Synthesis and vectorial functionalisation of pyrazolo[3,4- c ]pyridines

Heterocycles are a cornerstone of fragment-based drug discovery (FBDD) due to their prevalence in biologically active compounds. However, novel heterocyclic fragments are only valuable if they can be suitably elaborated to compliment a chosen target protein. Here we describe the synthesis of 5-halo-1H-pyrazolo[3,4-c]pyridine scaffolds and demonstrate how these compounds can be selectively elaborated along multiple growth-vectors. Specifically, N-1 and N-2 are accessed through protection-group and N-alkylation reactions; C-3 through tandem borylation and Suzuki–Miyaura cross-coupling reactions; C-5 through Pd-catalysed Buchwald–Hartwig amination; and C-7 through selective metalation with TMPMgCl.LiCl followed by reaction with electrophiles or transmetalation to ZnCl2 and Negishi cross-coupling. Linking multiple functionalisation strategies emulates a hit-to-lead pathway and demonstrates the utility of pyrazolo[3,4-c]pyridines to FBDD

Inhibition of HSP90 distinctively modulates the global phosphoproteome of Leishmania mexicana developmental stages

Heat shock protein 90 (HSP90) is an evolutionarily conserved chaperone protein that plays a central role in the folding and maturation of a large array of client proteins. In the unicellular parasite Leishmania, the etiological agent of the neglected tropical disease leishmaniasis, treatment with HSP90 inhibitors leads to differentiation from promastigote to amastigote stage, resembling the effects of established environmental triggers, low pH and heat shock. This indicates a crucial role for HSP90 in the life cycle control of Leishmania. However, the underlying molecular mechanisms remain unknown. Using a combination of treatment with the classical HSP90 inhibitor tanespimycin, phosphoproteome enrichment, and tandem mass tag (TMT) labeling-based quantitative proteomic mass spectrometry (MS), we systematically characterized the perturbing effect of HSP90 inhibition on the global phosphoproteome of Leishmania mexicana across its life cycle stages and showed that the HSP90 inhibition causes substantially distinct molecular effects in promastigote and amastigote forms.While phosphorylation of HSP90 and its co-chaperone HSP70 was decreased in amastigote, the opposite effect was observed in promastigotes. Our results showed that kinase activity and microtubule motor activity are highly represented in the negatively affected phosphoproteins of the promastigotes, whereas ribosomal proteins, protein folding, and proton channel activity are preferentially enriched in the perturbed amastigote phosphoproteome. Additionally, cross-comparison of our results with HSP90 inhibition-affected RNA-binding proteins showed that RNA helicase domains were distinctively enriched among the upregulated amastigote phosphoproteins. In addition to providing robust identification and quantification of 1,833 phosphorylated proteins across three life cycle stages of L. mexicana, this study reveals the dramatically different ways the HSP90 inhibition stress modulates the phosphoproteome of the pathogenic amastigote and provides in-depth insight into the scope of selective molecular targeting in the therapeutically relevant amastigote stage

Applications of Transition Metal-Catalyzed ortho-Fluorine-Directed C–H Functionalization of (Poly)fluoroarenes in Organic Synthesis

The synthesis of organic compounds efficiently via fewer steps but in higher yields is desirable as this reduces energy and reagent use, waste production, and thus environmental impact as well as cost. The reactivity of C–H bonds ortho to fluorine substituents in (poly)fluoroarenes with metal centers is enhanced relative to meta and para positions. Thus, direct C–H functionalization of (poly)fluoroarenes without prefunctionalization is becoming a significant area of research in organic chemistry. Novel and selective methodologies to functionalize (poly)fluorinated arenes by taking advantage of the reactivity of C–H bonds ortho to C–F bonds are continuously being developed. This review summarizes the reasons for the enhanced reactivity and the consequent developments in the synthesis of valuable (poly)fluoroarene-containing organic compounds

Iridium‐Catalysed C−H Borylation of Fluoroarenes: Insights into the Balance between Steric and Electronic Control of Regioselectivity

The iridium catalysed C−H borylation of polyfluorinated arenes and heteroarenes occurs rapidly and efficiently. As with other borylation reactions, whilst steric parameters dominate, an underlying electronic influence on reaction selectivity can be observed. Notably borylation regioselectivity in fluorinated (hetero)arenes is determined by purely electronic effects except for ortho-borylation between two fluorine atoms where steric effects of fluorine substituents become apparent. Borylation at the para position with respect to fluorine is disfavoured whereas a strong electronic preference for borylation para to the azinyl nitrogen of pyridine is observed. When these features co-operate high selectivity can be expected. For these reactions, computations based on transition state, rather than intermediate, energies in iridium geometries showed excellent agreement between predicted and observed selectivities

Assessing Dose-Exposure-Response Relationships of Miltefosine in Adults and Children using Physiologically-Based Pharmacokinetic Modeling Approach.

Miltefosine is the first and only oral medication to be successfully utilized as an antileishmanial agent. However, the drug is associated with differences in exposure patterns and cure rates among different population groups e.g. ethnicity and age (i.e., children v adults) in clinical trials. In this work, mechanistic population physiologically-based pharmacokinetic (PBPK) models have been developed to study the dose-exposure-response relationship of miltefosine in in silico clinical trials and evaluate the differences in population groups, particularly children and adults. The Simcyp population pharmacokinetics platform was employed to predict miltefosine exposure in plasma and peripheral blood mononuclear cells (PBMCs) in a virtual population under different dosing regimens. The cure rate of a simulation was based on the percentage of number of the individual virtual subjects with AUC  > 535 µg⋅day/mL in the virtual population. It is shown that both adult and paediatric PBPK models of miltefosine can be developed to predict the PK data of the clinical trials accurately. There was no significant difference in the predicted dose-exposure-response of the miltefosine treatment for different simulated ethnicities under the same dose regime and the dose-selection strategies determined the clinical outcome of the miltefosine treatment. A lower cure rate of the miltefosine treatment in paediatrics was predicted because a lower exposure of miltefosine was simulated in virtual paediatric in comparison with adult virtual populations when they received the same dose of the treatment. The mechanistic PBPK model suggested that the higher fraction of unbound miltefosine in plasma was responsible for a higher probability of failure in paediatrics because of the difference in the distribution of plasma proteins between adults and paediatrics. The developed PBPK models could be used to determine an optimal miltefosine dose regime in future clinical trials. [Abstract copyright: © 2023. The Author(s).

Navigating drug repurposing for Chagas disease: advances, challenges, and opportunities

Chagas disease is a vector-borne illness caused by the protozoan parasite Trypanosoma cruzi (T. cruzi). It poses a significant public health burden, particularly in the poorest regions of Latin America. Currently, there is no available vaccine, and chemotherapy has been the traditional treatment for Chagas disease. However, the treatment options are limited to just two outdated medicines, nifurtimox and benznidazole, which have serious side effects and low efficacy, especially during the chronic phase of the disease. Collectively, this has led the World Health Organization to classify it as a neglected disease. To address this problem, new drug regimens are urgently needed. Drug repurposing, which involves the use of existing drugs already approved for the treatment of other diseases, represents an increasingly important option. This approach offers potential cost reduction in new drug discovery processes and can address pharmaceutical bottlenecks in the development of drugs for Chagas disease. In this review, we discuss the state-of-the-art of drug repurposing approaches, including combination therapy with existing drugs, to overcome the formidable challenges associated with treating Chagas disease. Organized by original therapeutic area, we describe significant recent advances, as well as the challenges in this field. In particular, we identify candidates that exhibit potential for heightened efficacy and reduced toxicity profiles with the ultimate objective of accelerating the development of new, safe, and effective treatments for Chagas disease

Disruption of the inositol phosphorylceramide synthase gene affects Trypanosoma cruzi differentiation and infection capacity

Sphingolipids (SLs) are essential components of all eukaryotic cellular membranes. In fungi, plants and many protozoa, the primary SL is inositol-phosphorylceramide (IPC). Trypanosoma cruzi is a protozoan parasite that causes Chagas disease (CD), a chronic illness for which no vaccines or effective treatments are available. IPC synthase (IPCS) has been considered an ideal target enzyme for drug development because phosphoinositol-containing SL is absent in mammalian cells and the enzyme activity has been described in all parasite forms of T. cruzi. Furthermore, IPCS is an integral membrane protein conserved amongst other kinetoplastids, including Leishmania major, for which specific inhibitors have been identified. Using a CRISPR-Cas9 protocol, we generated T. cruzi knockout (KO) mutants in which both alleles of the IPCS gene were disrupted. We demonstrated that the lack of IPCS activity does not affect epimastigote proliferation or its susceptibility to compounds that have been identified as inhibitors of the L. major IPCS. However, disruption of the T. cruzi IPCS gene negatively affected epimastigote differentiation into metacyclic trypomastigotes as well as proliferation of intracellular amastigotes and differentiation of amastigotes into tissue culture-derived trypomastigotes. In accordance with previous studies suggesting that IPC is a membrane component essential for parasite survival in the mammalian host, we showed that T. cruzi IPCS null mutants are unable to establish an infection in vivo, even in immune deficient mice

Integral Role of Water in the Solid-State Behavior of the Antileishmanial Drug Miltefosine

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link. Open access articleMiltefosine is a repurposed anticancer drug and currently the only orally administered drug approved to treat the neglected tropical disease leishmaniasis. Miltefosine is hygroscopic and must be stored at sub-zero temperatures. In this work we report the X-ray structures of miltefosine monohydrate and methanol solvate, along with 12- and 14-carbon chain analogue hydrates and a solvate. The three hydrates are all isostructural and are conformational isomorphs with Z' = 2. The water bridges the gap between phosphocholine head groups caused by the interdigitated bilayer structure. The two methanol solvates are also mutually isostructural with the head groups adopting a more extended conformation. Again, the solvent bridges the gap between head groups in the bilayer. No anhydrous form of miltefosine or its analogues were isolated, with dehydration resulting in significantly reduced crystallinity. This arises as a result of the integral role that hydrogen bond donors (in the form of water or solvent molecules) play in the stability of the zwitterionic structures

Solar System Objects Observed in the SDSS Commissioning Data

We discuss measurements of the properties of about 10,000 asteroids detected in 500 deg2 of sky in the Sloan Digital Sky Survey (SDSS) commissioning data. The moving objects are detected in the magnitude range 14 < r < 21.5, with a baseline of 5 minutes. Extensive tests show that the sample is at least 98% complete, with the contamination rate of less than 3%. We find that the size distribution of asteroids resembles a broken power-law, independent of the heliocentric distance: D^{-2.3} for 0.4 km < D < 5 km, and D^{-4} for 5 km < D < 40 km. As a consequence of this break, the number of asteroids with r < 21.5 is ten times smaller than predicted by extrapolating the power-law relation observed for brighter asteroids (r < 18). The observed counts imply that there are about 530,000 objects with D>1 km in the asteroid belt, or about four times less than previous estimates. The distribution of main belt asteroids in the 4-dimensional SDSS color space is bimodal, and the two groups can be associated with S (rocky) and C (carbonaceous) asteroids. A strong bimodality is also seen in the heliocentric distribution of asteroids and suggests the existence of two distinct belts: the inner rocky belt, about 1 AU wide (FWHM) and centered at R~2.8 AU, and the outer carbonaceous belt, about 0.5 AU wide and centered at R~3.2 AU. The colors of Hungarias, Mars crossers, and near-Earth objects are more similar to the C-type than to S-type asteroids, suggesting that they originate in the outer belt. (abridged).Comment: 89 pages, 31 figures, submitted to A

Mapping the geographical distribution of podoconiosis in Cameroon using parasitological, serological, and clinical evidence to exclude other causes of lymphedema

Background Podoconiosis is a non-filarial elephantiasis, which causes massive swelling of the lower legs. It was identified as a neglected tropical disease by WHO in 2011. Understanding of the geographical distribution of the disease is incomplete. As part of a global mapping of podoconiosis, this study was conducted in Cameroon to map the distribution of the disease. This mapping work will help to generate data on the geographical distribution of podoconiosis in Cameroon and contribute to the global atlas of podoconiosis. Methods We used a multi‐stage sampling design with stratification of the country by environmental risk of podoconiosis. We sampled 76 villages from 40 health districts from the ten Regions of Cameroon. All individuals of 15-years old or older in the village were surveyed house-to-house and screened for lymphedema. A clinical algorithm was used to reliably diagnose podoconiosis, excluding filarial-associated lymphedema. Individuals with lymphoedema were tested for circulating Wuchereria bancrofti antigen and specific IgG4 in the field using the Alere Filariasis Test Strips (FTS) test and the Standard Diagnostics (SD) BIOLINE lymphatic filariasis IgG4 test (Wb123) respectively, in addition to thick blood films. Presence of DNA specific to W.bancrofti was checked on night blood using a qPCR technique. Principal Findings Overall, 10,178 individuals from 4,603 households participated in the study. In total, 83 individuals with lymphedema were identified. Of the 83 individuals with lymphedema, two were found to be FTS positive and all were negative using the Wb123 test. No microfilaria of W. bancrofti were found in the night blood of any individual with clinical lymphedema. None were found to be positive for W. bancrofti using qPCR. Of the two FTS positive cases, one was positive for Mansonella perstans DNA, while the other harbored Loa loa microfilaria. Overall, 52 people with podoconiosis were identified after applying the clinical algorithm. The overall prevalence of podoconiosis was found to be 0.5% (95% [confidence interval] CI; 0.4-0.7). At least one case of podoconiosis was found in every region of Cameroon except the two surveyed villages in Adamawa. Of the 40 health districts surveyed, 17 districts had no cases of podoconiosis; in 15 districts, mean prevalence was between 0.2% and 1.0%; and in the remaining eight, mean prevalence was between 1.2% and 2.7%. Conclusions Our investigation has demonstrated low prevalence but almost nationwide distribution of podoconiosis in Cameroon. Designing a podoconiosis control program is a vital next step. A health system response to the burden of podoconiosis is important, through case surveillance and morbidity management services