-insect activity against Diatraea saccharalis and Heliothis virescens larvae. The addition of 0.1% ILTI to the diet of D. saccharalis did not alter larval survival but decreased larval weight by 51%. The H. virescens larvae that were fed a diet containing 0.5% ILTI showed an 84% decrease in weight. ILTI was not digested by the midgut proteinases of either species of larvae. The trypsin levels were reduced by 55.3% in the feces of D. saccharalis and increased by 24.1% in the feces of H. virescens. The trypsin activity in both species fed with ILTI was sensitive to the inhibitor, suggesting that no novel proteinase resistant to ILTI was induced. Additionally, ILTI exhibited inhibitory activity against the proteinases present in the larval midgut of different species of Lepidoptera. The organization of the ilti gene was elucidated by analyzing its corresponding genomic sequence. The recombinant ILTI protein (reILTI) was expressed and purified, and its efficacy was evaluated. Both native ILTI and reILTI exhibited a similar strong inhibitory effect on bovine trypsin activity. These results suggest that ILTI presents insecticidal properties against both insects and may thus be a useful tool in the genetic engineering of plants. (c) 2012 Elsevier Inc. All rights reserved.

EVALUATION OF IN VITRO AND IN VIVO EFFECTS OF SEMIPURIFIED PROTEINASE INHIBITORS FROM THEOBROMA SEEDS ON MIDGUT PROTEASE ACTIVITY OF LEPIDOPTERAN PEST INSECTS

We have characterized in vitro and in vivo effects of trypsin inhibitors from Theobroma seeds on the activity of trypsin- and chymotrypsin-like proteins from Lepidopteran pest insects. The action of semipurified trypsin inhibitors from Theobroma was evaluated by the inhibition of bovine trypsin and chymotrypsin activities determined by the hydrolysis of N-Benzoyl-DL-Arginine-p-Nitroanilide (BAPA) and N-Succinyl-Ala-Ala-Pho-Phe p-Nitroanilide (S-(Ala)2ProPhe-pNA). Proteinase inhibitor activities from Theobroma cacao and T. obovatum seeds were the most effective in inhibiting trypsin-like proteins, whereas those from T. obovatum and T. sylvestre were the most efficient against chymotrypsin-like proteins. All larvae midgut extracts showed trypsin-like proteolytic activities, and the putative trypsin inhibitors from Theobroma seeds significantly inhibited purified bovine trypsin. With respect to the influence of Theobroma trypsin inhibitors on intact insects, the inclusion of T. cacao extracts in artificial diets of velvet bean caterpillars (Anticarsia gemmatalis) and sugarcane borer (Diatraea saccharalis) produced a significant increase in the percentage of adult deformation, which is directly related to both the survival rate of the insects and oviposition.

Lufaxin, a Novel Factor Xa Inhibitor From the Salivary Gland of the Sand Fly Lutzomyia longipalpis Blocks Protease-Activated Receptor 2 Activation and Inhibits Inflammation and Thrombosis In Vivo

Objective-Blood-sucking arthropods' salivary glands contain a remarkable diversity of antihemostatics. The aim of the present study was to identify the unique salivary anticoagulant of the sand fly Lutzomyia longipalpis, which remained elusive for decades. Methods and Results-Several L. longipalpis salivary proteins were expressed in human embryonic kidney 293 cells and screened for inhibition of blood coagulation. A novel 32.4-kDa molecule, named Lufaxin, was identified as a slow, tight, noncompetitive, and reversible inhibitor of factor Xa (FXa). Notably, Lufaxin's primary sequence does not share similarity to any physiological or salivary inhibitors of coagulation reported to date. Lufaxin is specific for FXa and does not interact with FX, Dansyl-Glu-Gly-Arg-FXa, or 15 other enzymes. In addition, Lufaxin blocks prothrombinase and increases both prothrombin time and activated partial thromboplastin time. Surface plasmon resonance experiments revealed that FXa binds Lufaxin with an equilibrium constant approximate to 3 nM, and isothermal titration calorimetry determined a stoichiometry of 1:1. Lufaxin also prevents protease-activated receptor 2 activation by FXa in the MDA-MB-231 cell line and abrogates edema formation triggered by injection of FXa in the paw of mice. Moreover, Lufaxin prevents FeCl3-induced carotid artery thrombus formation and prolongs activated partial thromboplastin time ex vivo, implying that it works as an anticoagulant in vivo. Finally, salivary gland of sand flies was found to inhibit FXa and to interact with the enzyme. Conclusion-Lufaxin belongs to a novel family of slow-tight FXa inhibitors, which display antithrombotic and anti-inflammatory activities. It is a useful tool to understand FXa structural features and its role in prohemostatic and proinflammatory events. (Arterioscler Thromb Vasc Biol. 2012;32:2185-2196.)