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Trypanosoma cruzi is a protozoan parasite and the etiological agent of Chagas disease, a debilitating and sometimes fatal disease that continues to spread to new areas. Yet, Chagas disease is still only treated with two related nitro compounds that are insufficiently effective and cause severe side effects. Nucleotide metabolism is one of the known vulnerabilities of T. cruzi, as they are auxotrophic for purines, and nucleoside analogues have been shown to have genuine promise against this parasite in vitro and in vivo. Since purine antimetabolites require efficient uptake through transporters, we here report a detailed characterisation of the T. cruzi NB1 nucleobase transporter with the aim of elucidating the interactions between TcrNB1 and its substrates and finding the positions that can be altered in the design of novel antimetabolites without losing transportability. Systematically determining the inhibition constants (K i ) of purine analogues for TcrNB1 yielded their Gibbs free energy of interaction, ΔG 0 . Pairwise comparisons of substrate (hypoxanthine, guanine, adenine) and analogues allowed us to determine that optimal binding affinity by TcrNB1 requires interactions with all four nitrogen residues of the purine ring, with N1 and N9, in protonation state, functioning as presumed hydrogen bond donors and unprotonated N3 and N7 as hydrogen bond acceptors. This is the same interaction pattern as we previously described for the main nucleobase transporters of Trypanosoma brucei spp. and Leishmania major and makes it the first of the ENT-family genes that is functionally as well as genetically conserved between the three main kinetoplast pathogens., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Vaginal foreign bodies represent a clinical and diagnostical challenge in pediatric gynecology. Several case reports, case series and retrospective studies have been published, highlighting rare or complex cases. A comprehensive systematic review is lacking., Methods: Published English-language articles on vaginal foreign objects in patients aged 16 years and younger, with full-text availability were included. Articles on adult patients and patients with an object migrating from the abdominal cavity into the vagina were excluded., Results: Out of the 215 screened articles 75 were included, comprising a total of 522 patients. The age ranged from 6 months to 16 years, with an average of 6 years and 3 months. The presenting symptoms were documented in 340 patients, with the two most common being vaginal bleeding (n = 172) and vaginal discharge (n = 134). Toilet paper or tissue was the most common object, in 155 out of 447 patients. Ultrasonography was the most utilized diagnostic method, with a sensitivity of 79.9 %. Radiography showed more false-negative than true-positive results, with a sensitivity of 33.3 %. Complications were reported in 35 patients. Evidence of sexual abuse was found in a small group of 16 patients. Vaginoscopy under sedation was the most frequently used therapeutic approach., Conclusion: A swift and accurate diagnosis is crucial, with clinical examination and ultrasonography playing pivotal roles. Vaginoscopy is the gold standard for definitive diagnosis and therapy. Attention should be given to a potential context of sexual abuse., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Although various types of mRNA-based vaccines have been explored, the optimal conditions for induction of both humoral and cellular immunity remain rather unknown. In this study, mRNA vaccines of nucleoside-modified mRNA in lipoplexes (LPXs) or lipid nanoparticles (LNPs) were evaluated after administration in mice through different routes, assessing mRNA delivery, tolerability and immunogenicity. In addition, we investigated whether mRNA vaccines could benefit from the inclusion of the adjuvant alpha-galactosylceramide (αGC), an invariant Natural Killer T (iNKT) cell ligand. Intramuscular (IM) vaccination with ovalbumin (OVA)-encoding mRNA encapsulated in LNPs adjuvanted with αGC showed the highest antibody- and CD8 + T cell responses. Furthermore, we observed that addition of signal peptides and endocytic sorting signals of either LAMP1 or HLA-B7 in the OVA-encoding mRNA sequence further enhanced CD8 + T cell activation although reducing the induction of IgG antibody responses. Moreover, mRNA LNPs with the ionizable lipidoid C12-200 exhibited higher pro-inflammatory- and reactogenic activity compared to mRNA LNPs with SM-102, correlating with increased T cell activation and antitumor potential. We also observed that αGC could further enhance the cellular immunity of clinically relevant mRNA LNP vaccines, thereby promoting therapeutic antitumor potential. Finally, a Listeria monocytogenes mRNA LNP vaccine supplemented with αGC showed synergistic protective effects against listeriosis, highlighting a key advantage of co-activating iNKT cells in antibacterial mRNA vaccines. Taken together, our study offers multiple insights for optimizing the design of mRNA vaccines for disease applications, such as cancer and intracellular bacterial infections., Competing Interests: Declaration of competing interest R.V., I.L. and S.D.S. are contributors to patent applications no. WO2020058239A1; Therapeutic nanoparticles and methods of use thereof. and no. WO2023209103; Prevention and treatment of infections with intracellular bacteria., together with I.A., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Leishmania is a trypanosomatid parasite that causes skin lesions in its cutaneous form. Current therapies rely on old and expensive drugs, against which the parasites have acquired considerable resistance. Trypanosomatids are unable to synthesize purines relying on salvaging from the host, and nucleoside analogues have emerged as attractive antiparasitic drug candidates. 4-Methyl-7-β-D-ribofuranosyl-7H-pyrrolo[2,3-d]pyrimidine (CL5564), an analogue of tubercidin in which the amine has been replaced by a methyl group, demonstrates activity against Trypanosoma cruzi and Leishmania infantum . Herein, we investigated its in vitro and in vivo activity against L. amazonensis . CL5564 was 6.5-fold ( P = 0.0002) more potent than milteforan™ (ML) against intracellular forms in peritoneal mouse macrophages, and highly selective, while combination with ML gave an additive effect. These results stimulated us to study the activity of CL5564 in mouse model of cutaneous Leishmania infection. BALB/c female and male mice infected by L. amazonensis treated with CL5564 (10 mg kg −1 , intralesional route for five days) presented a >93% reduction of paw lesion size likely ML given orally at 40 mg kg −1 , while the combination (10 + 40 mg kg −1 of CL5564 and ML, respectively) caused >96% reduction. The qPCR confirmed the suppression of parasite load, but only the combination approach reached 66% of parasitological cure. These results support additional studies with nucleoside derivatives.

Various purine-based nucleoside analogues have demonstrated unexpected affinity for nonpurinergic G protein-coupled receptors (GPCRs), such as opioid and serotonin receptors. In this work, we synthesized a small library of new 7-deazaadenosine and pyrazolo[3,4- d ]pyrimidine riboside analogues, featuring dual C7 and N 6 modifications and assessed their affinity for various GPCRs. During the course of the synthesis of 7-ethynyl pyrazolo[3,4- d ]pyrimidine ribosides, we observed the formation of an unprecedented tricyclic nucleobase, formed via a 6- endo-dig ring closure. The synthesis of this tricyclic nucleoside was optimized, and the substrate scope for such cyclization was further explored because it might avail further exploration in the nucleoside field. From displacement experiments on a panel of GPCRs and transporters, combining C7 and N 6 modifications afforded noncytotoxic nucleosides with micromolar and submicromolar affinity for different GPCRs, such as the 5-hydroxytryptamine (5-HT) 2B , κ-opioid (KOR), and σ 1/2 receptor. These results corroborate that the novel nucleoside analogues reported here are potentially useful starting points for the further development of modulators of GPCRs and transmembrane proteins., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

In the past decades an increasing number of studies revealed that G protein-coupled receptors (GPCRs) are capable of forming dimers or even higher-ordered oligomers, which may modulate receptor function and act as potential drug targets. In this review, we briefly summarized the design strategy of bivalent GPCR ligands and mainly focused on how to use them to study and/or detect GPCP dimerization in vitro and in vivo. Bivalent ligands show specific properties relative to their corresponding monomeric ligands because they are able to bind to GPCR homodimers or heterodimers simultaneously. For example, bivalent ligands with optimal length of spacers often exhibited higher binding affinities for dimers compared to that of monomers. Furthermore, bivalent ligands displayed specific signal transduction compared to monovalent ligands. Finally, we give our perspective on targeting GPCR dimers from traditional bivalent ligands to more drug-like small molecules., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Rationale: Although promising responses are obtained in patients treated with immune checkpoint inhibitors targeting programmed death ligand 1 (PD-L1) and its receptor programmed death-1 (PD-1), only a fraction of patients benefits from this immunotherapy. Cancer vaccination may be an effective approach to improve the response to immune checkpoint inhibitors anti-PD-L1/PD-1 therapy. However, there is a lack of research on the dynamics of PD-L1 expression in response to cancer vaccination. Methods: We performed non-invasive whole-body imaging to visualize PD-L1 expression at different timepoints after vaccination of melanoma-bearing mice. Mice bearing ovalbumin (OVA) expressing B16 tumors were i.v. injected with the Galsome mRNA vaccine: OVA encoding mRNA lipoplexes co-encapsulating a low or a high dose of the atypical adjuvant α-galactosylceramide (αGC) to activate invariant natural killer T (iNKT) cells. Serial non-invasive whole-body immune imaging was performed using a technetium-99m ( 99m Tc)-labeled anti-PD-L1 nanobody, single-photon emission computerized tomography (SPECT) and X-ray computed tomography (CT) images were quantified. Additionally, cellular expression of PD-L1 was evaluated with flow cytometry. Results: SPECT/CT-imaging showed a rapid and systemic upregulation of PD-L1 after vaccination. PD-L1 expression could not be correlated to the αGC-dose, although we observed a dose-dependent iNKT cell activation. Dynamics of PD-L1 expression were organ-dependent and most pronounced in lungs and liver, organs to which the vaccine was distributed. PD-L1 expression in lungs increased immediately after vaccination and gradually decreased over time, whereas in liver, vaccination-induced PD-L1 upregulation was short-lived. Flow cytometric analysis of these organs further showed myeloid cells as well as non-immune cells with elevated PD-L1 expression in response to vaccination. SPECT/CT imaging of the tumor demonstrated that the expression of PD-L1 remained stable over time and was overall not affected by vaccination although flow cytometric analysis at the cellular level demonstrated changes in PD-L1 expression in various immune cell populations following vaccination. Conclusion: Repeated non-invasive whole-body imaging using 99m Tc-labeled anti-PD-L1 nanobodies allows to document the dynamic nature of PD-L1 expression upon vaccination. Galsome vaccination rapidly induced systemic upregulation of PD-L1 expression with the most pronounced upregulation in lungs and liver while flow cytometry analysis showed upregulation of PD-L1 in the tumor microenvironment. This study shows that imaging using nanobodies may be useful for monitoring vaccine-mediated PD-L1 modulation in patients and could provide a rationale for combination therapy. To the best of our knowledge, this is the first report that visualizes PD-L1 expression upon cancer vaccination., Competing Interests: Competing Interests: Nick Devoogdt and Marleen Keyaerts are founders and shareholders in Abscint. Nick Devoogdt is a consultant for Precirix and reports grants, personal fees, and non-financial support from Precirix, and non-financial support from Abscint. Marleen Keyaerts and Karine Breckpot have ongoing collaborations with Precerix for which they receive non-financial support. Karine Breckpot, Katrijn Broos, Nick Devoogdt and Marleen Keyaerts hold a patent on 'Human pd-l1-binding immunoglobulins' (WO2019166622A1). Ine Lentacker, Stefaan De Smedt, Rein Verbeke and Heleen Dewitte have a patent application on 'Therapeutic nanoparticles and methods of use thereof”. (WO2020/058239)., (© The author(s).)

Acinetobacter baumannii causes a wide range of infections, including wound infections. Multidrug-resistant A. baumannii is a major healthcare concern and the development of novel treatments against these infections is needed. Fosmidomycin is a repurposed antimalarial drug targeting the non-mevalonate pathway, and several derivatives show activity toward A. baumannii . We evaluated the antimicrobial activity of CC366, a fosmidomycin prodrug, against a collection of A. baumannii strains, using various in vitro and in vivo models; emphasis was placed on the evaluation of its anti-biofilm activity. We also developed a 3D-printed wound dressing containing CC366, using melt electrowriting technology. Minimal inhibitory concentrations of CC366 ranged from 1 to 64 μg/mL, and CC366 showed good biofilm inhibitory and moderate biofilm eradicating activity in vitro . CC366 successfully eluted from a 3D-printed dressing, the dressings prevented the formation of A. baumannnii wound biofilms in vitro and reduced A. baumannii infection in an in vivo mouse model., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

African Animal Trypanosomiasis (AAT), caused predominantly by Trypanosoma brucei brucei , T. vivax and T. congolense , is a fatal livestock disease throughout Sub-Saharan Africa. Treatment options are very limited and threatened by resistance. Tubercidin (7-deazaadenosine) analogs have shown activity against individual parasites but viable chemotherapy must be active against all three species. Divergence in sensitivity to nucleoside antimetabolites could be caused by differences in nucleoside transporters. Having previously characterized the T. brucei nucleoside carriers, we here report the functional expression and characterization of the main adenosine transporters of T. vivax (TvxNT3) and T. congolense (TcoAT1/NT10), in a Leishmania mexicana cell line ('SUPKO') lacking adenosine uptake. Both carriers were similar to the T. brucei P1-type transporters and bind adenosine mostly through interactions with N3, N7 and 3'-OH. Expression of TvxNT3 and TcoAT1 sensitized SUPKO cells to various 7-substituted tubercidins and other nucleoside analogs although tubercidin itself is a poor substrate for P1-type transporters. Individual nucleoside EC 50 s were similar for T. b. brucei , T. congolense , T. evansi and T. equiperdum but correlated less well with T. vivax . However, multiple nucleosides including 7-halogentubercidines displayed pEC50>7 for all species and, based on transporter and anti-parasite SAR analyses, we conclude that nucleoside chemotherapy for AAT is viable.

Aurora kinase A (AURKA) is a well-established target in neuroblastoma (NB) due to both its catalytic functions during mitosis and its kinase-independent functions, including stabilization of the key oncoprotein MYCN. We present a structure-activity relationship (SAR) study of MK-5108-derived PROTACs against AURKA by exploring different linker lengths and exit vectors on the thalidomide moiety. PROTAC SK2188 induces the most potent AURKA degradation (DC 50,24h 3.9 nM, D max,24h 89%) and shows an excellent binding and degradation selectivity profile. Treatment of NGP neuroblastoma cells with SK2188 induced concomitant MYCN degradation, high replication stress/DNA damage levels and apoptosis. Moreover, SK2188 significantly outperforms the parent inhibitor MK-5108 in a cell proliferation screen and patient-derived organoids. Furthermore, altering the attachment point of the PEG linker to the 5-position of thalidomide allowed us to identify a potent AURKA degrader with a linker as short as 2 PEG units. With this, our SAR-study provides interesting lead structures for further optimization and validation of AURKA degradation as a potential therapeutic strategy in neuroblastoma., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Fosmidomycin is a natural antibiotic with potent IspC (DXR, 1-deoxy-d-xylulose-5-phosphate reductoisomerase) inhibitory activity. This enzyme catalyzes the first committed step of the non-mevalonate isoprenoid biosynthesis pathway, which is essential in most bacteria, including A. baumanii and M. tuberculosis, and apicomplexan parasites, including Plasmodium parasites. Mainly as a result of its high polarity, fosmidomycin displays suboptimal pharmacokinetic properties. Furthermore, fosmidomycin is inactive against A. baumannii and M. tuberculosis as a result of its inability to penetrate the bacterial cell wall. Temporarily masking the phosphonate moiety as a prodrug has the potential to solve both issues. We report on the expansion of the acyloxymethyl and alkoxycarbonyloxymethyl phosphonate ester prodrug series of a fosmidomycin surrogate. Prodrug promoieties were designed based on electronic, lipophilic and siderophoric properties. This investigation led to the discovery of derivatives with two-digit nanomolar and submicromolar IC 50 -values against P. falciparum and A. baumanii, respectively., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Chagas disease, caused by Trypanosoma cruzi (T. cruzi), is a serious public health problem. Current treatment is restricted to two drugs, benznidazole and nifurtimox, displaying serious efficacy and safety drawbacks. Nucleoside analogues represent a promising alternative as protozoans do not biosynthesize purines and rely on purine salvage from the hosts. Protozoan transporters often present different substrate specificities from mammalian transporters, justifying the exploration of nucleoside analogues as therapeutic agents. Previous reports identified nucleosides with potent trypanocidal activity; therefore, two 7-derivatized tubercidins (FH11706, FH10714) and a 3′-deoxytubercidin (FH8513) were assayed against T. cruzi. They were highly potent and selective, and the uptake of the tubercidin analogues appeared to be mediated by the nucleoside transporter TcrNT2. At 10 μM, the analogues reduced parasitemia >90% in 2D and 3D cardiac cultures. The washout assays showed that FH10714 sterilized the infected cultures. Given orally, the compounds did not induce noticeable mouse toxicity (50 mg/kg), suppressed the parasitemia of T. cruzi-infected Swiss mice (25 mg/kg, 5 days) and presented DNA amplification below the limit of detection. These findings justify further studies with longer treatment regimens, as well as evaluations in combination with nitro drugs, aiming to identify more effective and safer therapies for Chagas disease.

Among the scarce validated drug targets against Chagas disease (CD), caused by Trypanosoma cruzi , the parasite's nucleoside salvage system has recently attracted considerable attention. Although the trypanocidal activity of tubercidin (7-deazapurine) has long been known, the identification of a class of 7-substituted tubercidin analogs with potent in vitro and in vivo activity and much-enhanced selectivity has made nucleoside analogs among the most promising lead compounds against CD. Here, we investigate the recently identified TcrNT2 nucleoside transporter and its potential role in antimetabolite chemotherapy. TcrNT2, expressed in a Leishmania mexicana cell line lacking the NT1 nucleoside transporter locus, displayed very high selectivity and affinity for thymidine with a K m of 0.26 ± 0.05 µM. The selectivity was explained by interactions of 2-oxo, 4-oxo, 5-Me, 3'-hydroxy and 5'-hydroxy with the transporter binding pocket, whereas a hydroxy group at the 2' position was deleterious to binding. This made 5-halogenated 2'-deoxyuridine analogues good substrates but 5-F-2'-deoxyuridine displayed disappointing activity against T. cruzi trypomastigotes. By comparing the EC 50 values of tubercidin and its 7-substituted analogues against L. mexicana Cas9, Cas9 ΔNT1 and Cas9 ΔNT1+TcrNT2 it was shown that TcrNT2 can take up tubercidin and, at a minimum, a subset of the analogs.

We report the design, synthesis, and validation of the novel compound photocaged N 6 -cyclopentyladenosine (cCPA) to achieve precisely localized and timed release of the parent adenosine A1 receptor agonist CPA using 405 nm light. G i protein-coupled A 1 receptors (A 1 Rs) modulate neurotransmission via pre- and post-synaptic routes. The dynamics of the CPA-mediated effect on neurotransmission, characterized by fast activation and slow recovery, make it possible to implement a closed-loop control paradigm. The strength of neurotransmission is monitored as the amplitude of stimulus-evoked local field potentials. It is used for feedback control of light to release CPA. This system makes it possible to regulate neurotransmission to a pre-defined level in acute hippocampal brain slices incubated with 3 µM cCPA. This novel approach of closed-loop photopharmacology holds therapeutic potential for fine-tuned control of neurotransmission in diseases associated with neuronal hyperexcitability.

Animal trypanosomiasis (AT) is a parasitic disease with high socio-economic impact. Given the limited therapeutic options and problems of toxicity and drug resistance, this study assessed redirecting our previously identified antitrypanosomal nucleosides for the treatment of AT. Promising hits were identified with excellent in vitro activity across all important animal trypanosome species. Compound 7, an inosine analogue, and our previously described lead compound, 3'-deoxytubercidin (8), showed broad spectrum anti-AT activity, metabolic stability in the target host species and absence of toxicity, but with variable efficacy ranging from limited activity to full cure in mouse models of Trypanosoma congolense and T. vivax infection. Several compounds show promise against T. evansi (surra) and T. equiperdum (dourine). Given the preferred target product profile for a broad-spectrum compound against AT, this study emphasizes the need to include T. vivax in the screening cascade given its divergent susceptibility profile and provides a basis for lead optimization towards such broad spectrum anti-AT compound., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Leishmaniasis causes high mortality and morbidity in tropical and subtropical regions of Africa, Asia, the Americas and southern Europe, and is characterized by diverse clinical manifestations. As a neglected tropical disease, limited resources are allocated for antileishmanial drug discovery. The Leishmania parasite is deficient in de novo purine synthesis, and therefore acquires purines from the host and processes these using a purine salvage pathway. By making use of purine transport systems and interfering with this salvage pathway, purine (nucleoside) analogues might exert a selective detrimental impact on its growth and survival. In vitro screening of an in-house purine nucleoside library and analogue synthesis afforded the 6-methyl-7-(2-pyridyl)-7-deazapurine ribonucleoside analogue 18 as a promising hit. Optimization of the 7-substituent afforded 31 and 32 which displayed potent activity against wild-type and resistant L. infantum, intracellular amastigote and extracellular promastigote forms, and favorable selectivity versus primary mouse macrophages (Mφ) and MRC-5 cells. Encouraged by the favorable in vitro metabolic stability of 32, an in vivo study was performed using an early curative L. infantum hamster model. When orally administrated at 50 mg/kg once daily (s.i.d) for 10 days, 32 was devoid of side effects, however, it only poorly reduced amastigote burdens in the major target organs., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Profiling of the illicit fentanyl supply is invaluable from surveillance and intelligence perspectives. An important strategy includes the study of chemical attribution signatures (e.g., trace amounts of synthesis precursors, impurities/byproducts in seized material and metabolites in biological samples). This information provides valuable insight into the employed synthesis routes at the heart of illicit fentanyl manufacture (previously mainly the so-called Janssen or Siegfried methods), allowing to track and ultimately regulate crucial precursors. This report focuses on phenethyl-4-anilino-N-phenethylpiperidine (phenethyl-4-ANPP), a formerly unknown compound that was identified for the first time in a fentanyl powder sample seized in April 2019, followed by its identification in a biological sample in December 2019. Between 2019-Q4 and 2020-Q3, phenethyl-4-ANPP was detected in 25/1,054 fentanyl cases in the USA. There are currently no reports on how this compound may have ended up in illicit drug preparations and whether its presence may have potential in vivo relevance. We propose three possible fentanyl synthesis routes that, when badly executed in a single reaction vessel, may involve the formation of phenethyl-4-ANPP. We hypothesize that the presence of the latter is the result of a shift in fentanyl synthesis routes in an attempt to circumvent restrictions on previously used precursors. Using a cell-based µ-opioid receptor recruitment assay, we show that the extent of MOR activation caused by 100 µM phenethyl-4-ANPP is comparable to that exerted by a roughly 100,000-fold lower concentration of fentanyl (0.001 µM or 0.336 ng/mL). Negligible in vitro opioid activity, combined with its low abundance in fentanyl preparations, most likely renders phenethyl-4-ANPP biologically irrelevant in vivo. However, as clandestine operations are constantly changing shape, monitoring of fentanyl attributions remains pivotal in our understanding and control of illicit fentanyl manufacture and supply., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Chagas disease and leishmaniasis are two poverty-related neglected tropical diseases that cause high mortality and morbidity. Current treatments suffer from severe limitations and novel, safer and more effective drugs are urgently needed. Both Trypanosoma cruzi and Leishmania are auxotrophic for purines and absolutely depend on uptake and assimilation of host purines. This led us to successfully explore purine nucleoside analogues as chemotherapeutic agents against these and other kinetoplastid infections. This study extensively explored the modification of the 6-amino group of tubercidin, a natural product with trypanocidal activity but unacceptable toxicity for clinical use. We found that mono-substitution of the amine with short alkyls elicits potent and selective antitrypanosomal and antileishmanial activity. The methyl analogue 15 displayed the best in vitro activity against both T. cruzi and L. infantum and high selectivity versus host cells. Oral administration for five consecutive days in an acute Chagas disease mouse model resulted in significantly reduced peak parasitemia levels (75, 89 and 96% with 12.5, 25 and 50 mg/kg/day, respectively). as well as increased animal survival rates with the lower doses (83 and 67% for 12.5 and 25 mg/kg/day, respectively)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Toxoplasma gondii is unable to synthesize purines de novo, instead salvages them from its environment, inside the host cell, for which they need high affinity carriers. Here, we report the expression of a T. gondii Equilibrative Nucleoside Transporter, Tg244440, in a Trypanosoma brucei strain from which nucleobase transporters have been deleted. Tg244440 transported hypoxanthine and guanine with similar affinity ( K m ~1 µM), while inosine and guanosine displayed K i values of 4.05 and 3.30 µM, respectively. Low affinity was observed for adenosine, adenine, and pyrimidines, classifying Tg244440 as a high affinity oxopurine transporter. Purine analogues were used to probe the substrate-transporter binding interactions, culminating in quantitative models showing different binding modes for oxopurine bases, oxopurine nucleosides, and adenosine. Hypoxanthine and guanine interacted through protonated N1 and N9, and through unprotonated N3 and N7 of the purine ring, whereas inosine and guanosine mostly employed the ribose hydroxy groups for binding, in addition to N1H of the nucleobase. Conversely, the ribose moiety of adenosine barely made any contribution to binding. Tg244440 is the first gene identified to encode a high affinity oxopurine transporter in T. gondii and, to the best of our knowledge, the first purine transporter to employ different binding modes for nucleosides and nucleobases.

Mycobacterium tuberculosis thymidylate kinase (MtTMPK) has emerged as an attractive target for rational drug design. We recently investigated new families of non-nucleoside MtTMPK inhibitors in an effort to diversify MtTMPK inhibitor chemical space. We here report a new series of MtTMPK inhibitors by combining the Topliss scheme with rational drug design approaches, fueled by two co-crystal structures of MtTMPK in complex with developed inhibitors. These efforts furnished the most potent MtTMPK inhibitors in our assay, with two analogues displaying low micromolar MIC values against H37Rv Mtb. Prepared inhibitors address new sub-sites in the MtTMPK nucleotide binding pocket, thereby offering new insights into its druggability. We studied the role of efflux pumps as well as the impact of cell wall permeabilizers for selected compounds to potentially provide an explanation for the lack of correlation between potent enzyme inhibition and whole-cell activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Kinetoplastid parasites are the causative agents of Chagas disease (CD), leishmaniasis and human African trypanosomiasis (HAT). Despite a sustained decrease in the number of HAT cases, more efforts are needed to discover safe and effective therapies against CD and leishmaniasis. Kinetoplastid parasites lack the capability to biosynthesize purines de novo and thus critically depend on uptake and processing of purines from host cells. As such, modified purine nucleoside analogues may act as broad-spectrum antikinetoplastid agents. This study assessed the in vitro activity profile of 7-modified 6-methyl tubercidin derivatives against Trypanosoma cruzi, Leishmania infantum, Trypanosoma brucei brucei and T. b. rhodesiense, and led to the identification of analogues that display activity against all these species, such as 7-ethyl (13) and 7-chloro (7) analogues. These selected analogues also proved sufficiently stable in liver microsomes to warrant in vivo follow-up evaluation., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This paper reports on the identification and full chemical characterization of the substance colloquially called "etonitazepyne" or "N-pyrrolidino etonitazene" (2-(4-ethoxybenzyl)-5-nitro-1-(2-(pyrrolidin-1-yl)ethyl)-1H-benzo[d]imidazole), a potent NPS opioid of the 5-nitrobenzimidazole class. Identification of etonitazepyne was performed, on a sample purchased during routine monitoring of the drug market, using GC-MS, HRAM LC-MS/MS, 1 H NMR, and FTIR. The chromatographic data, together with the FTIR data, indicated the presence of a highly pure compound and already indicated a benzimidazole structure. The specific benzimidazole regio-isomer was confirmed using 1 H NMR spectroscopy, resulting in the unambiguous identification of etonitazepyne., (© 2021 John Wiley & Sons, Ltd.)

Chagas disease is a tropical infectious disease resulting in progressive organ-damage and currently lacks efficient treatment and vaccine options. The causative pathogen, Trypanosoma cruzi, requires uptake and processing of preformed purines from the host because it cannot synthesize these de novo, instigating the evaluation of modified purine nucleosides as potential trypanocides. By modifying the pyrimidine part of a previously identified 7-aryl-7-deazapurine nucleoside, we found that substitution of a 6-methyl for a 6-amino group allows retaining T. cruzi amastigote growth inhibitory activity but confers improved selectivity towards mammalian cells. By keeping the 6-methyl group unaltered, and introducing different 7-aryl groups, we identified several analogues with sub-micromolar antitrypanosomal activity. The 7-(4-chlorophenyl) analogue 14, which was stable in microsomes, was evaluated in an acute mouse model. Oral administration of 25 mg/kg b.i.d. suppressed peak parasitemia and protected mice from infection-related mortality, gave similar reductions as the reference drug of blood parasite loads determined by qPCR, but as benznidazole failed to induce sterile cure in the short time period of drug exposure (5 days)., (© 2021 Wiley-VCH GmbH.)

Trichomoniasis is the most common nonviral sexually transmitted disease in humans, but treatment options are limited. Here, we report a resorufin-based drug sensitivity assay for high-throughput microplate-based screening under hypoxic conditions. A 5203-compound enamine kinase library and several specialized compound series were tested for the inhibition of Trichomonas growth at 10 μM with Z ' values of >0.5. Hits were rescreened in serial dilution to establish an IC 50 concentration. A series of 7-substituted 7-deazaadenosine analogues emerged as highly potent anti- T . vaginalis agents, with EC 50 values in the low double digit nanomolar range. These analogues exhibited excellent selectivity indices. Follow-up medicinal chemistry efforts identified an optimal ribofuranose and C7 substituent. Several nucleosides rapidly cleared cultures of T . vaginalis at a concentrations of just 2 × EC 50 . Preliminary in vivo evaluation in a murine trichomoniasis model ( Tritrichomonas foetus ) revealed promising activity upon topical administration, validating purine nucleoside analogues as a new class of antitrichomonal agents.

Kinetoplastid parasites are the causative agents of neglected tropical diseases with an unmet medical need. These parasites are unable to synthesize the purine ring de novo, and therefore rely on purine salvage to meet their purine demand. Evaluating purine nucleoside analogs is therefore an attractive strategy to identify antikinetoplastid agents. Several anti-Trypanosoma cruzi and anti-Trypanosoma brucei 7-deazapurine nucleosides were previously discovered, with the removal of the 3'-hydroxyl group resulting in a significant boost in activity. In this work we therefore decided to assess the effect of the introduction of a 3'-fluoro substituent in 7-deazapurine nucleosides on the anti-kinetoplastid activities. Hence, we synthesized two series of 3'-deoxy-3'-fluororibofuranosyl and 3'-deoxy-3'-fluoroxylofuranosyl nucleosides comprising 7-deazaadenine and -hypoxanthine bases and assayed these for antiparasitic activity. Several analogs with potent activity against T. cruzi and T. brucei were discovered, indicating that a fluorine atom in the 3'-position is a promising modification for the discovery of antiparasitic nucleosides., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Human African trypanosomiasis is a neglected parasitic disease for which the current treatment options are quite limited. Trypanosomes are not able to synthesize purines de novo and thus solely depend on purine salvage from the host environment. This characteristic makes players of the purine salvage pathway putative drug targets. The activity of known nucleoside analogues such as tubercidin and cordycepin led to the development of a series of C7-substituted nucleoside analogues. Here, we use RNA interference (RNAi) libraries to gain insight into the mode-of-action of these novel nucleoside analogues. Whole-genome RNAi screening revealed the involvement of adenosine kinase and 4E interacting protein into the mode-of-action of certain antitrypanosomal nucleoside analogues. Using RNAi lines and gene-deficient parasites, 4E interacting protein was found to be essential for parasite growth and infectivity in the vertebrate host. The essential nature of this gene product and involvement in the activity of certain nucleoside analogues indicates that it represents a potential novel drug target.

Chagas disease and visceral leishmaniasis are two neglected tropical diseases responsible for numerous deaths around the world. For both, current treatments are largely inadequate, resulting in a continued need for new drug discovery. As both kinetoplastid parasites are incapable of de novo purine synthesis, they depend on purine salvage pathways that allow them to acquire and process purines from the host to meet their demands. Purine nucleoside analogues therefore constitute a logical source of potential antiparasitic agents. Earlier optimization efforts of the natural product tubercidin (7-deazaadenosine) involving modifications to the nucleobase 7-position and the ribofuranose 3'-position led to analogues with potent anti- Trypanosoma brucei and anti- Trypanosoma cruzi activities. In this work, we report the design and synthesis of pyrazolo[3,4- d ]pyrimidine nucleosides with 3'- and 7-modifications and assess their potential as anti- Trypanosoma cruzi and antileishmanial agents. One compound was selected for in vivo evaluation in an acute Chagas disease mouse model.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with inferior outcome compared with that of B cell ALL. Here, we show that Runt-related transcription factor 2 (RUNX2) was upregulated in high-risk T-ALL with KMT2A rearrangements (KMT2A-R) or an immature immunophenotype. In KMT2A-R cells, we identified RUNX2 as a direct target of the KMT2A chimeras, where it reciprocally bound the KMT2A promoter, establishing a regulatory feed-forward mechanism. Notably, RUNX2 was required for survival of immature and KMT2A-R T-ALL cells in vitro and in vivo. We report direct transcriptional regulation of CXCR4 signaling by RUNX2, thereby promoting chemotaxis, adhesion, and homing to medullary and extramedullary sites. RUNX2 enabled these energy-demanding processes by increasing metabolic activity in T-ALL cells through positive regulation of both glycolysis and oxidative phosphorylation. Concurrently, RUNX2 upregulation increased mitochondrial dynamics and biogenesis in T-ALL cells. Finally, as a proof of concept, we demonstrate that immature and KMT2A-R T-ALL cells were vulnerable to pharmacological targeting of the interaction between RUNX2 and its cofactor CBFβ. In conclusion, we show that RUNX2 acts as a dependency factor in high-risk subtypes of human T-ALL through concomitant regulation of tumor metabolism and leukemic cell migration.

Employing two different alkyne-modified dopamine agonists to construct bivalent compounds via click chemistry resulted in the identification of a bivalent ligand (11c) for dopamine D 2 receptor homodimer, which, compared to its parent monomeric alkyne, showed a 16-fold higher binding affinity for the dopamine D 2 receptor and a 5-fold higher potency in a cAMP assay in HEK 293T cells stably expressing D 2 R. Molecular modeling revealed that 11c can indeed bridge the orthosteric binding sites of a D 2 R homodimer in a relaxed conformation via the TM5-TM6 interface and allows to largely rationalize the results of the receptor assays., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

The kinetoplastid parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are the causative agents of neglected tropical diseases with a serious burden in several parts of the world. These parasites are incapable of synthesizing purines de novo, and therefore rely on ingenious purine salvage pathways to acquire and process purines from their host. Purine nucleoside analogs that may interfere with these pathways therefore constitute a privileged source of new antikinetoplastid agents. In this study, we synthetized a collection of C-nucleosides employing five different heterocyclic nucleobase surrogates. C-nucleosides are chemically and enzymatically stable and allow for extensive structural modification. Inspired by earlier 7-deazaadenosine nucleosides and known antileishmanial C-nucleosides, we introduced different modifications tailored towards antikinetoplastid activity. Both adenosine and inosine analogs were synthesized with the aim of discovering new antikinetoplastid hits and expanding knowledge of structure-activity relationships. Several promising hits with potent activity against Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum were discovered, and the nature of the nucleobase surrogate was found to have a profound influence on the selectivity profile of the compounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

In this paper, the identification and full characterization of a novel non-fentanyl opioid sourced online, which is a member of the 1-substituted-4-(1,2-diphenylethyl)piperazine derivatives related to MT-45, is reported. The sample was sold under the name "diphenpipenol," (3-[2-[4-(2-methoxyphenyl)piperazin-1-yl]-2-phenylethyl]phenol), although extensive NMR analysis showed that the product obtained was actually a diphenpipenol structural isomer, (2-[4-(2-methoxyphenyl)piperazin-1-yl]-1,2-diphenylethanol). Liquid chromatography time-of-flight mass spectrometry identified an exact mass for the protonated molecule of m/z 389.2264, with two prominent fragment ions (m/z 91.0567 and 150.0937), which were not reported in earlier literature describing MT-45 derivatives. The chemical characterization was finalized by gas chromatography-mass spectrometry, high-performance liquid chromatography diode array detector and Fourier-transform infrared spectroscopy analyses. This product is a clear example of the trend that new non-fentanyl opioids are reappearing on the recreational drug market to escape the recent changes in (inter)national legislation concerning fentanyl analogues. Although in this particular case, the product's potency and efficacy were relatively low, other new non-fentanyl opioids might possess stronger potencies and therefore pose greater health risks for ignorant users. The fact that the product was sold under the wrong name further demonstrates the well-known problematic issue of a mismatch between the adverted and true identity, confirming the irregularities of the online new psychoactive substances market., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

New psychoactive substances continue to appear on the drug market. Until recently, new synthetic opioids, which are among the most dangerous new psychoactive substances, primarily encompassed analogs of the potent analgesic fentanyl. Lately, also other new synthetic opioids have increasingly started to surface. This is the first report on the identification and full chemical characterization of brorphine, a novel potent synthetic opioid with a piperidine benzimidazolone structure. A powder, identified as brorphine, was obtained from a patient seeking medical help for detoxification. Brorphine was also found in a serum sample of the patient. Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) identified an exact mass of m/z 400.1020 and 402.1005 for the compound, corresponding to both bromine isotopes. Further chemical characterization was performed by gas chromatography-mass spectrometry, liquid chromatography-diode array detection and Fourier-transform infrared spectroscopy analyses. Finally, the structure was confirmed by performing 1H-NMR and 13C-NMR spectroscopy. In vitro biological activity of brorphine was determined by a cell-based µ-opioid receptor activation assay, resulting in an EC50 of 30.9 nM (13.5 ng/mL) and an Emax of 209% relative to hydromorphone, confirming the high potency and efficacy of this compound. In a serum sample of the patient, brorphine and a hydroxy-metabolite were found using the LC-HRMS screening method. The presence of opioid activity in the serum was also confirmed via the activity-based opioid screening assay. The occurrence of brorphine is yet another example of how the illicit drug market is continuously evolving in an attempt to escape international legislation. Its high potency poses a serious and imminent health threat for any user., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society of Forensic Toxicologists, Inc. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Swapping the substituents in positions 2 and 4 of the previously synthesized but yet undisclosed 5-cyano-4-(methylthio)-2-arylpyrimidin-6-ones 4 , ring closure, and further optimization led to the identification of the potent antitubercular 2-thio-substituted quinazolinone 26 . Structure-activity relationship (SAR) studies indicated a crucial role for both meta -nitro substituents for antitubercular activity, while the introduction of polar substituents on the quinazolinone core allowed reduction of bovine serum albumin (BSA) binding ( 63c , 63d ). While most of the tested quinazolinones exhibited no cytotoxicity against MRC-5, the most potent compound 26 was found to be mutagenic via the Ames test. This analogue exhibited moderate inhibitory potency against Mycobacterium tuberculosis thymidylate kinase, the target of the 3-cyanopyridones that lies at the basis of the current analogues, indicating that the whole-cell antimycobacterial activity of the present S -substituted thioquinazolinones is likely due to modulation of alternative or additional targets. Diminished antimycobacterial activity was observed against mutants affected in cofactor F 420 biosynthesis ( fbiC ), cofactor reduction ( fgd ), or deazaflavin-dependent nitroreductase activity ( rv3547 ), indicating that reductive activation of the 3,5-dinitrobenzyl analogues is key to antimycobacterial activity.

The C -nucleoside natural products formycin B and pyrazofurin were synthesized in seven steps employing a sydnone riboside as common intermediate. Sydnone ribosides were synthesized via a direct Lewis acid catalyzed dehydrative glycosylation reaction. We demonstrated that these can be used for the diversity-oriented synthesis of pyrazole C -nucleoside analogues via thermal 1,3-dipolar cycloaddition reactions with various alkynes, giving access to the pyrazole C -nucleoside natural products, as well as opening new avenues for exploring nucleoside chemical space.

Metachromatic leukodystrophy (MLD) is a rare genetic disease characterised by a dysfunction of the enzyme arylsulphatase A leading to the lysosomal accumulation of cerebroside sulphate (sulphatide) causing subsequent demyelination in patients. The enzyme galactosylceramide (cerebroside) sulphotransferase (CST) catalyses the transfer of a sulphate group from 3'-phosphoadenosine-5'-phosphosulphate (PAPS) to cerebrosides producing sulphatides. Substrate reduction therapy for arylsulphatase A by inhibition of CST was proposed as a promising therapeutic approach. To identify competitive CST inhibitors, we synthesised and investigated analogues of the substrate galactosylceramide with variations at the anomeric position, the acyl substituent and the carbohydrate moiety, and investigated their structure-activity relationships. While most of the compounds behaved as substrates, α-galactosylceramide 16 was identified as the first competitive CST inhibitor. Compound 16 can serve as a new lead structure for the development of drugs for the treatment of this devastating disease, MLD, for which small molecule therapeutics are currently not available.

As the last enzyme in nucleotide synthesis as precursors for DNA replication, thymidylate kinase of M. tuberculosis (MtbTMPK) attracts significant interest as a target in the discovery of new anti-tuberculosis agents. Earlier, we discovered potent MtbTMPK inhibitors, but these generally suffered from poor antimycobacterial activity, which we hypothesize is due to poor bacterial uptake. To address this, we herein describe our efforts to equip previously reported MtbTMPK inhibitors with targeting moieties to increase the whole cell activity of the hybrid analogues. Introduction of a simplified Fe-chelating siderophore motif gave rise to analogue 17 that combined favorable enzyme inhibitory activity with significant activity against M. tuberculosis (MIC of 12.5 μM). Conjugation of MtbTMPK inhibitors with an imidazo[1,2-a]pyridine or 3,5-dinitrobenzamide scaffold afforded analogues 26, 27 and 28, with moderate MtbTMPK enzyme inhibitory potency, but sub-micromolar activity against mycobacteria without significant cytotoxicity. These results indicate that conjugation with structural motifs known to favor mycobacterial uptake may be a valid approach for discovering new antimycobacterial agents., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Mycobacterium tuberculosis, the causative agent of tuberculosis, relies on thymidylate kinase (MtbTMPK) for the synthesis of thymidine triphosphates and thus also DNA synthesis. Therefore, this enzyme constitutes a potential Achilles heel of the pathogen. Based on a previously reported MtbTMPK 6-aryl-substituted pyridone inhibitor and guided by two co-crystal structures of MtbTMPK with pyridone- and thymine-based inhibitors, we report the synthesis of a series of aryl-shifted cyanopyridone analogues. These compounds generally lacked significant MtbTMPK inhibitory potency, but some analogues did exhibit promising antitubercular activity. Analogue 11i demonstrated a 10-fold increased antitubercular activity (MIC H37Rv, 1.2 μM) compared to literature compound 5. Many analogues with whole-cell antimycobacterial activity were devoid of significant cytotoxicity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Human African trypanosomiasis is a neglected tropical disease caused by Trypanosoma brucei parasites. These protists are unable to produce the purine ring, making them vulnerable to the effects of purine nucleoside analogues. Starting from 3'-deoxytubercidin ( 5 ), a lead compound with activity against central-nervous-stage human African trypanosomiasis, we investigate the structure-activity relationships of the purine and ribofuranose rings. The purine ring tolerated only modifications at C7, while from the many alterations of the 3'-deoxyribofuranosyl moiety only the arabino analogue 48 showed pronounced antitrypanosomal activity. Profiling of the most potent analogues against resistant T. brucei strains (resistant to pentamidine, diminazene, and isometamidium) showed reduced dependence on uptake mediated by the P2 aminopurine transporter relative to 5 . The introduction of a 7-substituent confers up to 10-fold increased affinity for the P1 nucleoside transporter while generally retaining high affinity for P2. Four of the most promising analogues were found to be metabolically stable, earmarking them as suitable backup analogues for lead 5 .

The metabolic alterations in tumors make it possible to visualize the latter by means of positron emission tomography, enabling diagnosis and providing metabolic information. The alanine serine cysteine transporter-2 (ASCT-2) is the main transporter of glutamine and is upregulated in several tumors. Therefore, a good positron emission tracer targeting this transport protein would have substantial value. Hence, the aim of this study is to develop a fluorine-18-labeled version of a V-9302 analogue, one of the most potent inhibitors of ASCT-2. The precursor was labeled with fluorine-18 via a nucleophilic substitution of the corresponding benzylic bromide. The cold reference product was subjected to in vitro assays with [ 3 H]glutamine in a PC-3 and F98 cell line to determine the affinity for both the human and rat ASCT-2. To evaluate the tracer potential dynamic μPET, images were acquired in a mouse xenograft model for prostate cancer. The tracer could be synthesized with an overall nondecay corrected yield of 3.66 ± 1.90%. in vitro experiments show inhibitor constants K i of 90 and 125 μM for the PC-3 and F98 cells, respectively. The experiments in the PC-3 xenograft demonstrate a low uptake in the tumor tissue. We have successfully synthesized the radiotracer [ 18 F]2-amino-4-((2-((3-fluorobenzyl)oxy)benzyl)(2-((3-(fluoromethyl)benzyl)oxy)benzyl)amino)butanoic acid. in vitro experiments show a good affinity for both the human and rat ASCT-2. However, the tracer suffers from poor in vivo tumor uptake in the PC-3 model. Briefly, we present the first fluorine-18-labeled derivative of compound V-9302, a promising novel ASCT-2 blocker used for inhibition of tumor growth., (© 2020 John Wiley & Sons, Ltd.)

A series of Mycobacterium tuberculosis TMPK ( Mtb TMPK) inhibitors based on a reported compound 3 were synthesized and evaluated for their capacity to inhibit Mtb TMPK catalytic activity and the growth of a virulent M. tuberculosis strain (H37Rv). Modifications of the scaffold of 3 failed to afford substantial improvements in Mtb TMPK inhibitory activity and antimycobacterial activity. Optimization of the substitution pattern of the D ring of 3 resulted in compound 21j with improved Mtb TMPK inhibitory potency (three-fold) and H37Rv growth inhibitory activity (two-fold). Moving the 3-chloro substituent of 21j to the para -position afforded isomer 21h , which, despite a 10-fold increase in IC 50 -value, displayed promising whole cell activity (minimum inhibitory concentration (MIC) = 12.5 μM).

Small nucleic acid (NA) therapeutics, such as small interfering RNA (siRNA), are generally formulated in nanoparticles (NPs) to overcome the multiple extra- and intracellular barriers upon in vivo administration. Interaction with target cells typically triggers endocytosis and sequesters the NPs in endosomes, thus hampering the pharmacological activity of the encapsulated siRNAs that occurs in the cytosol. Unfortunately, for most state-of-the-art NPs, endosomal escape is largely inefficient. As a result, the bulk of the endocytosed NA drug is rapidly trafficked toward the degradative lysosomes that are considered as a dead end for siRNA nanomedicines. In contrast to this paradigm, we recently reported that cationic amphiphilic drugs (CADs) could strongly promote functional siRNA delivery from the endolysosomal compartment via transient induction of lysosomal membrane permeabilization. However, many questions still remain regarding the broader applicability of such a CAD adjuvant effect on NA delivery. Here, we report a drug repurposing screen (National Institutes of Health Clinical Collection) that allowed identification of 56 CAD adjuvants. We furthermore demonstrate that the CAD adjuvant effect is dependent on the type of nanocarrier, with NPs that generate an appropriate pool of decomplexed siRNA in the endolysosomal compartment being most susceptible to CAD-promoted gene silencing. Finally, the CAD adjuvant effect was verified on human ovarian cancer cells and for antisense oligonucleotides. In conclusion, this study strongly expands our current knowledge on how CADs increase the cytosolic release of small NAs, providing relevant insights to more rationally combine CAD adjuvants with NA-loaded NPs for future therapeutic applications.

This paper reports on the identification and full chemical characterization of isotonitazene (N,N-diethyl-2-[5-nitro-2-({4-[(propan-2-yl)oxy]phenyl}methyl)-1H-benzimidazol-1-yl]ethan-1-amine), a potent NPS opioid and the first member of the benzimidazole class of compounds to be available on online markets. Interestingly, this compound was sold under the name etonitazene, a structural analog. Identification of isotonitazene was performed by gas chromatography mass spectrometry (GC-MS) and liquid chromatography time-of-flight mass spectrometry (LC-QTOF-MS), the latter identifying an exact-mass m/z value of 411.2398. All chromatographic data indicated the presence of a single, highly pure compound. Confirmation of the specific benzimidazole regio-isomer was performed using 1 H and 13 C NMR spectroscopy, after which the chemical characterization was finalized by recording Fourier-transform (FT-IR) spectra. A live cell-based reporter assay to assess the in vitro biological activity at the μ-opioid receptor (MOR) revealed that isotonitazene has a high potency (EC 50 of 11.1 nM) and efficacy (E max 180% of that of hydromorphone), thus confirming that this substance is a strong opioid. Isotonitazene has not been previously detected, either in powder form, or in biological fluids. The high potency and efficacy of isotonitazene, combined with the fact that this compound was being sold undiluted, represents an imminent danger to anyone aiming to use this powder., (© 2019 John Wiley & Sons, Ltd.)

In recent years, new drug discovery approaches based on novel pharmacological concepts have emerged. Allosteric modulators, for example, target receptors at sites other than the orthosteric binding sites and can modulate agonist-mediated activation. Interestingly, allosteric regulation may allow a fine-tuned regulation of unbalanced neurotransmitter' systems, thus providing safe and effective treatments for a number of central nervous system diseases. The metabotropic glutamate type 5 receptor (mGlu 5 R) has been shown to possess a druggable allosteric binding domain. Accordingly, novel allosteric ligands are being explored in order to finely regulate glutamate neurotransmission, especially in the brain. However, before testing the activity of these new ligands in the clinic or even in animal disease models, it is common to characterize their ability to bind mGlu 5 Rs in vitro. Here, we have developed a new series of fluorescent ligands that, when used in a new NanoBRET-based binding assay, will facilitate screening for novel mGlu 5 R allosteric modulators., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

African trypanosomiasis, a deadly infectious disease caused by the protozoan Trypanosoma brucei spp., is spread to new hosts by bites of infected tsetse flies. Currently approved therapies all have their specific drawbacks, prompting a search for novel therapeutic agents. T. brucei lacks the enzymes necessary to forge the purine ring from amino acid precursors, rendering them dependent on the uptake and interconversion of host purines. This dependency renders analogues of purines and corresponding nucleosides an interesting source of potential anti-T. brucei agents. In this study, we synthesized and evaluated a series of 7-substituted 7-deazainosine derivatives and found that 6-O-alkylated analogues in particular showed highly promising in vitro activity with EC 50 values in the mid-nanomolar range. SAR investigation of the O-alkyl chain showed that antitrypanosomal activity increased, and also cytotoxicity, with alkyl chain length, at least in the linear alkyl chain series. However, this could be attenuated by introducing a terminal branch point, resulting in the highly potent and selective analogues, 36, 37 and 38. No resistance related to transporter-mediated uptake could be identified, earmarking several of these analogues for further in vivo follow-up studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Synthetic materials capable of engineering the immune system are of great relevance in the fight against cancer to replace or complement the current monoclonal antibody and cell therapy-based immunotherapeutics. Here, we report on antibody recruiting glycopolymers (ARGPs). ARGPs consist of polymeric copies of a rhamnose motif, which can bind endogenous antirhamnose antibodies present in human serum. As a proof-of-concept, we have designed ARGPs with a lipophilic end group that efficiently inserts into cell-surface membranes. We validate the specificity of rhamnose to attract antibodies from human serum to the target cell surface and demonstrate that ARGPs outperform an analogous small-molecule compound containing only one single rhamnose motif. The ARGP concept opens new avenues for the design of potent immunotherapeutics that mark target cells for destruction by the immune system through antibody-mediated effector functions.

African trypanosomiasis is a disease caused by Trypanosoma brucei parasites with limited treatment options. Trypanosoma is unable to synthesize purines de novo and relies solely on their uptake and interconversion from the host, constituting purine nucleoside analogues a potential source of antitrypanosomal agents. Here we combine structural elements from known trypanocidal nucleoside analogues to develop a series of 3'-deoxy-7-deazaadenosine nucleosides, and investigate their effects against African trypanosomes. 3'-Deoxytubercidin is a highly potent trypanocide in vitro and displays curative activity in animal models of acute and CNS-stage disease, even at low doses and oral administration. Whole-genome RNAi screening reveals that the P2 nucleoside transporter and adenosine kinase are involved in the uptake and activation, respectively, of this analogue. This is confirmed by P1 and P2 transporter assays and nucleotide pool analysis. 3'-Deoxytubercidin is a promising lead to treat late-stage sleeping sickness.

A series of readily accessible 1-(piperidin-3-yl)thymine amides was designed, synthesised and evaluated as Mycobacterium tuberculosis TMPK ( Mtb TMPK) inhibitors. In line with the modelling results, most inhibitors showed reasonable Mtb TMPK inhibitory activity. Compounds 4b and 4i were slightly more potent than the parent compound 3 . Moreover, contrary to the latter, amide analogue 4g was active against the avirulent M. tuberculosis H37Ra strain (MIC 50 =35 µM). This finding opens avenues for future modifications.

Trypanosoma cruzi is the causative pathogen of Chagas disease and the main culprit for cardiac-related mortality in Latin-America triggered by an infective agent. Incapable of synthesizing purines de novo, this parasite depends on acquisition and processing of host-derived purines, making purine (nucleoside) analogues a potential source of antitrypanosomal agents. In this respect, hitherto 7-deazaadenosine (tubercidin) analogues attracted most attention. Here, we investigated analogues with an additional nitrogen (N1) removed. Structure-activity relationship investigation showed that C7 modification afforded analogues with potent antitrypanosomal activity. Halogens and small, linear carbon-based substituents were preferred. Compound 11 proved most potent in vitro, showed full suppression of parasitemia in a mouse model of acute infection, and elicited 100% animal survival after oral dosing at 25 mg/kg b.i.d. for 5 and 15 days. Cyclophosphamide-induced immunosuppression led to recrudescence. Washout experiments demonstrated a lack of complete clearance of infected cell cultures, potentially explaining the in vivo results.

Type I natural killer T (NKT) cells are a population of innate like T lymphocytes that rapidly respond to α-GalCer presented by CD1d via the production of both pro- and anti-inflammatory cytokines. While developing novel α-GalCer analogs that were meant to be utilized as potential adjuvants because of their production of pro-inflammatory cytokines (Th1 skewers), we generated α-galactosylsphingamides (αGSA). Surprisingly, αGSAs are not potent antigens in vivo despite their strong T-cell receptor (TCR)-binding affinities. Here, using surface plasmon resonance (SPR), antigen presentation assays, and X-ray crystallography (yielding crystal structures of 19 different binary (CD1d-glycolipid) or ternary (CD1d-glycolipid-TCR) complexes at resolutions between 1.67 and 2.85 Å), we characterized the biochemical and structural details of αGSA recognition by murine NKT cells. We identified a molecular switch within murine (m)CD1d that modulates NKT cell activation by αGSAs. We found that the molecular switch involves a hydrogen bond interaction between Tyr-73 of mCD1d and the amide group oxygen of αGSAs. We further established that the length of the acyl chain controls the positioning of the amide group with respect to the molecular switch and works synergistically with Tyr-73 to control NKT cell activity. In conclusion, our findings reveal important mechanistic insights into the presentation and recognition of glycolipids with polar moieties in an otherwise apolar milieu. These observations may inform the development αGSAs as specific NKT cell antagonists to modulate immune responses., (© 2019 Wang et al.)