Biology subjects

Siqueira-Neto, J. L.

Publications and source records attributed to Siqueira-Neto, J. L..

11 recordsLinked to original sources

bioRxiv2025

The Human Chk1 Inhibitor CHIR-124 Shows Multistage Activity Against Plasmodium falciparum via Dual Inhibition of PfArk1 and Hemozoin Formation

The high burden of malaria and growing resistance to frontline antimalarials demand new drug target combinations with reduced propensities for conferring parasite resistance. An attractive approach for circumventing antimalarial drug resistance is target repurposing in which known drugs that act through protein targets of human origin that are also active against the human malaria parasite Plasmodium falciparum are exploited to identify novel antimalarial drug targets. Here we show that the human checkpoint kinase 1 (Chk1) inhibitor CHIR-124 is active in vitro against both drug-sensitive and drug-resistant asexual blood stage parasites and competitively binds to several Plasmodium kinases. The compound also shows moderate activity against both the liver and gametocyte forms of the parasite. Further target investigation of CHIR-124 via conditional knockdown experiments confirmed that P. falciparum Aurora-related kinase 1 (PfArk1) is implicated in its parasiticidal activity. Notably, CHIR-124 also inhibits {beta}-hematin (synthetic hemozoin) formation and causes a dose-dependent increase in free heme that correlates with inhibition of parasite growth. These findings suggest that polypharmacology is involved in the activity of CHIR-124 against P. falciparum via the dual inhibition of Plasmodium PfArk1 and hemozoin formation, both essential for parasite proliferation. This is further supported by in vitro drug combination experiments, morphological studies and resistance generation attempts. This study validates the feasibility of dual Plasmodium kinase/hemozoin formation inhibitors active against resistant strains with decreased resistance risks in the fight against malaria.

microbiology↗

bioRxiv2022

Structure-based identification of naphthoquinones and derivatives as novel inhibitors of main protease Mpro and papain-like protease PLpro of SARS-CoV-2

The worldwide COVID-19 pandemic caused by the coronavirus SARS-CoV-2 urgently demands novel direct antiviral treatments. The main protease (Mpro) and papain-like protease (PLpro) are attractive drug targets among coronaviruses due to their essential role in processing the polyproteins translated from the viral RNA. In the present work, we virtually screened 688 naphthoquinoidal compounds and derivatives against Mpro of SARS-CoV-2. Twenty-four derivatives were selected and evaluated in biochemical assays against Mpro using a novel fluorogenic substrate. In parallel, these compounds were also assayed with SARS-CoV-2 PLpro. Four compounds inhibited Mpro with half-maximal inhibitory concentration (IC50) values between 0.41 {micro}M and 66 {micro}M. In addition, eight compounds inhibited PLpro with IC50 ranging from 1.7 {micro}M to 46 {micro}M. Molecular dynamics simulations suggest stable binding modes for Mpro inhibitors with frequent interactions with residues in the S1 and S2 pockets of the active site. For two PLpro inhibitors, interactions occur in the S3 and S4 pockets. In summary, our structure-based computational and biochemical approach identified novel naphthoquinonal scaffolds that can be further explored as SARS-CoV-2 antivirals.

biochemistry↗

bioRxiv2021

Leveraging Allele-Specific Expression for Therapeutic Response Gene Discovery in Glioblastoma.

Glioblastoma is the most prevalent primary malignant brain tumor in adults and is characterized by poor prognosis and universal tumor recurrence. Effective glioblastoma treatments are lacking, in part due to somatic mutations and epigenetic reprogramming that alter gene expression and confer drug resistance. Here, we interrogated allele-specific expression (ASE) in 43 patient-derived glioblastoma stem cells (GSCs) to identify recurrently dysregulated genes in glioblastoma. We identified 118 genes with recurrent ASE preferentially found in GSCs compared to normal tissues. These genes were enriched for apoptotic regulators, including Schlafen Family Member 11 (SLFN11). Loss of SLFN11 gene expression was associated with aberrant promoter methylation and conferred resistance to chemotherapy and poly ADP ribose polymerase inhibition. Conversely, low SLFN11 expression rendered GSCs susceptible to the oncolytic flavivirus Zika, which suggests a potential alternative treatment strategy for chemotherapy resistant GBMs.

cancer biology↗

bioRxiv2021

Identification of Leucinostatins from Ophiocordyceps sp. as Antiparasitic Agents Against Trypanosoma cruzi

Safe and effective treatments for Chagas disease, a potentially fatal parasitic infection associated with cardiac and gastrointestinal pathology and caused by the kinetoplastid parasite Trypanosoma cruzi, have yet to be developed. Benznidazole and nifurtimox, which are currently the only available drugs against T. cruzi, are associated with severe adverse effects and questionable efficacy in the late stage of the disease. Natural products have proven to be a rich source of new chemotypes for other infectious agents. We utilized a microscopy-based high-throughput phenotypic screen to identify inhibitors of T. cruzi from a library of natural product samples obtained from fungi procured through a Citizen Science Soil Collection Program (https://whatsinyourbackyard.org/), and the Great Lakes (USA) benthic environment. We identified five leucinostatins (A, B, F, NPDG C and NPDG D) as potent inhibitors of the intracellular amastigote form of T. cruzi. Leucinostatin B also showed in vivo efficacy in a mouse model of Chagas disease. Given prior reports that leucinostatins A and B have antiparasitic activity against the related kinetoplastid T. brucei, our findings suggest a potential cross-trypanocidal compound class and provide a platform for further chemical derivatization of a potent chemical scaffold against T. cruzi.

microbiology↗

bioRxiv2020

A cysteine protease inhibitor blocks SARS-CoV-2 infection of human and monkey cells

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 M 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 M. 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. SIGNIFICANCEThe virus causing COVID-19 is highly infectious and has resulted in a global pandemic. We confirm that a cysteine protease inhibitor, approved by the FDA as a clinical-stage compound, inhibits SARS-CoV-2 infection of several human and monkey cell lines with notable(nanomolar) efficacy. The mechanism of action of this inhibitor is identified as a specific inhibition of host cell cathepsin L. This in turn inhibits host cell processing of the coronaviral spike protein, a step required for cell entry. Neither of the coronaviral proteases are inhibited, and the cleavage site of spike protein processing is different from that reported in other coronaviruses. Hypotheses to explain the differential activity of the inhibitor with different cell types are discussed.

biochemistry↗

bioRxiv2020

Spatial metabolomics identifies localized chemical changes in heart tissue during chronic cardiac Chagas disease

Chagas disease (CD) is one of thirteen neglected tropical diseases caused by the parasite Trypanosoma cruzi. CD is a vector-borne disease transmitted by triatomines but CD can also be transmitted through blood transfusions, organ transplants and congenital transmission. While endemic to Latin America, 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 disease pathogenesis are not fully understood. Using spatial metabolomics (chemical cartography), we sought to understand the localized impact of infection on the cardiac metabolome of mice chronically 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 select sites in chronic CD. These sites were distinct from the sites of highest parasite burden. In addition, we identified acylcarnitines and phosphocholines as discriminatory chemical families within each heart region, comparing infected and uninfected samples. Overall, our study indicated overall 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 spatial perspective to understand infectious disease tropism.Author Summary Chagas disease (CD) is a tropical disease caused by the parasite Trypanosoma cruzi. CD originated in South America; however, there are now 7-8 million people infected worldwide due to population movements. CD is transmitted through a triatomine vector, organ transplants, blood transfusions and congenital transmission. It occurs in two stages, an acute stage (usually asymptomatic) and the chronic stage. Chronic stage CD presents with severe cardiac symptoms such as heart failure, localized aneurysms and cardiomyopathy. Unfortunately, what causes severe cardiac symptoms in some individuals in chronic CD is not fully understood. Therefore, we used liquid chromatography-tandem mass spectrometry to analyze the heart tissue of chronically T. cruzi-infected and uninfected mice, to understand the impact of infection on the tissue metabolome. We identified discriminatory small molecules related to T. cruzi infection. We also determined that regions with the highest parasite burden are distinct from the regions with the largest changes in overall metabolite profile; these locations of high metabolic perturbation provide a molecular mechanism to why localized cardiac symptoms occur in CD. Overall, our work gives insight to chronic cardiac CD symptom development and shapes a framework for novel treatment and biomarker development.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2020

Machine Learning Models Identify Inhibitors of SARS-CoV-2

With the ongoing SARS-CoV-2 pandemic there is an urgent need for the discovery of a treatment for the coronavirus disease (COVID-19). Drug repurposing is one of the most rapid strategies for addressing this need and numerous compounds have been selected for in vitro testing by several groups already. These have led to a growing database of molecules with in vitro activity against the virus. Machine learning models can assist drug discovery through prediction of the best compounds based on previously published data. Herein we have implemented several machine learning methods to develop predictive models from recent SARS-CoV-2 in vitro inhibition data and used them to prioritize additional FDA approved compounds for in vitro testing selected from our in-house compound library. From the compounds predicted with a Bayesian machine learning model, CPI1062 and CPI1155 showed antiviral activity in HeLa-ACE2 cell-based assays and represent potential repurposing opportunities for COVID-19. This approach can be greatly expanded to exhaustively virtually screen available molecules with predicted activity against this virus as well as a prioritization tool for SARS-CoV-2 antiviral drug discovery programs. The very latest model for SARS-CoV-2 is available at www.assaycentral.org.Competing Interest StatementSE is CEO and owner of Collaborations Pharmaceuticals, Inc. DHF, KMZ, TRL, AP are employees of Collaborations Pharmaceuticals, Inc.View Full Text

pharmacology and toxicology↗

bioRxiv2019

High-throughput screening of the ReFRAME library identifies potential drug repurposing candidates for Trypanosoma cruzi

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 ~ 12,000 compounds from the ReFRAME library, a collection of drugs or compounds with confirmed clinical safety, against T. cruzi. We identified 7 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.

microbiology↗

bioRxiv2019

Activity of Selected Nucleoside Analogue ProTides against Zika Virus in Human Neural Stem Cells

Zika virus (ZIKV), an emerging flavivirus which causes neurodevelopmental impairment to fetuses and has been linked to Guillain-Barre syndrome, continues to threaten global health due to the absence of targeted prophylaxis or treatment. Nucleoside analogues are good examples of efficient anti-viral inhibitors, and prodrug strategies using phosphate masking groups (ProTides) have been employed to improve the bioavailability of ribonucleoside analogues. Here, we synthesized and tested a library of 13 ProTides against ZIKV in human neural stem cells. Strong activity was observed for 2'-C-methyluridine and 2'-C-ethynyluridine ProTides with an aryloxyl phosphoramidate masking group. Conversion of the aryloxyl phosphoramidate ProTide group of 2'-C-methyluridine to a 2-(methylthio)ethyl phosphoramidate completely abolished antiviral activity of the compound. The aryloxyl phosphoramidate ProTide of 2'-C-methyluridine outperformed the hepatitis C virus (HCV) drug sofosbuvir in suppression of viral titers and protection from cytopathic effect, while the former compounds triphosphate active metabolite was better incorporated by purified ZIKV NS5 polymerase over time. Molecular superpositioning revealed different orientations of residues opposite the 2'-fluoro group of sofosbuvir. These findings suggest both a nucleobase and ProTide group bias for the anti-ZIKV activity of nucleoside analogue ProTides in a disease-relevant cell model.

microbiology↗

bioRxiv2018

Mechanism of action of methotrexate against Zika virus

Zika virus (ZIKV), which is associated with microcephaly in infants and Guillain-Barre syndrome, reemerged as a serious public health threat in Latin America in recent years. Previous high-throughput screening (HTS) campaigns have revealed several potential hit molecules against ZIKV, including methotrexate (MTX), which is clinically used as an anti-cancer chemotherapy and anti-rheumatoid agent. We studied the mechanism of action of MTX against ZIKV in relation to its inhibition of dihydrofolate reductase (DHFR) in vitro using Vero and human neural stem cells (hNSCs). As expected, an antiviral effect for MTX against ZIKV was observed, showing up to ten-fold decrease in virus titer during MTX treatment. We also observed that addition of leucovorin (a downstream metabolite of DHFR pathway) rescued the ZIKV replication impaired by MTX treatment in ZIKV-infected cells, explaining the antiviral effect of MTX through inhibition of DHFR. We also found that addition of adenosine to ZIKV-infected cells was able to rescue ZIKV replication inhibited by MTX, suggesting that restriction of de novo synthesis adenosine triphosphate (ATP) pools suppresses viral replication. These results confirm that the DFHR pathway can be targeted to inhibit replication of ZIKV, similar to other published results showing this effect in related flaviviruses.

microbiology↗

bioRxiv2018

Development and validation of a phenotypic high-content imaging assay for assessing the antiviral activity of small-molecule inhibitors targeting the Zika virus

Zika virus (ZIKV) has been linked to the development of microcephaly in newborns, as well as Guillain-Barre syndrome. There are currently no drugs available to treat infection, and accordingly there is an unmet medical need for discovery of new therapies. High-throughput drug screening efforts focusing on indirect readouts of cell viability are prone to a higher frequency of false positives in cases where the virus is viable in the cell but the cytopathic effect is reduced or delayed. Here, we describe a fast and label-free phenotypic high-content imaging assay used to detect cells affected by the viral-induced cytopathic effect (CPE) using automated imaging and analysis. Protection from CPE correlates with a decrease in viral antigen production as observed by immunofluorescence. We trained our assay using a collection of nucleoside analogues against ZIKV; the previously reported antiviral activities of 2-C-methylribonucleosides and ribavirin against the Zika virus in Vero cells were confirmed using our developed method. Profiling of a novel library of 24 natural product derivatives using our assay revealed compound 1 as an inhibitor of ZIKV-induced cytopathic effect; activity of the compound was confirmed in human fetal neural stem cells (NSCs). The described technique can be easily leveraged as a primary screening assay for profiling large compound libraries against ZIKV, and can be expanded to other ZIKV strains and other cell lines displaying morphological changes upon ZIKV infection.

microbiology↗