Intercepting fleeting cyclic allenes with asymmetric nickel catalysis.

Strained cyclic organic molecules, such as arynes, cyclic alkynes and cyclic allenes, have intrigued chemists for more than a century with their unusual structures and high chemical reactivity1. The considerable ring strain (30-50 kilocalories per mole)2,3 that characterizes these transient intermediates imparts high reactivity in many reactions, including cycloadditions and nucleophilic trappings, often generating structurally complex products4. Although strategies to control absolute stereochemistry in these reactions have been reported using stoichiometric chiral reagents5,6, catalytic asymmetric variants to generate enantioenriched products have remained difficult to achieve. Here we report the interception of racemic cyclic allene intermediates in a catalytic asymmetric reaction and provide evidence for two distinct mechanisms that control absolute stereochemistry in such transformations: kinetic differentiation of allene enantiomers and desymmetrization of intermediate π-allylnickel complexes. Computational studies implicate a catalytic mechanism involving initial kinetic differentiation of the cyclic allene enantiomers through stereoselective olefin insertion, loss of the resultant stereochemical information, and subsequent introduction of absolute stereochemistry through desymmetrization of an intermediate π-allylnickel complex. These results reveal reactivity that is available to cyclic allenes beyond the traditional cycloadditions and nucleophilic trappings previously reported, thus expanding the types of product accessible from this class of intermediates. Additionally, our computational studies suggest two potential strategies for stereocontrol in reactions of cyclic allenes. Combined, these results lay the foundation for the development of catalytic asymmetric reactions involving these classically avoided strained intermediates.

The obesity-linked human lncRNA AATBC stimulates mitochondrial function in adipocytes.

Adipocytes are critical regulators of metabolism and energy balance. While white adipocyte dysfunction is a hallmark of obesity-associated disorders, thermogenic adipocytes are linked to cardiometabolic health. As adipocytes dynamically adapt to environmental cues by functionally switching between white and thermogenic phenotypes, a molecular understanding of this plasticity could help improving metabolism. Here, we show that the lncRNA Apoptosis associated transcript in bladder cancer (AATBC) is a human-specific regulator of adipocyte plasticity. Comparing transcriptional profiles of human adipose tissues and cultured adipocytes we discovered that AATBC was enriched in thermogenic conditions. Using primary and immortalized human adipocytes we found that AATBC enhanced the thermogenic phenotype, which was linked to increased respiration and a more fragmented mitochondrial network. Expression of AATBC in adipose tissue of mice led to lower plasma leptin levels. Interestingly, this association was also present in human subjects, as AATBC in adipose tissue was inversely correlated with plasma leptin levels, BMI, and other measures of metabolic health. In conclusion, AATBC is a novel obesity-linked regulator of adipocyte plasticity and mitochondrial function in humans.

Development of a peptide drug restoring AMPK and adipose tissue functionality in cancer cachexia.

Cancer cachexia is a severe systemic wasting disease that negatively affects quality of life and survival in patients with cancer. To date, treating cancer cachexia is still a major unmet clinical need. We recently discovered the destabilization of the AMP-activated protein kinase (AMPK) complex in adipose tissue as a key event in cachexia-related adipose tissue dysfunction and developed an adeno-associated virus (AAV)-based approach to prevent AMPK degradation and prolong cachexia-free survival. Here, we show the development and optimization of a prototypic peptide, Pen-X-ACIP, where the AMPK-stabilizing peptide ACIP is fused to the cell-penetrating peptide moiety penetratin via a propargylic glycine linker to enable late-stage functionalization using click chemistry. Pen-X-ACIP was efficiently taken up by adipocytes, inhibited lipolysis, and restored AMPK signaling. Tissue uptake assays showed a favorable uptake profile into adipose tissue upon intraperitoneal injection. Systemic delivery of Pen-X-ACIP into tumor-bearing animals prevented the progression of cancer cachexia without affecting tumor growth and preserved body weight and adipose tissue mass with no discernable side effects in other peripheral organs, thereby achieving proof of concept. As Pen-X-ACIP also exerted its anti-lipolytic activity in human adipocytes, it now provides a promising platform for further (pre)clinical development toward a novel, first-in-class approach against cancer cachexia.

Adipocyte function and the development of cardiometabolic disease.

Obesity is a medical disorder caused by multiple mechanisms of dysregulated energy balance. A major consequence of obesity is an increased risk to develop diabetes, diabetic complications and cardiovascular disease. While a better understanding of the molecular mechanisms linking obesity, insulin resistance and cardiovascular disease is needed, translational research of the human pathology is hampered by the available cellular and rodent model systems. Major barriers are the species-specific differences in energy balance, vascular biology and adipose tissue physiology, especially related to white and brown adipocytes, and adipose tissue browning. In rodents, non-shivering thermogenesis is responsible for a large part of energy expenditure, but humans possess much less thermogenic fat, which means temperature is an important variable in translational research. Mouse models with predisposition to dyslipidaemia housed at thermoneutrality and fed a high-fat diet more closely reflect human physiology. Also, adipocytes play a key role in the endocrine regulation of cardiovascular function. Adipocytes secrete a variety of hormones, lipid mediators and other metabolites that directly influence the local microenvironment as well as distant tissues. This is specifically apparent in perivascular depots, where adipocytes modulate vascular function and inflammation. Altogether, these mechanisms highlight the critical role of adipocytes in the development of cardiometabolic disease.

HAND2 is a novel obesity-linked adipogenic transcription factor regulated by glucocorticoid signalling.

AIMS/HYPOTHESIS: Adipocytes are critical cornerstones of energy metabolism. While obesity-induced adipocyte dysfunction is associated with insulin resistance and systemic metabolic disturbances, adipogenesis, the formation of new adipocytes and healthy adipose tissue expansion are associated with metabolic benefits. Understanding the molecular mechanisms governing adipogenesis is of great clinical potential to efficiently restore metabolic health in obesity. Here we investigate the role of heart and neural crest derivatives-expressed 2 (HAND2) in adipogenesis. METHODS: Human white adipose tissue (WAT) was collected from two cross-sectional studies of 318 and 96 individuals. In vitro, for mechanistic experiments we used primary adipocytes from humans and mice as well as human multipotent adipose-derived stem (hMADS) cells. Gene silencing was performed using siRNA or genetic inactivation in primary adipocytes from loxP and or tamoxifen-inducible Cre-ERT2 mouse models with Cre-encoding mRNA or tamoxifen, respectively. Adipogenesis and adipocyte metabolism were measured by Oil Red O staining, quantitative PCR (qPCR), microarray, glucose uptake assay, western blot and lipolysis assay. A combinatorial RNA sequencing (RNAseq) and ChIP qPCR approach was used to identify target genes regulated by HAND2. In vivo, we created a conditional adipocyte Hand2 deletion mouse model using Cre under control of the Adipoq promoter (Hand2AdipoqCre) and performed a large panel of metabolic tests. RESULTS: We found that HAND2 is an obesity-linked white adipocyte transcription factor regulated by glucocorticoids that was necessary but insufficient for adipocyte differentiation in vitro. In a large cohort of humans, WAT HAND2 expression was correlated to BMI. The HAND2 gene was enriched in white adipocytes compared with brown, induced early in differentiation and responded to dexamethasone (DEX), a typical glucocorticoid receptor (GR, encoded by NR3C1) agonist. Silencing of NR3C1 in hMADS cells or deletion of GR in a transgenic conditional mouse model results in diminished HAND2 expression, establishing that adipocyte HAND2 is regulated by glucocorticoids via GR in vitro and in vivo. Furthermore, we identified gene clusters indirectly regulated by the GR-HAND2 pathway. Interestingly, silencing of HAND2 impaired adipocyte differentiation in hMADS and primary mouse adipocytes. However, a conditional adipocyte Hand2 deletion mouse model using Cre under control of the Adipoq promoter did not mirror these effects on adipose tissue differentiation, indicating that HAND2 was required at stages prior to Adipoq expression. CONCLUSIONS/INTERPRETATION: In summary, our study identifies HAND2 as a novel obesity-linked adipocyte transcription factor, highlighting new mechanisms of GR-dependent adipogenesis in humans and mice. DATA AVAILABILITY: Array data have been submitted to the GEO database at NCBI (GSE148699).

A miR-29a-driven negative feedback loop regulates peripheral glucocorticoid receptor signaling.

The glucocorticoid receptor (GR) represents the crucial molecular mediator of key endocrine, glucocorticoid hormone-dependent regulatory circuits, including control of glucose, protein, and lipid homeostasis. Consequently, aberrant glucocorticoid signaling is linked to severe metabolic disorders, including insulin resistance, obesity, and hyperglycemia, all of which also appear upon chronic glucocorticoid therapy for the treatment of inflammatory conditions. Of note, long-term glucocorticoid exposure under these therapeutic conditions typically induces glucocorticoid resistance, requiring higher doses and consequently triggering more severe metabolic phenotypes. However, the molecular basis of acquired glucocorticoid resistance remains unknown. In a screen of differential microRNA expression during glucocorticoid-dependent adipogenic differentiation of human multipotent adipose stem cells, we identified microRNA 29a (miR-29a) as one of the most down-regulated transcripts. Overexpression of miR-29a impaired adipogenesis. We found that miR-29a represses GR in human adipogenesis by directly targeting its mRNA, and downstream analyses revealed that GR mediates most of miR-29a's anti-adipogenic effects. Conversely, miR-29a expression depends on GR activation, creating a novel miR-29-driven feedback loop. miR-29a and GR expression were inversely correlated both in murine adipose tissue and in adipose tissue samples obtained from human patients. In the latter, miR-29a levels were additionally strongly negatively correlated with body mass index and adipocyte size. Importantly, inhibition of miR-29 in mice partially rescued the down-regulation of GR during dexamethasone treatment. We discovered that, in addition to modulating GR function under physiologic conditions, pharmacologic glucocorticoid application in inflammatory disease also induced miR-29a expression, correlating with reduced GR levels. This effect was abolished in mice with impaired GR function. In summary, we uncovered a novel GR-miR-29a negative feedback loop conserved between mice and humans, in health and disease. For the first time, we elucidate a microRNA-related mechanism that might contribute to GR dysregulation and resistance in peripheral tissues.-Glantschnig, C., Koenen, M., Gil-Lozano, M., Karbiener, M., Pickrahn, I., Williams-Dautovich, J., Patel, R., Cummins, C. L., Giroud, M., Hartleben, G., Vogl, E., Blüher, M., Tuckermann, J., Uhlenhaut, H., Herzig, S., Scheideler, M. A miR-29a-driven negative feedback loop regulates peripheral glucocorticoid receptor signaling.

Impact of dietary ω3 polyunsaturated fatty acid supplementation on brown and brite adipocyte function.

The recent characterization of functional brown adipose tissue in adult humans has opened new perspectives for regulation of energy expenditure with respect to obesity and diabetes. Furthermore, dietary recommendations have taken into account the insufficient dietary intake of ω3 PUFAs and the concomitant excessive intake of ω6 PUFA associated with the occurrence of overweight/obesity. We aimed to study whether ω3 PUFAs could play a role in the recruitment and function of energy-dissipating brown/brite adipocytes. We show that ω3 PUFA supplementation has a beneficial effect on the thermogenic function of adipocytes. In vivo, a low dietary ω6:ω3 ratio improved the thermogenic response of brown and white adipose tissues to ß3-adrenergic stimulation. This effect was recapitulated in vitro by PUFA treatment of hMADS adipocytes. We pinpointed the ω6-derived eicosanoid prostaglandin (PG)F2α as the molecular origin because the effects were mimicked with a specific PGF2α receptor agonist. PGF2α level in hMADS adipocytes was reduced in response to ω3 PUFA supplementation. The recruitment of thermogenic adipocytes is influenced by the local quantity of individual oxylipins, which is controlled by the ω6:ω3 ratio of available lipids. In human nutrition, energy homeostasis may thus benefit from the implementation of a more balanced dietary ω6:ω3 ratio.

Norepinephrine triggers an immediate-early regulatory network response in primary human white adipocytes.

BACKGROUND: Norepinephrine (NE) signaling has a key role in white adipose tissue (WAT) functions, including lipolysis, free fatty acid liberation and, under certain conditions, conversion of white into brite (brown-in-white) adipocytes. However, acute effects of NE stimulation have not been described at the transcriptional network level. RESULTS: We used RNA-seq to uncover a broad transcriptional response. The inference of protein-protein and protein-DNA interaction networks allowed us to identify a set of immediate-early genes (IEGs) with high betweenness, validating our approach and suggesting a hierarchical control of transcriptional regulation. In addition, we identified a transcriptional regulatory network with IEGs as master regulators, including HSF1 and NFIL3 as novel NE-induced IEG candidates. Moreover, a functional enrichment analysis and gene clustering into functional modules suggest a crosstalk between metabolic, signaling, and immune responses. CONCLUSIONS: Altogether, our network biology approach explores for the first time the immediate-early systems level response of human adipocytes to acute sympathetic activation, thereby providing a first network basis of early cell fate programs and crosstalks between metabolic and transcriptional networks required for proper WAT function.

Arynes and Cyclic Alkynes as Synthetic Building Blocks for Stereodefined Quaternary Centers.

We report a facile method to synthesize stereodefined quaternary centers from reactions of arynes and related strained intermediates using ß-ketoester-derived substrates. The conversion of ß-ketoesters to chiral enamines is followed by reaction with in situ generated strained arynes or cyclic alkynes. Hydrolytic workup provides the arylated or alkenylated products in enantiomeric excesses as high as 96%. We also describe the one-pot conversion of a ß-ketoester substrate to the corresponding enantioenriched α-arylated product. Computations show how chirality is transferred from the N-bound chiral auxiliary to the final products. These are the first theoretical studies of aryne trapping by chiral nucleophiles to set new stereocenters. Our approach provides a solution to the challenging problem of stereoselective ß-ketoester arylation/alkenylation, with formation of a quaternary center.

IP-receptor and PPARs trigger the conversion of human white to brite adipocyte induced by carbaprostacyclin.

Brite adipocytes recently discovered in humans are of considerable importance in energy expenditure by converting energy excess into heat. This property could be useful in the treatment of obesity, and nutritional aspects are relevant to this important issue. Using hMADS cells as a human cell model which undergoes a white to a brite adipocyte conversion, we had shown previously that arachidonic acid, the major metabolite of the essential nutrient Ω6-linoleic acid, plays a major role in this process. Its metabolites PGE2 and PGF2 alpha inhibit this process via a calcium-dependent pathway, whereas in contrast carbaprostacyclin (cPGI2), a stable analog of prostacyclin, activates white to brite adipocyte conversion. Herein, we show that cPGI2 generates via its cognate cell-surface receptor IP-R, a cyclic AMP-signaling pathway involving PKA activity which in turn induces the expression of UCP1. In addition, cPGI2 activates the pathway of nuclear receptors of the PPAR family, i.e. PPARα and PPARγ, which act separately from IP-R to up-regulate the expression of key genes involved in the function of brite adipocytes. Thus dual pathways are playing in concert for the occurrence of a browning process of human white adipocytes. These results make prostacyclin analogs as a new class of interesting molecules to treat obesity and associated diseases.

The K+ channel TASK1 modulates ß-adrenergic response in brown adipose tissue through the mineralocorticoid receptor pathway.

Brown adipose tissue (BAT) is essential for adaptive thermogenesis and dissipation of caloric excess through the activity of uncoupling protein (UCP)-1. BAT in humans is of great interest for the treatment of obesity and related diseases. In this study, the expression of Twik-related acid-sensitive K(+) channel (TASK)-1 [a pH-sensitive potassium channel encoded by the potassium channel, 2-pore domain, subfamily K, member 3 (Kcnk3) gene] correlated highly with Ucp1 expression in obese and cold-exposed mice. In addition, Task1-null mice, compared with their controls, became overweight, mainly because of an increase in white adipose tissue mass and BAT whitening. Task1(-/-)-mouse-derived brown adipocytes, compared with wild-type mouse-derived brown adipocytes, displayed an impaired ß3-adrenergic receptor response that was characterized by a decrease in oxygen consumption, Ucp1 expression, and lipolysis. This phenotype was thought to be caused by an exacerbation of mineralocorticoid receptor (MR) signaling, given that it was mimicked by corticoids and reversed by an MR inhibitor. We concluded that the K(+) channel TASK1 controls the thermogenic activity in brown adipocytes through modulation of ß-adrenergic receptor signaling.

Long Non-Coding RNAs in Metabolic Organs and Energy Homeostasis.

Single cell organisms can surprisingly exceed the number of human protein-coding genes, which are thus not at the origin of the complexity of an organism. In contrast, the relative amount of non-protein-coding sequences increases consistently with organismal complexity. Moreover, the mammalian transcriptome predominantly comprises non-(protein)-coding RNAs (ncRNA), of which the long ncRNAs (lncRNAs) constitute the most abundant part. lncRNAs are highly species- and tissue-specific with very versatile modes of action in accordance with their binding to a large spectrum of molecules and their diverse localization. lncRNAs are transcriptional regulators adding an additional regulatory layer in biological processes and pathophysiological conditions. Here, we review lncRNAs affecting metabolic organs with a focus on the liver, pancreas, skeletal muscle, cardiac muscle, brain, and adipose organ. In addition, we will discuss the impact of lncRNAs on metabolic diseases such as obesity and diabetes. In contrast to the substantial number of lncRNA loci in the human genome, the functionally characterized lncRNAs are just the tip of the iceberg. So far, our knowledge concerning lncRNAs in energy homeostasis is still in its infancy, meaning that the rest of the iceberg is a treasure chest yet to be discovered.

Replacement of water molecules in a phosphate binding site by furanoside-appended lin-benzoguanine ligands of tRNA-guanine transglycosylase (TGT).

The enzyme tRNA-guanine transglycosylase has been identified as a drug target for the foodborne illness shigellosis. A key challenge in structure-based design for this enzyme is the filling of the polar ribose-34 pocket. Herein, we describe a novel series of ligands consisting of furanoside-appended lin-benzoguanines. They were designed to replace a conserved water cluster and differ by the functional groups at C(2) and C(3) of the furanosyl moiety being either OH or OMe. The unfavorable desolvation of Asp102 and Asp280, which are located close to the ribose-34 pocket, had a significant impact on binding affinity. While the enzyme has tRNA as its natural substrate, X-ray co-crystal structures revealed that the furanosyl moieties of the ligands are not accommodated in the tRNA ribose-34 site, but at the location of the adjacent phosphate group. A remarkable similarity of the position of the oxygen atoms in these two structures suggests furanosides as a potential phosphate isoster.

Drug Discovery Efforts to Identify Novel Treatments for Neglected Tropical Diseases - Cysteine Protease Inhibitors.

BACKGROUND: Neglected tropical diseases are a severe burden for mankind, affecting an increasing number of people around the globe. Many of those diseases are caused by protozoan parasites in which cysteine proteases plays a key role in the parasite's pathogenesis. OBJECTIVE: In this review article, we summarize the drug discovery efforts of the research community from 2017 - 2022 with a special focus on activities such as the optimization of small molecule cysteine protease inhibitors in terms of selectivity profiles or drug-like properties as well as in vivo studies. The cysteine proteases evaluated by this methodology include Cathepsin B1 from Schistosoma mansoni, papain, cruzain, falcipain, and rhodesain. METHODS: Exhaustive literature searches were performed using the keywords "Cysteine Proteases" and "Neglected Tropical Diseases" including the years 2017 - 2022. Overall, approximately 3'000 scientific papers were retrieved, which were filtered using specific keywords enabling the focus on drug discovery efforts. CONCLUSION: Potent and selective cysteine protease inhibitors to treat neglected tropical diseases were identified, which progressed to pharmacokinetic and in vivo efficacy studies. As far as the authors are aware of, none of those inhibitors reached the stage of active clinical development. Either the inhibitor's potency or pharmacokinetic properties or safety profile or a combination thereof prevented further development of the compounds. More efforts with particular emphasis on optimizing pharmacokinetic and safety properties are needed, potentially by collaborations of academic and industrial research groups with complementary expertise. Furthermore, new warheads reacting with the catalytic cysteine should be exploited to advance the research field in order to make a meaningful impact on society.

Structural characterization of the highly cyclized lantibiotic paenicidin A via a partial desulfurization/reduction strategy.

Lantibiotics are ribosomally synthesized antimicrobial peptides produced by bacteria that are increasingly of interest for food preservation and possible therapeutic uses. These peptides are extensively post-translationally modified, and are characterized by lanthionine and methyllanthionine thioether cross-links. Paenibacillus polymyxa NRRL B-30509 was found to produce polymyxins and tridecaptins, in addition to a novel lantibiotic termed paenicidin A. A bacteriocin termed SRCAM 602 previously reported to be produced by this organism and claimed to be responsible for inhibition of Campylobacter jejuni could not be detected either directly or by genomic analysis. The connectivities of the thioether cross-links of paenicidin A were solved using a novel partial desulfurization/reduction strategy in combination with tandem mass spectrometry. This approach overcame the limitations of NMR-based structural characterization that proved mostly unsuccessful for this peptide. Paenicidin A is a highly cyclized lantibiotic, containing six lanthionine and methyllanthionine rings, three of which are interlocking.

Trip13 Depletion in Liver Cancer Induces a Lipogenic Response Contributing to Plin2-Dependent Mitotic Cell Death.

Aberrant energy metabolism and cell cycle regulation both critically contribute to malignant cell growth and both processes represent targets for anticancer therapy. It is shown here that depletion of the AAA+-ATPase thyroid hormone receptor interacting protein 13 (Trip13) results in mitotic cell death through a combined mechanism linking lipid metabolism to aberrant mitosis. Diminished Trip13 levels in hepatocellular carcinoma cells result in insulin-receptor-/Akt-pathway-dependent accumulation of lipid droplets, which act as functional acentriolar microtubule organizing centers disturbing mitotic spindle polarity. Specifically, the lipid-droplet-coating protein perilipin 2 (Plin2) is required for multipolar spindle formation, induction of DNA damage, and mitotic cell death. Plin2 expression in different tumor cells confers susceptibility to cell death induced by Trip13 depletion as well as treatment with paclitaxel, a spindle-interfering drug commonly used against different cancers. Thus, assessment of Plin2 levels enables the stratification of tumor responsiveness to mitosis-targeting drugs, including clinically approved paclitaxel and Trip13 inhibitors currently under development.

Chemoenzymatic conversion of amides to enantioenriched alcohols in aqueous medium.

One-pot reactions that combine non-enzymatic and biocatalytic transformations represent an emerging strategy in chemical synthesis. Some of the most powerful chemoenzymatic methodologies, although uncommon, are those that form a carbon-carbon (C-C) bond and a stereocenter at one of the reacting carbons, thereby streamlining traditional retrosynthetic disconnections. Here we report the one-pot, chemoenzymatic conversion of amides to enantioenriched alcohols. This transformation combines a nickel-catalyzed Suzuki-Miyaura coupling of amides in aqueous medium with an asymmetric, biocatalytic reduction to provide diarylmethanol derivatives in high yields and enantiomeric excesses. The synthetic utility of this platform is underscored by the formal syntheses of both antipodes of the pharmaceutical orphenadrine, which rely on ketoreductase enzymes that instill complementary stereoselectivities. We provide an explanation for the origins of stereoselectivity based on an analysis of the enzyme binding pockets.

Mitochondrial fission is associated with UCP1 activity in human brite/beige adipocytes.

OBJECTIVE: Thermogenic adipocytes (i.e. brown or brite/beige adipocytes) are able to burn large amounts of lipids and carbohydrates as a result of highly active mitochondria and enhanced uncoupled respiration, due to UCP1 activity. Although mitochondria are the key organelles for this thermogenic function, limited human data are available. METHODS/RESULTS: We characterized changes in the mitochondrial function of human brite adipocytes, using hMADS cells as a model of white- to brite-adipocyte conversion. We found that profound molecular modifications were associated with morphological changes in mitochondria. The fission process was partly driven by the DRP1 protein, which also promoted mitochondrial uncoupling. CONCLUSION: Our data demonstrate that white-to-brite conversion of human adipocytes relies on molecular, morphological and functional changes in mitochondria, which enable brite/beige cells to carry out thermogenesis.

2 H-1,2,3-Triazole-Based Dipeptidyl Nitriles: Potent, Selective, and Trypanocidal Rhodesain Inhibitors by Structure-Based Design.

Macrocyclic inhibitors of rhodesain (RD), a parasitic cysteine protease and drug target for the treatment of human African trypanosomiasis, have shown low metabolic stability at the macrocyclic ether bridge. A series of acyclic dipeptidyl nitriles was developed using structure-based design (PDB ID: 6EX8 ). The selectivity against the closely related cysteine protease human cathepsin L (hCatL) was substantially improved, up to 507-fold. In the S2 pocket, 3,4-dichlorophenylalanine residues provided high trypanocidal activities. In the S3 pocket, aromatic residues provided enhanced selectivity against hCatL. RD inhibition ( Ki values) and in vitro cell-growth of Trypanosoma brucei rhodesiense (IC50 values) were measured in the nanomolar range. Triazole-based ligands, obtained by a safe, gram-scale flow production of ethyl 1 H-1,2,3-triazole-4-carboxylate, showed excellent metabolic stability in human liver microsomes and in vivo half-lives of up to 1.53 h in mice. When orally administered to infected mice, parasitaemia was reduced but without complete removal of the parasites.

Repurposing a Library of Human Cathepsin L Ligands: Identification of Macrocyclic Lactams as Potent Rhodesain and Trypanosoma brucei Inhibitors.

Rhodesain (RD) is a parasitic, human cathepsin L (hCatL) like cysteine protease produced by Trypanosoma brucei ( T. b.) species and a potential drug target for the treatment of human African trypanosomiasis (HAT). A library of hCatL inhibitors was screened, and macrocyclic lactams were identified as potent RD inhibitors ( Ki < 10 nM), preventing the cell-growth of Trypanosoma brucei rhodesiense (IC50 < 400 nM). SARs addressing the S2 and S3 pockets of RD were established. Three cocrystal structures with RD revealed a noncovalent binding mode of this ligand class due to oxidation of the catalytic Cys25 to a sulfenic acid (Cys-SOH) during crystallization. The P-glycoprotein efflux ratio was measured and the in vivo brain penetration in rats determined. When tested in vivo in acute HAT model, the compounds permitted up to 16.25 (vs 13.0 for untreated controls) mean days of survival.