Collective and Diverted Total Synthesis of the Strasseriolides: A Family of Macrolides Endowed with Potent Antiplasmodial and AntitrypanosomalActivity.

The strasseriolide macrolides show promising in vitro and in vivo activities against P. falciparum and T. cruzi, the parasites causing malaria and Chagas disease, respectively. However, the as yet poor understanding of structure/activity relationships and the fact that one family member proved systemically toxic for unknown reasons render a more detailed assessment of these potential lead compounds difficult. To help overcome these issues, a collective total synthesis was devised. The key steps consisted of a ring closing alkyne metathesis (RCAM) reaction to forge a common macrocyclic intermediate followed by a hydroxy-directed ruthenium catalyzed trans-hydrostannation of the propargyl alcohol site thus formed. The resulting alkenyltin derivative served as the central node of the synthesis blueprint, which could be elaborated into the natural products themselves as well as into a set of non-natural analogues according to the concept of diverted total synthesis. The recorded biological data confirmed the potency of the compounds and showed the lack of noticeable cytotoxicity. The "northern" allylic alcohol subunit was recognized as an integral part of the pharmacophore, yet it provides opportunities for chemical modification.

Corrigendum: Identification of novel anti-amoebic pharmacophores from kinase inhibitor chemotypes.

[This corrects the article DOI: 10.3389/fmicb.2023.1149145.].

A nuclear orthologue of the dNTP triphosphohydrolase SAMHD1 controls dNTP homeostasis and genomic stability in Trypanosoma brucei.

Maintenance of dNTPs pools in Trypanosoma brucei is dependent on both biosynthetic and degradation pathways that together ensure correct cellular homeostasis throughout the cell cycle which is essential for the preservation of genomic stability. Both the salvage and de novo pathways participate in the provision of pyrimidine dNTPs while purine dNTPs are made available solely through salvage. In order to identify enzymes involved in degradation here we have characterized the role of a trypanosomal SAMHD1 orthologue denominated TbHD82. Our results show that TbHD82 is a nuclear enzyme in both procyclic and bloodstream forms of T. brucei. Knockout forms exhibit a hypermutator phenotype, cell cycle perturbations and an activation of the DNA repair response. Furthermore, dNTP quantification of TbHD82 null mutant cells revealed perturbations in nucleotide metabolism with a substantial accumulation of dATP, dCTP and dTTP. We propose that this HD domain-containing protein present in kinetoplastids plays an essential role acting as a sentinel of genomic fidelity by modulating the unnecessary and detrimental accumulation of dNTPs.

Strasseriolides display in vitro and in vivo activity against trypanosomal parasites and cause morphological and size defects in Trypanosoma cruzi.

Neglected diseases caused by kinetoplastid parasites are a health burden in tropical and subtropical countries. The need to create safe and effective medicines to improve treatment remains a priority. Microbial natural products are a source of chemical diversity that provides a valuable approach for identifying new drug candidates. We recently reported the discovery and bioassay-guided isolation of a novel family of macrolides with antiplasmodial activity. The novel family of four potent antimalarial macrolides, strasseriolides A-D, was isolated from cultures of Strasseria geniculata CF-247251, a fungal strain obtained from plant tissues. In the present study, we analyze these strasseriolides for activity against kinetoplastid protozoan parasites, namely, Trypanosoma brucei brucei, Leishmania donovani and Trypanosoma cruzi. Compounds exhibited mostly low activities against T. b. brucei, yet notable growth inhibition and selectivity were observed for strasseriolides C and D in the clinically relevant intracellular T. cruzi and L. donovani amastigotes with EC50 values in the low micromolar range. Compound C is fast-acting and active against both intracellular and trypomastigote forms of T. cruzi. While cell cycle defects were not identified, prominent morphological changes were visualized by differential interference contrast microscopy and smaller and rounded parasites were visualized upon exposure to strasseriolide C. Moreover, compound C lowers parasitaemia in vivo in acute models of infection of Chagas disease. Hence, strasseriolide C is a novel natural product active against different forms of T. cruzi in vitro and in vivo. The study provides an avenue for blocking infection of new cells, a strategy that could additionally contribute to avoid treatment failure.

Stepwise emergence of the neuronal gene expression program in early animal evolution.

The assembly of the neuronal and other major cell type programs occurred early in animal evolution. We can reconstruct this process by studying non-bilaterians like placozoans. These small disc-shaped animals not only have nine morphologically described cell types and no neurons but also show coordinated behaviors triggered by peptide-secreting cells. We investigated possible neuronal affinities of these peptidergic cells using phylogenetics, chromatin profiling, and comparative single-cell genomics in four placozoans. We found conserved cell type expression programs across placozoans, including populations of transdifferentiating and cycling cells, suggestive of active cell type homeostasis. We also uncovered fourteen peptidergic cell types expressing neuronal-associated components like the pre-synaptic scaffold that derive from progenitor cells with neurogenesis signatures. In contrast, earlier-branching animals like sponges and ctenophores lacked this conserved expression. Our findings indicate that key neuronal developmental and effector gene modules evolved before the advent of cnidarian/bilaterian neurons in the context of paracrine cell signaling.

Identification of novel anti-amoebic pharmacophores from kinase inhibitor chemotypes.

Acanthamoeba species, Naegleria fowleri, and Balamuthia mandrillaris are opportunistic pathogens that cause a range of brain, skin, eye, and disseminated diseases in humans and animals. These pathogenic free-living amoebae (pFLA) are commonly misdiagnosed and have sub-optimal treatment regimens which contribute to the extremely high mortality rates (>90%) when they infect the central nervous system. To address the unmet medical need for effective therapeutics, we screened kinase inhibitor chemotypes against three pFLA using phenotypic drug assays involving CellTiter-Glo 2.0. Herein, we report the activity of the compounds against the trophozoite stage of each of the three amoebae, ranging from nanomolar to low micromolar potency. The most potent compounds that were identified from this screening effort were: 2d (A. castellanii EC50: 0.92 ± 0.3 µM; and N. fowleri EC50: 0.43 ± 0.13 µM), 1c and 2b (N. fowleri EC50s: <0.63 µM, and 0.3 ± 0.21 µM), and 4b and 7b (B. mandrillaris EC50s: 1.0 ± 0.12 µM, and 1.4 ± 0.17 µM, respectively). With several of these pharmacophores already possessing blood-brain barrier (BBB) permeability properties, or are predicted to penetrate the BBB, these hits present novel starting points for optimization as future treatments for pFLA-caused diseases.

Antiparasitic Meroterpenoids Isolated from Memnoniella dichroa CF-080171.

Memnoniella is a fungal genus from which a wide range of diverse biologically active compounds have been isolated. A Memnoniella dichroa CF-080171 extract was identified to exhibit potent activity against Plasmodium falciparum 3D7 and Trypanosoma cruzi Tulahuen whole parasites in a high-throughput screening (HTS) campaign of microbial extracts from the Fundación MEDINA's collection. Bioassay-guided isolation of the active metabolites from this extract afforded eight new meroterpenoids of varying potencies, namely, memnobotrins C-E (1-3), a glycosylated isobenzofuranone (4), a tricyclic isobenzofuranone (5), a tetracyclic benzopyrane (6), a tetracyclic isobenzofuranone (7), and a pentacyclic isobenzofuranone (8). The structures of the isolated compounds were established by (+)-ESI-TOF high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. Compounds 1, 2, and 4 exhibited potent antiparasitic activity against P. falciparum 3D7 (EC50 0.04-0.243 µM) and T. cruzi Tulahuen (EC50 0.266-1.37 µM) parasites, as well as cytotoxic activity against HepG2 tumoral liver cells (EC50 1.20-4.84 µM). The remaining compounds (3, 5-8) showed moderate or no activity against the above-mentioned parasites and cells.

Antiparasitic Activities of Compounds Isolated from Aspergillus fumigatus Strain Discovered in Northcentral Nigeria.

In this study, we explored a fungal strain UIAU-3F identified as Aspergillus fumigatus isolated from soil samples collected from the River Oyun in Kwara State, Nigeria. In order to explore its chemical diversity, the fungal strain UIAU-3F was cultured in three different fermentation media, which resulted in different chemical profiles, evidenced by LC-ESI-MS-based metabolomics and multivariate analysis. The methanolic extract afforded two known compounds, fumitremorgin C (1) and pseurotin D (2). The in vitro antiparasitic assays of 1 against Trypanosoma cruzi and Plasmodium falciparum showed moderate activity with IC50 values of 9.6 µM and 2.3 µM, respectively, while 2 displayed IC50 values > 50 µM. Molecular docking analysis was performed on major protein targets to better understand the potential mechanism of the antitrypanosomal and antiplasmodial activities of the two known compounds.

Adar-mediated A-to-I editing is required for embryonic patterning and innate immune response regulation in zebrafish.

Adenosine deaminases (ADARs) catalyze the deamination of adenosine to inosine, also known as A-to-I editing, in RNA. Although A-to-I editing occurs widely across animals and is well studied, new biological roles are still being discovered. Here, we study the role of A-to-I editing in early zebrafish development. We demonstrate that Adar, the zebrafish orthologue of mammalian ADAR1, is essential for establishing the antero-posterior and dorso-ventral axes and patterning. Genome-wide editing discovery reveals pervasive editing in maternal and the earliest zygotic transcripts, the majority of which occurred in the 3'-UTR. Interestingly, transcripts implicated in gastrulation as well as dorso-ventral and antero-posterior patterning are found to contain multiple editing sites. Adar knockdown or overexpression affect gene expression by 12 hpf. Analysis of adar-/- zygotic mutants further reveals that the previously described role of Adar in mammals in regulating the innate immune response is conserved in zebrafish. Our study therefore establishes distinct maternal and zygotic functions of RNA editing by Adar in embryonic patterning along the zebrafish antero-posterior and dorso-ventral axes, and in the regulation of the innate immune response, respectively.

A phylogenetic and proteomic reconstruction of eukaryotic chromatin evolution.

Histones and associated chromatin proteins have essential functions in eukaryotic genome organization and regulation. Despite this fundamental role in eukaryotic cell biology, we lack a phylogenetically comprehensive understanding of chromatin evolution. Here, we combine comparative proteomics and genomics analysis of chromatin in eukaryotes and archaea. Proteomics uncovers the existence of histone post-translational modifications in archaea. However, archaeal histone modifications are scarce, in contrast with the highly conserved and abundant marks we identify across eukaryotes. Phylogenetic analysis reveals that chromatin-associated catalytic functions (for example, methyltransferases) have pre-eukaryotic origins, whereas histone mark readers and chaperones are eukaryotic innovations. We show that further chromatin evolution is characterized by expansion of readers, including capture by transposable elements and viruses. Overall, our study infers detailed evolutionary history of eukaryotic chromatin: from its archaeal roots, through the emergence of nucleosome-based regulation in the eukaryotic ancestor, to the diversification of chromatin regulators and their hijacking by genomic parasites.

Hepatitis C eradication improves cognitive function in patients with or without cirrhosis: A prospective real-life study.

BACKGROUND AND PURPOSE: Hepatitis C virus (HCV) infection is associated with neuropsychiatric disturbances that impact on functioning and health-related quality of life (HRQoL). Reversibility at different liver disease stages is unknown, particularly in cirrhosis. We aimed to evaluate cognition, functioning, and HRQoL following HCV eradication at different liver disease stages. METHODS: A random sample (n = 152) of consecutive patients treated with direct-acting antiviral agents (DAAs) between April 2015 and March 2017 were included. A comprehensive neuropsychological assessment, functioning and HRQoL questionnaires were applied at baseline, and 12 and 48 weeks after the end of antivirals. RESULTS: One-hundred thirty-five patients who achieved virological response completed the follow-up, of whom 44 had cirrhosis (27% decompensated). Twenty-one percent had cognitive impairment before starting DAAs (34.1% cirrhotic vs. 14.4% noncirrhotic, p < 0.011). Viral eradication was associated with a decrease in cognitive impairment to 23% of cirrhotic and 6% of noncirrhotic patients (p < 0.05). Interestingly, older patients (B = 0.11, 95% confidence interval [CI] = 0.03-0.19) with baseline cognitive impairment (B = 3.58, 95% CI = 1.54-5.62) were those with higher cognitive benefit, regardless of liver disease. Persistent cognitive impairment was associated with having higher cardiovascular risk, cirrhosis, lower education, and higher anxiety and depression scores. Functioning and HRQoL also improved after eradication but remained worse in the cirrhotic group. CONCLUSIONS: Viral eradication decreases the prevalence of cognitive impairment and improves functioning and HRQoL. Patients with lower brain reserve (older patients) and baseline cognitive impairment may benefit the most. Identification and treatment of HCV patients through screening programs may reduce the burden of cognitive disturbances beyond the prevention of liver disease progression.

New Compounds with Bioisosteric Replacement of Classic Choline Kinase Inhibitors Show Potent Antiplasmodial Activity.

In the fight against Malaria, new strategies need to be developed to avoid resistance of the parasite to pharmaceutics and other prevention barriers. Recently, a Host Directed Therapy approach based on the suppression of the starting materials uptake from the host by the parasite has provided excellent results. In this article, we propose the synthesis of bioisosteric compounds that are capable of inhibiting Plasmodium falciparum Choline Kinase and therefore to reduce choline uptake, which is essential for the development of the parasite. Of the 41 bioisosteric compounds reported herein, none showed any influence of the linker on the antimalarial and enzyme inhibitory activity, whereas an effect of the type of cationic heads used could be observed. SARs determined that the thienopyrimidine substituted in 4 by a pyrrolidine is the best scaffold, independently of the chosen linker. The decrease in lipophilicity seems to improve the antimalarial activity but to cause an opposite effect on the inhibition of the enzyme. While potent compounds with similar good inhibitory values have been related to the proposed mechanism of action, some of them still show discrepancies and further studies are needed to determine their specific molecular target.

HCV eradication in recurrent hepatitis C after liver transplantation normalizes enhanced endothelial activation.

The increased risk of cardiovascular disease (CVD) conferred by hepatitis C virus (HCV) is especially relevant after liver transplantation (LT), but its mechanism is still not well defined. This study aimed to evaluate the influence of HCV eradication in inflammatory and endothelial activation markers after LT. We evaluated inflammatory (TNF-alfa, IL-6, IL-8, and MCP-1) and endothelial activation (E-selectin, ICAM-1, VCAM-1, and MMP-9) markers before and after eradication in 45 LT recipients with HCV infection (LT+/HCV+) and 44 non-transplanted HCV-infected patients (LT-/HCV+). We also considered an additional group of 40 LT recipients without HCV infection (LT+/HCV-). LT+/HCV+ patients presented a higher endothelial activation status before eradication compared with LT+/HCV- patients. However, levels of E-selectin, ICAM-1, VCAM-1, and MMP-9 were comparable between LT+/HCV+ and LT-/HCV+ patients before eradication. HCV eradication decreased ICAM-1 (5466.55 pg/ml vs. 3354.88 pg/ml, P < 0.001) and VCAM-1 (10456.52 pg/ml vs. 6658.85 pg/ml, P < 0.001) levels in LT+/HCV+ and LT-/HCV+ patients. Remarkably, HCV eradication restored levels of endothelial activation markers of LT+/HCV+ patients compared with that of LT+/HCV- patients. HCV plays a major role in endothelial dysfunction after LT. Furthermore, HCV eradication restores endothelial activation despite the exposure to immunosuppressive therapy.

Lead Optimization of 3,5-Disubstituted-7-Azaindoles for the Treatment of Human African Trypanosomiasis.

Neglected tropical diseases such as human African trypanosomiasis (HAT) are prevalent primarily in tropical climates and among populations living in poverty. Historically, the lack of economic incentive to develop new treatments for these diseases has meant that existing therapeutics have serious shortcomings in terms of safety, efficacy, and administration, and better therapeutics are needed. We now report a series of 3,5-disubstituted-7-azaindoles identified as growth inhibitors of Trypanosoma brucei, the parasite that causes HAT, through a high-throughput screen. We describe the hit-to-lead optimization of this series and the development and preclinical investigation of 29d, a potent antitrypanosomal compound with promising pharmacokinetic (PK) parameters. This compound was ultimately not progressed beyond in vivo PK studies due to its inability to penetrate the blood-brain barrier (BBB), critical for stage 2 HAT treatments.

Pipecolisporin, a Novel Cyclic Peptide with Antimalarial and Antitrypanosome Activities from a Wheat Endophytic Nigrospora oryzae.

A novel cyclic antimalarial and antitrypanosome hexapeptide, pipecolisporin (1), was isolated from cultures of Nigrospora oryzae CF-298113, a fungal endophyte isolated from roots of Triticum sp. collected in a traditional agricultural land of Montefrío, Granada, Spain. The structure of this compound, including its absolute configuration, was elucidated by HRMS, 1-D and 2-D NMR spectroscopy, and Marfey's analysis. This metabolite displayed interesting activity against Plasmodium falciparum and Trypanosoma cruzi, with IC50 values in the micromolar range, and no significant cytotoxicity against the human cancer cell lines A549, A2058, MCF7, MIA PaCa-2, and HepG2.

Huntington's disease brain-derived small RNAs recapitulate associated neuropathology in mice.

Progressive motor alterations and selective death of striatal medium spiny neurons (MSNs) are key pathological hallmarks of Huntington's disease (HD), a neurodegenerative condition caused by a CAG trinucleotide repeat expansion in the coding region of the huntingtin (HTT) gene. Most research has focused on the pathogenic effects of the resultant protein product(s); however, growing evidence indicates that expanded CAG repeats within mutant HTT mRNA and derived small CAG repeat RNAs (sCAG) participate in HD pathophysiology. The individual contribution of protein versus RNA toxicity to HD pathophysiology remains largely uncharacterized and the role of other classes of small RNAs (sRNA) that are strongly perturbed in HD is uncertain. Here, we demonstrate that sRNA produced in the putamen of HD patients (HD-sRNA-PT) are sufficient to induce HD pathology in vivo. Mice injected with HD-sRNA-PT show motor abnormalities, decreased levels of striatal HD-related proteins, disruption of the indirect pathway, and strong transcriptional abnormalities, paralleling human HD pathology. Importantly, we show that the specific blockage of sCAG mitigates HD-sRNA-PT neurotoxicity only to a limited extent. This observation prompted us to identify other sRNA species enriched in HD putamen with neurotoxic potential. We detected high levels of tRNA fragments (tRFs) in HD putamen, and we validated the neurotoxic potential of an Alanine derived tRF in vitro. These results highlight that HD-sRNA-PT are neurotoxic, and suggest that multiple sRNA species contribute to striatal dysfunction and general transcriptomic changes, favoring therapeutic strategies based on the blockage of sRNA-mediated toxicity.

A Mitochondrial Orthologue of the dNTP Triphosphohydrolase SAMHD1 Is Essential and Controls Pyrimidine Homeostasis in Trypanosoma brucei.

The maintenance of deoxyribonucleotide triphosphate (dNTP) homeostasis through synthesis and degradation is critical for accurate genomic and mitochondrial DNA replication fidelity. Trypanosoma brucei makes use of both the salvage and de novo pathways for the provision of pyrimidine dNTPs. In this respect, the sterile α motif and histidine-aspartate domain-containing protein 1 (SAMHD1) appears to be the most relevant dNTPase controlling dNTP/deoxynucleoside homeostasis in mammalian cells. Here, we have characterized the role of a unique trypanosomal SAMHD1 orthologue denominated TbHD52. Our results show that TbHD52 is a mitochondrial enzyme essential in bloodstream forms of T. brucei. Knockout cells are pyrimidine auxotrophs that exhibit strong defects in genomic integrity, cell cycle progression, and nuclear DNA and kinetoplast segregation in the absence of extracellular thymidine. The lack of TbHD52 can be counteracted by the overexpression of human dCMP deaminase, an enzyme that is directly involved in dUMP formation yet absent in trypanosomes. Furthermore, the cellular dNTP quantification and metabolomic analysis of TbHD52 null mutants revealed perturbations in the nucleotide metabolism with a substantial accumulation of dCTP and cytosine-derived metabolites while dTTP formation was significantly reduced. We propose that this HD-domain-containing protein unique to kinetoplastids plays an essential role in pyrimidine dNTP homeostasis and contributes to the provision of deoxycytidine required for cellular dTTP biosynthesis.

An epitope-based approach of HLA-matched platelets for transfusion: a noninferiority crossover randomized trial.

Platelet transfusion refractoriness results in adverse outcomes and increased health care costs. Managing refractoriness resulting from HLA alloimmunization necessitates the use of HLA antigen-matched platelets but requires a large platelet donor pool and does not guarantee full matching. We report the first randomized, double-blind, noninferiority, crossover trial comparing HLA epitope-matched (HEM) platelets with HLA standard antigen-matched (HSM) platelet transfusions. Alloimmunized, platelet-refractory, thrombocytopenic patients with aplastic anemia, myelodysplastic syndrome, or acute myeloid leukemia were eligible. HEM platelets were selected using HLAMatchMaker epitope (specifically eplet) matching. Patients received up to 8 prophylactic HEM and HSM transfusions provided in random order. The primary outcome was 1-hour posttransfusion platelet count increment (PCI). Forty-nine patients were randomized at 14 UK hospitals. For intention to treat, numbers of evaluable transfusions were 107 and 112 for HEM and HSM methods, respectively. Unadjusted mean PCIs for HEM and HSM methods were 23.9 (standard deviation [SD], 15) and 23.5 (SD, 14.1), respectively (adjusted mean difference, -0.1; 95% confidence interval [CI], -2.9 to 2.8). Because the lower limit of the 95% CI was not greater than the predefined noninferiority limit, the HEM approach was declared noninferior to the HSM approach. There were no differences in secondary outcomes of platelet counts, transfusion requirements, and bleeding events. Adequate 1-hour PCI was more frequently observed, with a mean number of 3.2 epitope mismatches, compared with 5.5 epitope mismatches for inadequate 1-hour increments. For every additional epitope mismatch, the likelihood of an adequate PCI decreased by 15%. Epitope-matched platelets should be considered to support HLA alloimmunized patients. This trial was registered at www.isrctn.com as #ISRCTN23996532.

Next generation sequencing of 11 HLA loci characterises a diverse UK cord blood bank.

The introduction of next generation sequencing (NGS) for stem cell donor registry typing has contributed to faster identification of compatible stem cell donors. However, the successful search for a matched unrelated donor for some patient groups is still affected by their ethnicity. In this study, DNA samples from 714 National Health Service (NHS) Cord Blood Bank donors were typed for HLA-A, -B, -C, -DRB1, -DRB345, -DQA1, -DQB1, -DPA1 and -DPB1 by NGS. Analysis of the ethnic diversity showed a high level of diversity, with the cohort comprising of 62.3% European and 37.7% of either multi-ethnic or non-European donors, of which 12.3% were multi-ethnic. The HLA diversity was further confirmed using PyPop analysis, 405 distinct alleles were observed in the overall NHS-CBB cohort, of which 37 alleles are non-CWD, including A*31:14N, B*35:68:02, C*14:23 and DQA1*05:10. Furthermore, HLA-DQA1 and HLA-DPA1 analysis showed 12% and 10%, respectively, of the alleles currently submitted to IMGT, confirming further diversity of the NHS-CBB cohort. The application of 11 HLA loci resolution by NGS revealed a high level of diversity in the NHS-CBB cohort. The incorporation of this data coupled with ethnicity data could lead to improved donor selection, contributing to better clinical outcomes for patients.

Allele-specific expression changes dynamically during T cell activation in HLA and other autoimmune loci.

Genetic studies have revealed that autoimmune susceptibility variants are over-represented in memory CD4+ T cell regulatory elements1-3. Understanding how genetic variation affects gene expression in different T cell physiological states is essential for deciphering genetic mechanisms of autoimmunity4,5. Here, we characterized the dynamics of genetic regulatory effects at eight time points during memory CD4+ T cell activation with high-depth RNA-seq in healthy individuals. We discovered widespread, dynamic allele-specific expression across the genome, where the balance of alleles changes over time. These genes were enriched fourfold within autoimmune loci. We found pervasive dynamic regulatory effects within six HLA genes. HLA-DQB1 alleles had one of three distinct transcriptional regulatory programs. Using CRISPR-Cas9 genomic editing we demonstrated that a promoter variant is causal for T cell-specific control of HLA-DQB1 expression. Our study shows that genetic variation in cis-regulatory elements affects gene expression in a manner dependent on lymphocyte activation status, contributing to the interindividual complexity of immune responses.