Search

IntroductionLeishmaniasis is a parasitic disease that affects more than 1 million people worldwide annually, predominantly in resource-limited settings. The challenge in compound development is to exhibit potent activity against the intracellular stage of the parasite (the stage present in the mammalian host) without harming the infected host cells. We have identified a compound series (pyrazolopyrrolidinones) active against the intracellular parasites of Leishmania donovani and L. major; the causative agents of visceral and cutaneous leishmaniasis in the Old World, respectively.MethodsIn this study, we performed medicinal chemistry on a newly-discovered antileishmanial chemotype, with over 100 analogs tested. Studies included assessments of antileishmanial potency, toxicity towards host cells, and in vitro ADME screening of key drug properties.Results and discussionMembers of the series showed high potency against the deadliest form, visceral leishmaniasis (approximate EC50 ≥ 0.01 µM without harming the host macrophage up to 10.0 µM). In comparison, the most efficient monotherapy treatment for visceral leishmaniasis is amphotericin B, which presents similar activity in the same assay (EC50 = 0.2 µM) while being cytotoxic to the host cell at 5.0 µM. Continued development of this compound series with the Discovery Partnership with Academia (DPAc) program at the GlaxoSmithKline Diseases of the Developing World (GSK DDW) laboratories found that the compounds passed all of GSK’s criteria to be defined as a potential lead drug series for leishmaniasis.ConclusionHere, we describe preliminary structure-activity relationships for antileishmanial pyrazolopyrrolidinones, and our progress towards the identification of candidates for future in vivo assays in models of visceral and cutaneous leishmaniasis.

Infections of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have triggered a global pandemic with millions of deaths worldwide. Herein, the synthesis of functionalized Re(i) tricarbonyl complexes as inhibitors of the SARS-CoV-2 main protease, also referred to as the 3-chymotrypsin-like protease (3CLpro), is presented. The metal complexes were found to inhibit the activity of the enzyme with IC50 values in the low micromolar range. Mass spectrometry revealed that the metal complexes formed a coordinate covalent bond with the enzyme. Chiral separation of the enantiomers of the lead compound showed that one enantiomer was significantly more active than the other, consistent with specific binding and much like that observed for conventional organic small molecule inhibitors and druglike compounds. Evaluation of the lead compound against SARS-CoV-2 in a cell-based infection assay confirmed enantiospecific inhibition against the virus. This study represents a significant advancement in the use of metal complexes as coordinate covalent inhibitors of enzymes, as well as a novel starting point for the development of novel SARS-CoV-2 inhibitors.

Trypanosoma cruzi (Chagas diseases – also named American trypanosomiasis) and T. brucei (human African trypanosomiasis – HAT) negatively impact public health, being endemic in several countries and leading to thousands of deaths per year.

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 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.

There are currently no FDA approved inhibitors of SARS-CoV-2 viral proteases with specific treatment for post-exposure of SARS-CoV-2. Here, we discovered inhibitors containing thiuram disulfide or dithiobis-(thioformate) tested against three key proteases in SARS CoV-2 replication including SARS CoV-2 Main Protease (Mpro), SARS CoV-2 Papain Like Protease (PLpro), and human cathepsin L. The use of thiuram disulfide and dithiobis-(thioformate) covalent inhibitor warheads was inspired by disulfiram, a currently prescribed drug commonly used to treat chronic alcoholism that at the present time is in Phase 2 clinical trials against SARS-CoV-2. At the maximal allowed dose, disulfiram is associated with adverse effects. Our goal was to find more potent inhibitors that target both viral proteases and one essential human protease to reduce the dosage and minimize the adverse effects associated with these agents. We found that compounds coded as RI175, JX 06, and RI172 are the most potent inhibitors from an enzymatic assay against SARS-CoV-2 Mpro, SARS-CoV-2 PLpro, and human cathepsin L with IC50s of 330, 250 nM, and 190 nM about 4.5, 17, and 11.5-fold more potent than disulfiram, respectively. The identified protease inhibitors in this series were also tested against SARS CoV-2 in a cell-based and toxicity assay and were shown to have similar or greater antiviral effect than disulfiram. The identified triple protease inhibitors and their derivatives are promising candidates for treatment of the Covid-19 virus and related variants.

Cysteine proteases comprise an important class of drug targets, especially for infectious diseases such as Chagas disease (cruzain) and COVID-19 (3CL protease, cathepsin L). Peptide aldehydes have proven to be potent inhibitors for all of these proteases. However, the intrinsic, high electrophilicity of the aldehyde group is associated with safety concerns and metabolic instability, limiting the use of aldehyde inhibitors as drugs. We have developed a novel class of self-masked aldehyde inhibitors (SMAIs) for cruzain, the major cysteine protease of the causative agent of Chagas disease-Trypanosoma cruzi. These SMAIs exerted potent, reversible inhibition of cruzain (Ki* = 18-350 nM) while apparently protecting the free aldehyde in cell-based assays. We synthesized prodrugs of the SMAIs that could potentially improve their pharmacokinetic properties. We also elucidated the kinetic and chemical mechanism of SMAIs and applied this strategy to the design of anti-SARS-CoV-2 inhibitors.

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, 50 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.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.

Ergosterol biosynthesis inhibitors, such as posaconazole and ravuconazole, have been proposed as drug candidates for Chagas disease, a neglected infectious tropical disease caused by the protozoan parasite Trypanosoma cruzi. To understand better the mechanism of action and resistance to these inhibitors, a clone of the T. cruzi Y strain was cultured under intermittent and increasing concentrations of ravuconazole until phenotypic stability was achieved. The ravuconazole-selected clone exhibited loss in fitness in vitro when compared to the wild-type parental clone, as observed in reduced invasion capacity and slowed population growth in both mammalian and insect stages of the parasite. In drug activity assays, the resistant clone was above 300-fold more tolerant to ravuconazole than the sensitive parental clone, when the half-maximum effective concentration (EC50) was considered. The resistant clones also showed reduced virulence in vivo, when compared to parental sensitive clones. Cross-resistance to posaconazole and other CYP51 inhibitors, but not to other antichagasic drugs that act independently of CYP51, such as benznidazole and nifurtimox, was also observed. A novel amino acid residue change, T297M, was found in the TcCYP51 gene in the resistant but not in the sensitive clones. The structural effects of the T297M, and of the previously described P355S residue changes, were modelled to understand their impact on interaction with CYP51 inhibitors., Graphical abstract Image 1, Highlights • A ravuconazole-resistant T. cruzi clone presented reduced in vitro and in vivo fitness. • The ravuconazole-resistant clone presented cross-resistance to other CYP51 inhibitors. • There was no cross-resistance to benznidazole and nifurtimox. • Resistance is associated with a novel structural mutation in the TcCYP51 protein.

Telomeres are protein–DNA complexes that protect chromosome ends from degradation and fusion. In Leishmania spp., telomeric DNA comprises a conserved TTAGGG repeat and is maintained by telomerase. Telomerase is a multisubunit enzymatic complex that ensures the complete DNA replication by adding new telomeric repeats to the G-rich strand. In this report we aimed to purify and study the biochemical properties of Leishmani amazonensis telomerase. In a first trial we used affinity chromatography with antisense 2′-O-methyl oligonucleotide without success since the Leishmania telomerase, similarly to Trypanosoma cruzi enzyme, was not eluted by competition, but instead, it remained bound to the column. Partially purified L. amazonensis telomerase activity was achieved by fractionation of extracts on complementary ion exchange and Heparin columns. Further purification of these fractions on a G-rich telomeric DNA affinity chromatography enriched for telomerase activity. The knowledge of telomerase characteristics in Leishmania could help to develop new strategies to overcome leishmaniasis.

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in different events of DNA metabolism. In yeast, subunits 1 (RPA-1) and 2 (RPA-2) work also as telomerase recruiters and, in humans, the complex unfolds G-quartet structures formed by the 3' G-rich telomeric strand. In most eukaryotes, RPA-1 and RPA-2 bind DNA using multiple OB fold domains. In trypanosomatids, including Leishmania, RPA-1 has a canonical OB fold and a truncated RFA-1 structural domain. In Leishmania amazonensis, RPA-1 alone can form a complex in vitro with the telomeric G-rich strand. In this work, we show that LaRPA-1 is a nuclear protein that associates in vivo with Leishmania telomeres. We mapped the boundaries of the OB fold DNA-binding domain using deletion mutants. Since Leishmania and other trypanosomatids lack homologues of known telomere end binding proteins, our results raise questions about the function of RPA-1 in parasite telomeres.

Telomeres are the physical ends of eukaryotic linear chromosomes. Telomeres form special structures that cap chromosome ends to prevent degradation by nucleolytic attack and to distinguish chromosome termini from DNA double-strand breaks. With few exceptions, telomeres are composed primarily of repetitive DNA associated with proteins that interact specifically with double- or single-stranded telomeric DNA or with each other, forming highly ordered and dynamic complexes involved in telomere maintenance and length regulation. In proliferative cells and unicellular organisms, telomeric DNA is replicated by the actions of telomerase, a specialized reverse transcriptase. In the absence of telomerase, some cells employ a recombination-based DNA replication pathway known as alternative lengthening of telomeres. However, mammalian somatic cells that naturally lack telomerase activity show telomere shortening with increasing age leading to cell cycle arrest and senescence. In another way, mutations or deletions of telomerase components can lead to inherited genetic disorders, and the depletion of telomeric proteins can elicit the action of distinct kinases-dependent DNA damage response, culminating in chromosomal abnormalities that are incompatible with life. In addition to the intricate network formed by the interrelationships among telomeric proteins, long noncoding RNAs that arise from subtelomeric regions, named telomeric repeat-containing RNA, are also implicated in telomerase regulation and telomere maintenance. The goal for the next years is to increase our knowledge about the mechanisms that regulate telomere homeostasis and the means by which their absence or defect can elicit telomere dysfunction, which generally results in gross genomic instability and genetic diseases.

DNDi Institut Pasteur Korea (IPK) Reconstruction Credit Institution-Federal Ministry of Education and Research (KfW-BMBF)/Germany Medecins Sans Frontieres (Doctors without Borders)/International Korean government (MSIP), Gyeonggi-do KISTI Advocacy for better drugs and access to treatment has boosted the interest in drug discovery and development for Chagas disease, a chronic infection caused by the genetically heterogeneous parasite, Trypanosoma cruzi. in this work new in vitro assays were used to gain a better understanding of the antitrypanosomal properties of the most advanced antichagasic lead and clinical compounds, the nitroheterocyclics benznidazole, nifurtimox and fexinidazole sulfone, the oxaborole AN4169, and four ergosterol biosynthesis inhibitors -posaconazole, ravuconazole, EPL-BS967 and EPL-BS1246. Two types of assays were developed: one for evaluation of potency and efficacy in dose-response against a panel of T. cruzi stocks representing all current discrete typing units (DTUs), and a time-kill assay. Although less potent, the nitroheterocyclics and the oxaborole showed broad efficacy against all T. cruzi tested and were rapidly trypanocidal, whilst ergosterol biosynthesis inhibitors showed variable activity that was both compoundand strain-specific, and were unable to eradicate intracellular infection even after 7 days of continuous compound exposure at most efficacious concentrations. These findings contest previous reports of variable responses to nitroderivatives among different T. cruzi strains and further challenge the introduction of ergosterol biosynthesis inhibitors as new single chemotherapeutic agents for the treatment of Chagas disease. Inst Pasteur Korea, Ctr Neglected Dis Drug Discovery CND3, Songnam, South Korea Ctr Nacl Pesquisa Energia & Mat, Lab Nacl Biociencias LNBio, Campinas, SP, Brazil Universidade Federal de São Paulo UNIFESP, Depto Microbiol Imunol & Parasitol, São Paulo, Brazil Drugs Neglected Dis Initiat DNDi, Geneva, Switzerland Universidade Federal de São Paulo UNIFESP, Depto Microbiol Imunol & Parasitol, São Paulo, Brazil Korean government (MSIP), Gyeonggi-do: 2007-00559 Web of Science

Orientador: Maria Isabel Nogueira Cano Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: Telômeros são complexos DNA-proteínas que protegem os cromossomos eucarióticos da degradação, garantindo estabilidade genômica. As seqüências teloméricas são ricas em G e apresentam uma protrusão 3¿ simples-fita que se estende em direção ao terminal cromossômico. Em Leishmania, os telômeros são compostos pela seqüência repetida 5¿-TTAGGG-3¿ e são replicados pela telomerase, a principal responsável pela manutenção dos terminais cromossômicos em eucariotos. Além de replicar os telômeros, o complexo holoenzimático da telomerase, composto pela transcriptase reversa da telomerase (TERT), pelo RNA da telomerase (TER) e por proteínas associadas, também atua como parte de um complexo de ordem maior que protege os terminais teloméricos. O entendimento do mecanismo de regulação da manutenção telomérica será de grande valor científico e poderá levar ao descobrimento de algum alvo potencial para o desenvolvimento de novas drogas anti-leishmania. Com esse objetivo, identificamos, clonamos e caracterizamos o gene que codifica o componente TERT em Leishmania spp.. O alinhamento múltiplo das seqüências através do programa ClustalW demonstrou que as telomerases de Leishmania apresentam muito mais homologia entre si do que com as proteínas de outros kinetoplastídeos e eucariotos. Experimentos de caracterização indicaram que a seqüência putativa do gene da telomerase de Leishmania localiza-se provavelmente em cópia única nos maiores cromossomos. Um único transcrito de RNA mensageiro foi encontrado nos promastigotas. Análises filogenéticas sugeriram que a telomerase de Leishmania pode representar uma ligação entre os mais antigos e os mais novos ramos das telomerases. Além disso, proteínas recombinantes foram expressas em sistema bacteriano, tornando possível a produção de anticorpos policlonais em coelhos. Experimentos de ¿Western blotting¿ e imunoprecipitação de cromatina indicaram que o anticorpo foi capaz de reconhecer a proteína nativa e que a telomerase de L. amazonensis interage in vivo com a seqüência telomérica rica em G. A atividade de telomerase de L. amazonensis foi purificada utilizando-se uma combinação de colunas cromatográficas. Testou-se a atividade enzimática em cada passo da purificação utilizando-se o ensaio ¿Two-tube TRAP¿. Os resultados mostraram que a atividade enzimática é encontrada nas frações purificadas pelas cromatografias de troca iônica, de afinidade por Heparina e de gel filtração. A atividade foi altamente enriquecida após a purificação por afinidade utilizando um oligonucleotídeo de DNA telomérico rico em G. Quando foi utilizado um oligorribonucleotídeo 2¿O-metil complementar à putativa seqüência molde do TER de Leishmania como ligante na cromatografia de afinidade, pouca ou nenhuma atividade enzimática foi eluída da resina, sugerindo que a interação entre a telomerase de L. amazonensis e este oligorribonucleotídeo é tão forte que não permite sua dissociação nas condições de eluição gentis necessárias para manter a atividade enzimática. Formas procíclicas de Trypanosoma brucei foram utilizadas para a construção do sistema ¿PTP-tagging¿, no intuito de futuramente purificar o complexo holoenzimático da telomerase. Em paralelo, ensaios de ¿primer extension¿ foram padronizados e identificou-se uma seqüência candidata ao gene do RNA da telomerase de T. brucei. Também foi identificada e clonada em L. amazonensis, uma seqüência homóloga à PinX1 humana, descrita como uma proteína que interage diretamente com a TERT humana e considerada um inibidor natural da atividade de telomerase Abstract: Telomeres are protein-DNA complexes that protect linear chromosomes from degradation, providing genomic stability. The telomeric sequences are G-rich and contain a 3¿ single-stranded region that protrudes toward the chromosome end. In Leishmania, the telomeric DNA is composed by the conserved 5¿-TTAGGG-3¿ repeated sequence and it is replicated by telomerase. Telomerase is responsible for maintaining chromosome ends in most eucaryotes by adding new telomeric sequences to the G-rich strand. Besides replicating telomeres, the telomerase holoenzyme complex, composed by the reverse transcriptase component (TERT), the telomerase RNA (TER) and associated proteins, also works as part of the higher order complex that protects telomeric ends. Understanding the regulation of the telomeric maintainance mechanism may allow the discovery of potential targets to the development of new antileishmania drugs. Therefore, we identified, cloned and characterized the TERT gene in Leishmania spp.. A ClustalW multiple-sequence alignment demonstrated that the Leishmania telomerases show greater homology with each other than with the proteins of other kinetoplastids and eukaryotes. Characterization experiments indicated that the putative Leishmania TERT gene was probably located in single copy at the largest chromosomes. A single messenger RNA transcript was found in promastigotes. Phylogenetic analysis suggested that Leishmania telomerase might represent a liaison between the oldest and the newest branches of telomerases. Besides that, recombinant proteins were expressed in bacterial system, allowing production of anti-LaTERT polyclonal serum in rabbits. Western blotting and chromatin immunoprecipitation assays indicated that the anti-LaTERT serum was able to recognize a native protein in nuclear and total extracts of the parasite and that L. amazonensis telomerase interacts in vivo with the G-richtelomeric sequence. We have also purified the L. amazonensis telomerase activity in order to better understand its biochemical features. Protein extracts of L. amazonensis containing telomerase activity were purified using combined chromatographic columns. Enzyme activity was tested in each purification step using the ¿Two-tube TRAP¿ assay. The results showed that enzyme activity is found in fractions purified by ion exchange (DEAE), Heparin affinity and gel filtration chromatographic methods. The activity was greatly enriched after affinity purification using a G rich telomeric DNA oligonucleotide as the ligand. When a 2¿O-methyl oligoribonucleotide complementary to the putative L. amazonensis TER template was used as a ligand in the affinity purification, little or no enzyme activity was eluted from resin, suggesting that the interaction between L. amazonensis telomerase and this oligoribonucleotide is too strong that disables its dissociation under the gentle elution conditions necessary to maintain enzyme activity. In order to identify the telomerase holoenzyme components, procyclic forms of Trypanosoma brucei were used to construct the PTP-tagging system. ¿Primer extension¿ reactions were also done in order to isolate and sequence an RNA candidate for the telomerase RNA gene in T. brucei. In addition, we have cloned a L. amazonensis homologue of the human PinX1 protein, previously known as a hTERT-interacting factor and as a potent telomerase inhibitor Doutorado Genética de Microorganismos Doutor em Genética e Biologia Molecular

Studies of telomere structure and maintenance in trypanosomatids have provided insights into the evolutionary origin and conservation of some telomeric components shared by trypanosomes and vertebrates. For example, trypanosomatid telomeres are maintained by telomerase and consist of the canonical TTAGGG repeats, which in Trypanosoma brucei can form telomeric loops (t-loops). However, the telomeric chromatin of trypanosomatids is composed of organism-specific proteins and other proteins that share little sequence similarity with their vertebrate counterparts. Because telomere maintenance mechanisms are essential for genome stability, we propose that the particular features shown by the trypanosome telomeric chromatin hold the key for the design of antiparasitic drugs.

The Leishmania amazonensis telomerase gene was cloned by a polymerase chain reaction-based strategy using primers designed from a Leishmania major sequence that shared similarities with conserved telomerase motifs. The genes from three other species were cloned for comparative purposes. A ClustalW multiple-sequence alignment demonstrated that the Leishmania telomerases show greater homology with each other than with the proteins of other kinetoplastids and eukaryotes. Characterization experiments indicated that the putative Leishmania telomerase gene was probably in single copy and located in the largest chromosomes. A single messenger ribonucleic acid transcript was found in promastigotes. Phylogenetic analysis suggested that Leishmania telomerase might represent a liaison between the oldest and the newest branches of telomerases.