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1.
PLoS Negl Trop Dis ; 15(10): e0009819, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34606502

RESUMO

Chagas disease (CD), caused by the parasite Trypanosoma cruzi, is one of nineteen neglected tropical diseases. CD is a vector-borne disease transmitted by triatomines, but CD can also be transmitted through blood transfusions, organ transplants, T. cruzi-contaminated food and drinks, and congenital transmission. While endemic to the Americas, T. cruzi infects 7-8 million people worldwide and can induce severe cardiac symptoms including apical aneurysms, thromboembolisms and arrhythmias during the chronic stage of CD. However, these cardiac clinical manifestations and CD pathogenesis are not fully understood. Using spatial metabolomics (chemical cartography), we sought to understand the localized impact of chronic CD on the cardiac metabolome of mice infected with two divergent T. cruzi strains. Our data showed chemical differences in localized cardiac regions upon chronic T. cruzi infection, indicating that parasite infection changes the host metabolome at specific sites in chronic CD. These sites were distinct from the sites of highest parasite burden. In addition, we identified acylcarnitines and glycerophosphocholines as discriminatory chemical families within each heart region, comparing infected and uninfected samples. Overall, our study indicated global and positional metabolic differences common to infection with different T. cruzi strains and identified select infection-modulated pathways. These results provide further insight into CD pathogenesis and demonstrate the advantage of a systematic spatial perspective to understand infectious disease tropism.

2.
Pathogens ; 10(5)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068119

RESUMO

Cutaneous leishmaniasis (CL) is the most common disease form caused by a Leishmania parasite infection and considered a neglected tropical disease (NTD), affecting 700,000 to 1.2 million new cases per year in the world. Leishmania major is one of several different species of the Leishmania genus that can cause CL. Current CL treatments are limited by adverse effects and rising resistance. Studying disease metabolism at the site of infection can provide knowledge of new targets for host-targeted drug development. In this study, tissue samples were collected from mice infected in the ear or footpad with L. major and analyzed by untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS). Significant differences in overall metabolite profiles were noted in the ear at the site of the lesion. Interestingly, lesion-adjacent, macroscopically healthy sites also showed alterations in specific metabolites, including selected glycerophosphocholines (PCs). Host-derived PCs in the lower m/z range (m/z 200-799) showed an increase with infection in the ear at the lesion site, while those in the higher m/z range (m/z 800-899) were decreased with infection at the lesion site. Overall, our results expanded our understanding of the mechanisms of CL pathogenesis through host metabolism and may lead to new curative measures against infection with Leishmania.

3.
Bioorg Med Chem ; 41: 116213, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33992862

RESUMO

Chagas disease and Human African Trypanosomiasis (HAT) are caused by Trypanosoma cruzi and T. brucei parasites, respectively. Cruzain (CRZ) and Rhodesain (RhD) are cysteine proteases that share 70% of identity and play vital functions in these parasites. These macromolecules represent promising targets for designing new inhibitors. In this context, 26 CRZ and 5 RhD 3D-structures were evaluated by molecular redocking to identify the most accurate one to be utilized as a target. Posteriorly, a virtual screening of a library containing 120 small natural and nature-based compounds was performed on both of them. In total, 14 naphthoquinone-based analogs were identified, synthesized, and biologically evaluated. In total, five compounds were active against RhD, being three of them also active on CRZ. A derivative of 1,4-naphthoquinonepyridin-2-ylsulfonamide was found to be the most active molecule, exhibiting IC50 values of 6.3 and 1.8 µM for CRZ and RhD, respectively. Dynamic simulations at 100 ns demonstrated good stability and do not alter the targets' structures. MM-PBSA calculations revealed that it presents a higher affinity for RhD (-25.3 Kcal mol-1) than CRZ, in which van der Waals interactions were more relevant. A mechanistic hypothesis (via C3-Michael-addition reaction) involving a covalent mode of inhibition for this compound towards RhD was investigated by covalent molecular docking and DFT B3LYP/6-31 + G* calculations, exhibiting a low activation energy (ΔG‡) and providing a stable product (ΔG), with values of 7.78 and - 39.72 Kcal mol-1, respectively; similar to data found in the literature. Nevertheless, a reversibility assay by dilution revealed that JN-11 is a time-dependent and reversible inhibitor. Finally, this study applies modern computer-aided techniques to identify promising inhibitors from a well-known chemical class of natural products. Then, this work could inspire other future studies in the field, being useful for designing potent naphthoquinones as RhD inhibitors.

4.
Expert Rev Anti Infect Ther ; : 1-15, 2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-33929276

RESUMO

Introduction: Acanthamoeba encompasses several species of free-living ameba encountered commonly throughout the environment. Unfortunately, these species of ameba can cause opportunistic infections that result in Acanthamoeba keratitis, granulomatous amebic encephalitis, and occasionally systemic infection.Areas covered: This review discusses relevant literature found through PubMed and Google scholar published as of January 2021. The review summarizes current common Acanthamoeba keratitis treatments, drug discovery methodologies available for screening potential anti-Acanthamoeba compounds, and the anti-Acanthamoeba activity of various azole antifungal agents.Expert opinion: While several biguanide and diamidine antimicrobial agents are available to clinicians to effectively treat Acanthamoeba keratitis, no singular treatment can effectively treat every Acanthamoeba keratitis case.Efforts to identify new anti-Acanthamoeba agents include trophozoite cell viability assays, which are amenable to high-throughput screening. Cysticidal assays remain largely manual and would benefit from further automation development. Additionally, the existing literature on the effectiveness of various azole antifungal agents for treating Acanthamoeba keratitis is incomplete or contradictory, suggesting the need for a systematic review of all azoles against different pathogenic Acanthamoeba strains.

5.
ACS Chem Biol ; 16(4): 642-650, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33787221

RESUMO

Host-cell cysteine proteases play an essential role in the processing of the viral spike protein of SARS coronaviruses. K777, an irreversible, covalent inactivator of cysteine proteases that has recently completed phase 1 clinical trials, reduced SARS-CoV-2 viral infectivity in several host cells: Vero E6 (EC50< 74 nM), HeLa/ACE2 (4 nM), Caco-2 (EC90 = 4.3 µM), and A549/ACE2 (<80 nM). Infectivity of Calu-3 cells depended on the cell line assayed. If Calu-3/2B4 was used, EC50 was 7 nM, but in the ATCC Calu-3 cell line without ACE2 enrichment, EC50 was >10 µM. There was no toxicity to any of the host cell lines at 10-100 µM K777 concentration. Kinetic analysis confirmed that K777 was a potent inhibitor of human cathepsin L, whereas no inhibition of the SARS-CoV-2 cysteine proteases (papain-like and 3CL-like protease) was observed. Treatment of Vero E6 cells with a propargyl derivative of K777 as an activity-based probe identified human cathepsin B and cathepsin L as the intracellular targets of this molecule in both infected and uninfected Vero E6 cells. However, cleavage of the SARS-CoV-2 spike protein was only carried out by cathepsin L. This cleavage was blocked by K777 and occurred in the S1 domain of the SARS-CoV-2 spike protein, a different site from that previously observed for the SARS-CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of cathepsin L-mediated viral spike protein processing.


Assuntos
Antivirais/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Fenilalanina/farmacologia , Piperazinas/farmacologia , SARS-CoV-2/efeitos dos fármacos , Compostos de Tosil/farmacologia , Animais , Catepsina L/antagonistas & inibidores , Catepsina L/metabolismo , Linhagem Celular Tumoral , Chlorocebus aethiops , Humanos , Testes de Sensibilidade Microbiana , Domínios Proteicos , Proteólise , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Internalização do Vírus/efeitos dos fármacos
6.
Pathogens ; 10(1)2021 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-33466870

RESUMO

Filarial worms cause multiple debilitating diseases in millions of people worldwide, including river blindness. Currently available drugs reduce transmission by killing larvae (microfilariae), but there are no effective cures targeting the adult parasites (macrofilaricides) which survive and reproduce in the host for very long periods. To identify effective macrofilaricides, we carried out phenotypic screening of a library of 2121 approved drugs for clinical use against adult Brugia pahangi and prioritized the hits for further studies by integrating those results with a computational prioritization of drugs and associated targets. This resulted in the identification of 18 hits with anti-macrofilaricidal activity, of which two classes, azoles and aspartic protease inhibitors, were further expanded upon. Follow up screening against Onchocerca spp. (adult Onchocerca ochengi and pre-adult O. volvulus) confirmed activity for 13 drugs (the majority having IC50 < 10 µM), and a counter screen of a subset against L. loa microfilariae showed the potential to identify selective drugs that prevent adverse events when co-infected individuals are treated. Stage specific activity was also observed. Many of these drugs are amenable to structural optimization, and also have known canonical targets, making them promising candidates for further optimization that can lead to identifying and characterizing novel anti-macrofilarial drugs.

7.
PLoS One ; 16(1): e0246084, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33503071

RESUMO

The Nicaraguan COVID-19 situation is exceptional for Central America. The government restricts testing and testing supplies, and the true extent of the coronavirus crisis remains unknown. Dozens of deaths have been reported among health-care workers. However, statistics on the crisis' effect on health-care workers and their risk of being infected with SARS-CoV-2 are lacking. We aimed to estimate the prevalence of SARS-CoV-2 infection in health-care workers and to examine correlations with risk factors such as age, sex and comorbidities. Study participants (N = 402, median age 38.48 years) included physicians, nurses and medical assistants, from public and private hospitals, independent of symptom presentation. SARS-CoV-2 was detected on saliva samples using the loop-mediated isothermal amplification assay. A questionnaire was employed to determine subjects' COVID-19-associated symptoms and their vulnerability to complications from risk factors such as age, sex, professional role and comorbidities. The study was performed five weeks into the exponential growth period in Nicaragua. We discovered that 30.35% of health-care workers participating in our study had been infected with SARS-CoV-2. A large percentage (54.92%) of those who tested positive were asymptomatic and were still treating patients. Nearly 50% of health-care workers who tested positive were under 40, an astonishing 30.33% reported having at least one comorbidity. In our study, sex and age are important risk factors for the probability of testing positive for SARS-CoV-2 with significance being greatest among those between 30 and 40 years of age. In general, being male resulted in higher risk. Our data are the first non-governmental data obtained in Nicaragua. They shed light on several important aspects of COVID-19 in an underdeveloped nation whose government has implemented a herd-immunity strategy, while lacking an adequate healthcare system and sufficient PPE for health-care workers. These data are important for creating policies for containing the spread of SARS-CoV-2.


Assuntos
COVID-19/epidemiologia , Pessoal de Saúde/estatística & dados numéricos , Adulto , Idoso , Doenças Assintomáticas/epidemiologia , COVID-19/transmissão , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Feminino , Humanos , Transmissão de Doença Infecciosa do Paciente para o Profissional/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Nicarágua/epidemiologia , Prevalência , Fatores de Risco , SARS-CoV-2/isolamento & purificação , Saliva/virologia
8.
bioRxiv ; 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-33140046

RESUMO

K777 is a di-peptide analog that contains an electrophilic vinyl-sulfone moiety and is a potent, covalent inactivator of cathepsins. Vero E6, HeLa/ACE2, Caco-2, A549/ACE2, and Calu-3, cells were exposed to SARS-CoV-2, and then treated with K777. K777 reduced viral infectivity with EC50 values of inhibition of viral infection of: 74 nM for Vero E6, <80 nM for A549/ACE2, and 4 nM for HeLa/ACE2 cells. In contrast, Calu-3 and Caco-2 cells had EC50 values in the low micromolar range. No toxicity of K777 was observed for any of the host cells at 10-100 µM inhibitor. K777 did not inhibit activity of the papain-like cysteine protease and 3CL cysteine protease, encoded by SARS-CoV-2 at concentrations of ≤ 100 µM. These results suggested that K777 exerts its potent anti-viral activity by inactivation of mammalian cysteine proteases which are essential to viral infectivity. Using a propargyl derivative of K777 as an activity-based probe, K777 selectively targeted cathepsin B and cathepsin L in Vero E6 cells. However only cathepsin L cleaved the SARS-CoV-2 spike protein and K777 blocked this proteolysis. The site of spike protein cleavage by cathepsin L was in the S1 domain of SARS-CoV-2 , differing from the cleavage site observed in the SARS CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of viral spike protein processing.

9.
PLoS Negl Trop Dis ; 14(9): e0008726, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32970692

RESUMO

Chagas' Disease, caused by the protozoan parasite Trypanosoma cruzi, is responsible for up to 41% of the heart failures in endemic areas in South America and is an emerging infection in regions of North America, Europe, and Asia. Treatment is suboptimal due to two factors. First, the lack of an adequate biomarker to predict disease severity and response to therapy; and second, up to 120-days treatment course coupled with a significant incidence of adverse effects from the drug currently used. Because the disease can manifest itself clinically a few years to decades after infection, controversy remains concerning the suitability of current drug treatment (benznidazole), and the efficacy of alternative drugs (e.g. posaconazole). We therefore followed the clinical course, and PCR detection of parasite burden, in a mouse model of infection for a full year following treatment with benznidazole or posaconazole. Efficacy of the two drugs depended on whether the treatment was performed during the acute model or the chronic model of infection. Posaconazole was clearly superior in treatment of acute disease whereas only benznidazole had efficacy in the chronic model. These results have important implications for the design and analysis of human clinical trials, and the use of specific drugs in specific clinical settings.


Assuntos
Doença de Chagas/tratamento farmacológico , Nitroimidazóis/farmacologia , Triazóis/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Administração Oral , Animais , Doença de Chagas/parasitologia , Modelos Animais de Doenças , Seguimentos , Masculino , Camundongos Endogâmicos C57BL , Nitroimidazóis/administração & dosagem , Reação em Cadeia da Polimerase , Triazóis/administração & dosagem , Tripanossomicidas/farmacologia , Trypanosoma cruzi/isolamento & purificação
10.
PLoS Negl Trop Dis ; 14(5): e0007980, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32433643

RESUMO

Chagas disease, the clinical presentation of T. cruzi infection, is a major human health concern. While the acute phase of Chagas disease is typically asymptomatic and self-resolving, chronically infected individuals suffer numerous sequelae later in life. Cardiomyopathies in particular are the most severe consequence of chronic Chagas disease and cannot be reversed solely by parasite load reduction. To prioritize new therapeutic targets, we unbiasedly interrogated the host signaling events in heart tissues isolated from a Chagas disease mouse model using quantitative, multiplexed proteomics. We defined the host response to infection at both the proteome and phospho-proteome levels. The proteome showed an increase in the immune response and a strong repression of several mitochondrial proteins. Complementing the proteome studies, the phospho-proteomic survey found an abundance of phospho-site alterations in plasma membrane and cytoskeletal proteins. Bioinformatic analysis of kinase activity provided substantial evidence for the activation of NDRG2 and JNK/p38 kinases during Chagas disease. A significant activation of DYRK2 and AMPKA2 and the inhibition of casein family kinases were also predicted. We concluded our analyses by linking the diseased heart proteome profile to known therapeutic interventions, uncovering a potential to target mitochondrial proteins, secreted immune effectors and core kinases for the treatment of chronic Chagas disease. Together, this study provides molecular insight into host proteome and phospho-proteome responses to T. cruzi infection in the heart for the first time, highlighting pathways that can be further validated for functional contributions to disease and suitability as drug targets.


Assuntos
Cardiomiopatia Chagásica/metabolismo , Animais , Cardiomiopatia Chagásica/genética , Cardiomiopatia Chagásica/imunologia , Cardiomiopatia Chagásica/parasitologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteoma/genética , Proteoma/metabolismo , Proteômica , Transdução de Sinais , Trypanosoma cruzi/fisiologia
11.
Microorganisms ; 8(4)2020 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-32224991

RESUMO

Chagas disease, caused by the kinetoplastid parasite Trypanosoma cruzi, affects between 6 and 7 million people worldwide, with an estimated 300,000 to 1 million of these cases in the United States. In the chronic phase of infection, T. cruzi can cause severe gastrointestinal and cardiac disease, which can be fatal. Currently, only benznidazole is clinically approved by the FDA for pediatric use to treat this infection in the USA. Toxicity associated with this compound has driven the search for new anti-Chagas agents. Drug repurposing is a particularly attractive strategy for neglected diseases, as pharmacological parameters and toxicity are already known for these compounds, reducing costs and saving time in the drug development pipeline. Here, we screened 7680 compounds from the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library, a collection of drugs or compounds with confirmed clinical safety, against T. cruzi. We identified seven compounds of interest with potent in vitro activity against the parasite with a therapeutic index of 10 or greater, including the previously unreported activity of the antiherpetic compound 348U87. These results provide the framework for further development of new T. cruzi leads that can potentially move quickly to the clinic.

12.
J Med Chem ; 63(6): 3298-3316, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32125159

RESUMO

Cruzain, an essential cysteine protease of the parasitic protozoan, Trypanosoma cruzi, is an important drug target for Chagas disease. We describe here a new series of reversible but time-dependent inhibitors of cruzain, composed of a dipeptide scaffold appended to vinyl heterocycles meant to provide replacements for the irreversible reactive "warheads" of vinyl sulfone inactivators of cruzain. Peptidomimetic vinyl heterocyclic inhibitors (PVHIs) containing Cbz-Phe-Phe/homoPhe scaffolds with vinyl-2-pyrimidine, vinyl-2-pyridine, and vinyl-2-(N-methyl)-pyridine groups conferred reversible, time-dependent inhibition of cruzain (Ki* = 0.1-0.4 µM). These cruzain inhibitors exhibited moderate to excellent selectivity versus human cathepsins B, L, and S and showed no apparent toxicity to human cells but were effective in cell cultures of Trypanosoma brucei brucei (EC50 = 1-15 µM) and eliminated T. cruzi in infected murine cardiomyoblasts (EC50 = 5-8 µM). PVHIs represent a new class of cruzain inhibitors that could progress to viable candidate compounds to treat Chagas disease and human sleeping sickness.


Assuntos
Inibidores de Cisteína Proteinase/farmacologia , Peptidomiméticos/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Tripanossomicidas/farmacologia , Compostos de Vinila/farmacologia , Animais , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/metabolismo , Desenho de Fármacos , Ensaios Enzimáticos , Humanos , Cinética , Camundongos , Simulação de Acoplamento Molecular , Mioblastos Cardíacos/efeitos dos fármacos , Peptidomiméticos/síntese química , Peptidomiméticos/metabolismo , Ligação Proteica , Proteínas de Protozoários/metabolismo , Piridinas/síntese química , Piridinas/metabolismo , Piridinas/farmacologia , Pirimidinas/síntese química , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Tripanossomicidas/síntese química , Tripanossomicidas/metabolismo , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos , Compostos de Vinila/síntese química , Compostos de Vinila/metabolismo
13.
Artigo em Inglês | MEDLINE | ID: mdl-32094126

RESUMO

Current treatments for Acanthamoeba keratitis rely on a combination of chlorhexidine gluconate, propamidine isethionate, and polyhexamethylene biguanide. These disinfectants are nonspecific and inherently toxic, which limits their effectiveness. Furthermore, in 10% of cases, recurrent infection ensues due to the difficulty in killing both trophozoites and double-walled cysts. Therefore, development of efficient, safe, and target-specific drugs which are capable of preventing recurrent Acanthamoeba infection is a critical unmet need for averting blindness. Since both trophozoites and cysts contain specific sets of membrane sterols, we hypothesized that antifungal drugs targeting sterol 14-demethylase (CYP51), known as conazoles, would have deleterious effects on A. castellanii trophozoites and cysts. To test this hypothesis, we first performed a systematic screen of the FDA-approved conazoles against A. castellanii trophozoites using a bioluminescence-based viability assay adapted and optimized for Acanthamoeba The most potent drugs were then evaluated against cysts. Isavuconazole and posaconazole demonstrated low nanomolar potency against trophozoites of three clinical strains of A. castellanii Furthermore, isavuconazole killed trophozoites within 24 h and suppressed excystment of preformed Acanthamoeba cysts into trophozoites. The rapid action of isavuconazole was also evident from the morphological changes at nanomolar drug concentrations causing rounding of trophozoites within 24 h of exposure. Given that isavuconazole has an excellent safety profile, is well tolerated in humans, and blocks A. castellanii excystation, this opens an opportunity for the cost-effective repurposing of isavuconazole for the treatment of primary and recurring Acanthamoeba keratitis.


Assuntos
Acanthamoeba castellanii/efeitos dos fármacos , Amebicidas/farmacologia , Antifúngicos/farmacologia , Cistos/tratamento farmacológico , Nitrilas/farmacologia , Piridinas/farmacologia , Triazóis/farmacologia , Inibidores de 14-alfa Desmetilase/farmacologia , Inibidores de 14-alfa Desmetilase/uso terapêutico , Acanthamoeba castellanii/crescimento & desenvolvimento , Amebíase/tratamento farmacológico , Amebíase/parasitologia , Amebicidas/uso terapêutico , Animais , Antifúngicos/uso terapêutico , Reposicionamento de Medicamentos , Humanos , Testes de Sensibilidade Microbiana , Nitrilas/uso terapêutico , Piridinas/uso terapêutico , Triazóis/uso terapêutico , Trofozoítos/efeitos dos fármacos
14.
bioRxiv ; 2020 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-33398273

RESUMO

The emergence of SARS-CoV-2 in late 2019, and the subsequent COVID-19 pandemic, has led to substantial mortality, together with mass global disruption. There is an urgent need for novel antiviral drugs for therapeutic or prophylactic application. Cathepsin L is a key host cysteine protease utilized by coronaviruses for cell entry and is recognized as a promising drug target. The marine natural product, gallinamide A and several synthetic analogues, were identified as potent inhibitors of cathepsin L activity with IC 50 values in the picomolar range. Lead molecules possessed selectivity over cathepsin B and other related human cathepsin proteases and did not exhibit inhibitory activity against viral proteases Mpro and PLpro. We demonstrate that gallinamide A and two lead analogues potently inhibit SARS-CoV-2 infection in vitro , with EC 50 values in the nanomolar range, thus further highlighting the potential of cathepsin L as a COVID-19 antiviral drug target.

15.
ACS Infect Dis ; 5(12): 2029-2038, 2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31583882

RESUMO

Naegleria fowleri is a free-living amoeba causing primary amoebic meningoencephalitis, a rapid-onset brain infection in humans with over 97% mortality rate. Despite some progress in the treatment of the disease, there is no single, proven, evidence-based treatment with a high probability of cure. Here we report the chemical library screening and experimental identification of four new compounds with amoebicidal effects against N. fowleri. The chemical library was screened by molecular docking against a homology model of sterol Δ8-Δ7 isomerase (NfERG2). Thirty top-ranking hits were then tested in a cell-based assay for antiproliferative/amoebicidal activities. Eight chemicals exhibited nearly 100% inhibition of N. fowleri at 50 µM, with the EC50 values ranging from 6 to 25 µM. A cell toxicity assay using human HEK-293 cells was also performed. Four of the compounds preferentially kill amoeba cells with no apparent human cell toxicities. These compounds fall into two distinct chemical scaffolds with druglike properties.


Assuntos
Amebicidas/farmacologia , Isomerases/química , Naegleria fowleri/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Amebicidas/química , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Isomerases/efeitos dos fármacos , Isomerases/genética , Modelos Moleculares , Simulação de Acoplamento Molecular , Naegleria fowleri/efeitos dos fármacos , Naegleria fowleri/genética , Fenótipo , Conformação Proteica , Homologia de Sequência , Bibliotecas de Moléculas Pequenas/química
16.
PLoS Negl Trop Dis ; 12(10): e0006923, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30379807

RESUMO

BACKGROUND: Angiostrongylus costaricensis is a relatively uncharacterized nematode that causes abdominal angiostrongyliasis in Latin America, a human parasitic disease. Currently, no effective pharmacological treatment for angiostrongyliasis exists. Peptidases are known to be druggable targets for a variety of diseases and are essential for several biological processes in parasites. Therefore, this study aimed to systematically characterize the peptidase activity of A. costaricensis in different developmental stages of this parasitic nematode. METHODOLOGY/PRINCIPAL FINDINGS: A library of diverse tetradecapeptides was incubated with cellular lysates from adult worms and from first-stage larvae (L1) and cleaved peptide products were identified by mass spectrometry. Lysates were also treated with class specific peptidase inhibitors to determine which enzyme class was responsible for the proteolytic activity. Peptidase activity from the four major mechanistic classes (aspartic, metallo, serine and cysteine) were detected in adult worm lysate, whereas aspartic, metallo and serine-peptidases were found in the larval lysates. In addition, the substrate specificity profile was found to vary at different pH values. CONCLUSIONS/SIGNIFICANCE: The proteolytic activities in adult worm and L1 lysates were characterized using a highly diversified library of peptide substrates and the activity was validated using a selection of fluorescent substrates. Taken together, peptidase signatures for different developmental stages of this parasite has improved our understanding of the disease pathogenesis and may be useful as potential drug targets or vaccine candidates.


Assuntos
Angiostrongylus/enzimologia , Peptídeo Hidrolases/análise , Animais , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Larva/enzimologia , Peptídeo Hidrolases/química , Proteólise , Especificidade por Substrato
17.
Sci Rep ; 8(1): 13092, 2018 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-30166569

RESUMO

Infection by the human blood fluke, Schistosoma mansoni involves a variety of cross-species protein- protein interactions. The pathogen expresses a diverse arsenal of proteins that facilitate the breach of physical and biochemical barriers present in skin evasion of the immune system, and digestion of human plasma proteins including albumin and hemoglobin, allowing schistosomes to reside in the host for years. However, only a small number of specific interactions between S. mansoni and human proteins have been identified. We present and apply a protocol that generates testable predictions of S. mansoni-human protein interactions. In this study, we have preliminary predictions of novel interactions between schistosome and human proteins relevant to infection and the ability of the parasite to evade the immune system. We applied a computational whole-genome comparative approach to predict potential S. mansoni-human protein interactions based on similarity to known protein complexes. We first predict S. mansoni -human protein interactions based on similarity to known protein complexes. Putative interactions were then scored and assessed using several contextual filters, including the use of annotation automatically derived from literature using a simple natural language processing methodology. Next, in vitro experiments were carried out between schistosome and host proteins to validate several prospective predictions. Our method predicted 7 out of the 10 previously known cross-species interactions involved in pathogenesis between S. mansoni and its human host. Interestingly, two novel putative interactions involving Schistosoma proteins, the cercarial elastase SmCE, and the adult tegument surface protein Sm29, were also predicted and experimentally characterized. Preliminary data suggest that elafin, a host endogenous serine protease inhibitor, may be a novel substrate for SmCE. Additionally, CD59, an inhibitor of the membrane attack complex, could interact with Sm29. Furthermore, the application framework provides an integrated methodology for investigation of host-pathogen interactions and an extensive source of orthogonal data for experimental analysis. We have made the predictions available for community perusal.


Assuntos
Proteínas de Helminto/metabolismo , Mapeamento de Interação de Proteínas , Schistosoma mansoni/metabolismo , Animais , Antígenos de Helmintos/metabolismo , Antígenos CD59/metabolismo , Cercárias/enzimologia , Humanos , Estágios do Ciclo de Vida , Mesocricetus , Modelos Moleculares , Elastase Pancreática/metabolismo , Schistosoma mansoni/crescimento & desenvolvimento , Esquistossomose mansoni/imunologia , Especificidade por Substrato , Vacinas/imunologia
20.
Front Mol Biosci ; 5: 40, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29725596

RESUMO

Cysteine proteases are widespread in all life kingdoms, being central to diverse physiological processes based on a broad range of substrate specificity. Paralogous Fasciola hepatica cathepsin L proteases are essential to parasite invasion, tissue migration and reproduction. In spite of similarities in their overall sequence and structure, these enzymes often exhibit different substrate specificity. These preferences are principally determined by the amino acid composition of the active site's S2 subsite (pocket) of the enzyme that interacts with the substrate P2 residue (Schetcher and Berger nomenclature). Although secreted FhCL1 accommodates aliphatic residues in the S2 pocket, FhCL2 is also efficient in cleaving proline in that position. To understand these differences, we engineered the FhCL1 S2 subsite at three amino acid positions to render it identical to that present in FhCL2. The substitutions did not produce the expected increment in proline accommodation in P2. Rather, they decreased the enzyme's catalytic efficiency toward synthetic peptides. Nonetheless, a change in the P3 specificity was associated with the mutation of Leu67 to Tyr, a hinge residue between the S2 and S3 subsites that contributes to the accommodation of Gly in S3. Molecular dynamic simulations highlighted changes in the spatial distribution and secondary structure of the S2 and S3 pockets of the mutant FhCL1 enzymes. The reduced affinity and catalytic efficiency of the mutant enzymes may be due to a narrowing of the active site cleft that hinders the accommodation of substrates. Because the variations in the enzymatic activity measured could not be exclusively allocated to those residues lining the active site, other more external positions might modulate enzyme conformation, and, therefore, catalytic activity.

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