Synthesis and biological evaluation of natural Lachnophyllum methyl ester, Lachnophyllum lactone and their synthetic analogs.

(2Z)-Lachnophyllum methyl ester and (4Z)-Lachnophyllum lactone were recently identified as major components in essential oils and extracts of Conyza bonariensis from Togo. Extended biological evaluation of these acetylenic compounds was however hampered by the reduced amounts isolated. A synthetic route was designed providing access to larger quantities of these two natural products as well as to original non-natural analogs with the prospect of exploring for the first time the structure-activity relationships in this series. Using LC/MS analysis, synthetic samples allowed confirming the presence of the two previously isolated natural products in plant extracts obtained by the accelerated solvent extraction technique. The nematocidal activity of the synthesized compounds confirmed the potency of the natural products, which remain the most active among all analogs tested. The synthesized compounds were also assessed against Leishmania infantum axenic amastigotes and the Mycobacterium tuberculosis H37Rv pathogenic strain. (2Z)-Lachnophyllum methyl ester, (4Z)-Lachnophyllum lactone and lactone analogs exhibited the strongest antileishmanial potency. As expected, a longer alkyl chain was necessary to observe significant antimycobacterial activity. The lactone analog bearing a C10 lipophilic appendage displayed the highest antimycobacterial potency. The notable activities of lactones, naturally occurring or analogs, either nematicidal, antileishmanial or antimycobacterial, were compared to their cytotoxicity for mammalian cells and revealed moderate selectivity index values. In this regard, the innocuous (2Z)-Lachnophyllum methyl ester and its analogs open up more promising perspectives for the discovery of bioactive agents to protect both agricultural crops and human health.

Natural and Bioinspired Lipidic Alkynylcarbinols as Leishmanicidal, Antiplasmodial, Trypanocidal, Fungicidal, Antibacterial, and Antimycobacterial Agents.

The present review article recapitulates for the first time the antipathogenic biological data of a series of lipidic natural products and synthetic analogues thereof characterized by the presence in their structure of an alkynylcarbinol unit. The cytotoxic properties of such natural and bioinspired compounds have been covered by several literature overviews, but to date, no review article detailing their activity against pathogens has been proposed. This article thus aims at providing a comprehensive overview of the field including early studies from the 1970s and 1980s with a specific focus on results published from the late 1990s until nowadays. Publications presenting the data of almost 50 different natural products are reported. Detailed activities encompass the fields of leishmanicidal, antiplasmodial, trypanocidal, fungicidal, and mainly antibacterial and antimycobacterial compounds. The few published studies aimed at exploring the structure-activity relationship in these series are also described. Around 15 different synthetic analogues of natural products, selected among the most active reported, are also presented. The rare data available regarding the antipathogenic mode of action of these products are recalled, and finally, a comparative analysis of the available biological data is proposed with the aim of identifying the key structural determinants for the bioactivity against pathogens of these unusual compounds.

Multivalent pyrrolidines acting as pharmacological chaperones against Gaucher disease.

A concise asymmetric synthesis of clickable enantiomeric pyrrolidines was achieved using Crabbé-Ma allenation. The synthesized iminosugars were grafted by copper-free strain-promoted alkyne-azide cycloaddition onto phosphorus dendrimers. The hexavalent and dodecavalent pyrrolidines were evaluated as ß-glucocerebrosidase inhibitors. The level of inhibition suggests that monofluorocyclooctatriazole group may contribute to the affinity for the protein leading to potent multivalent inhibitors. Docking studies were carried out to rationalize these results. Then, the iminosugars clusters were evaluated as pharmacological chaperones in Gaucher patients' fibroblasts. An increase in ß-glucocerebrosidase activity was observed with hexavalent and dodecavalent pyrrolidines at concentrations as low as 1 µM and 0.1 µM, respectively. These iminosugar clusters constitute the first example of multivalent pyrrolidines acting as pharmacological chaperones against Gaucher disease.

Phosphorus Dendrimers for Metal-Free Ligation: Design of Multivalent Pharmacological Chaperones against Gaucher Disease.

The first phosphorus dendrimers built on a cyclotriphosphazene core and decorated with six or twelve monofluorocyclooctyne units were prepared. A simple stirring allowed the grafting of N-hexyl deoxynojirimycin inhitopes onto their surface by copper-free strain promoted alkyne-azide cycloaddition click reaction. The synthesized iminosugars clusters were tested as multivalent inhibitors of the biologically relevant enzymes ß-glucocerebrosidase and acid α-glucosidase, involved in Gaucher and Pompe lysosomal storage diseases, respectively. For both enzymes, all the multivalent compounds were more potent than the reference N-hexyl deoxynojirimycin. Remarkably, the final dodecavalent compound proved to be one of the best ß-glucocerebrosidase inhibitors described to date. These cyclotriphosphazene-based deoxynojirimycin dendrimers were then evaluated as pharmacological chaperones against Gaucher disease. Not only did these multivalent constructs cross the cell membranes but they were also able to increase ß-glucocerebrosidase activity in Gaucher cells. Notably, dodecavalent compound allowed a 1.4-fold enzyme activity enhancement at a concentration as low as 100 nM. These new monofluorocyclooctyne-presenting dendrimers may further find numerous applications in the synthesis of multivalent objects for biological and pharmacological purposes.

Pharmacological Chaperone Therapy for Pompe Disease.

Pompe disease (PD), a lysosomal storage disease, is caused by mutations of the GAA gene, inducing deficiency in the acid alpha-glucosidase (GAA). This enzymatic impairment causes glycogen burden in lysosomes and triggers cell malfunctions, especially in cardiac, smooth and skeletal muscle cells and motor neurons. To date, the only approved treatment available for PD is enzyme replacement therapy (ERT) consisting of intravenous administration of rhGAA. The limitations of ERT have motivated the investigation of new therapies. Pharmacological chaperone (PC) therapy aims at restoring enzymatic activity through protein stabilization by ligand binding. PCs are divided into two classes: active site-specific chaperones (ASSCs) and the non-inhibitory PCs. In this review, we summarize the different pharmacological chaperones reported against PD by specifying their PC class and activity. An emphasis is placed on the recent use of these chaperones in combination with ERT.

N,O-Dialkyl deoxynojirimycin derivatives as CERT START domain ligands.

Dysregulation of the ceramide transport protein CERT is associated to diseases such as cancer. In search for new CERT START domain ligands, N-dodecyl-deoxynojirimycin (N-dodecyl-DNJ) iminosugar was found to display, as a ceramide mimic, significant protein recognition. To reinforce the lipophilic interactions and strengthen this protein binding, a docking study was carried out in order to select the optimal position on which to introduce an additional O-alkyl chain on N-dodecyl-DNJ. Analysis of the calculated poses for three different regioisomers indicated an optimal calculated interaction pattern for N,O3-didodecyl-DNJ. The two most promising regioisomers were prepared by a divergent route and their binding to the CERT START domain was evaluated with fluorescence intensity (FLINT) binding assay. N,O3-didodecyl-DNJ was confirmed to be a new binder prototype with level of protein recognition in the FLINT assay comparable to the best known ligands from the alkylated HPA-12 series. This work opens promising perspectives for the development of new inhibitors of CERT-mediated ceramide trafficking.

Concise asymmetric synthesis of new enantiomeric C-alkyl pyrrolidines acting as pharmacological chaperones against Gaucher disease.

A concise and asymmetric synthesis of the enantiomeric pyrrolidines 2 and ent-2 are herein reported. Both enantiomers were assessed as ß-GCase inhibitors. While compound ent-2 acted as a poor competitive inhibitor, its enantiomer 2 proved to be a potent non-competitive inhibitor. Docking studies were carried out to substantiate their respective protein binding mode. Both pyrrolidines were also able to enhance lysosomal ß-GCase residual activity in N370S homozygous Gaucher fibroblasts. Notably, the non-competitive inhibitor 2 displayed an enzyme activity enhancement comparable to that of reference compounds IFG and NN-DNJ. This work highlights the impact of inhibitors chirality on their protein binding mode and shows that, beyond competitive inhibitors, the study of non-competitive ones can lead to the identification of new relevant parmacological chaperones.

Second-Generation Pharmacological Chaperones: Beyond Inhibitors.

Protein misfolding induced by missense mutations is the source of hundreds of conformational diseases. The cell quality control may eliminate nascent misfolded proteins, such as enzymes, and a pathological loss-of-function may result from their early degradation. Since the proof of concept in the 2000s, the bioinspired pharmacological chaperone therapy became a relevant low-molecular-weight compound strategy against conformational diseases. The first-generation pharmacological chaperones were competitive inhibitors of mutant enzymes. Counterintuitively, in binding to the active site, these inhibitors stabilize the proper folding of the mutated protein and partially rescue its cellular function. The main limitation of the first-generation pharmacological chaperones lies in the balance between enzyme activity enhancement and inhibition. Recent research efforts were directed towards the development of promising second-generation pharmacological chaperones. These non-inhibitory ligands, targeting previously unknown binding pockets, limit the risk of adverse enzymatic inhibition. Their pharmacophore identification is however challenging and likely requires a massive screening-based approach. This review focuses on second-generation chaperones designed to restore the cellular activity of misfolded enzymes. It intends to highlight, for a selected set of rare inherited metabolic disorders, the strategies implemented to identify and develop these pharmacologically relevant small organic molecules as potential drug candidates.

Alkyne-Tagged Analogue of Jaspine†B: New Tool for Identifying Jaspine†B Mode of Action.

The first biologically relevant clickable probe related to the antitumor marine lipid jaspine B is reported. The concise synthetic route to both enantiomers relied on the supercritical fluid chromatography (SFC) enantiomeric resolution of racemic materials. The eutomeric dextrogyre derivative represents the first jaspine B analogue with enhanced cytotoxicity with IC50 down to 30 nm. These enantiomeric probes revealed a chiralitydependent cytoplasmic imaging of U2OS cancer cells by in situ click labeling.

Iminosugar-based ceramide mimicry for the design of new CERT START domain ligands.

The enigmatical dichotomy between the two CERT/GPBP protein isoforms, their vast panel of biological implications and the scarcity of known antagonist series call for new ligand chemotypes identification. We report the design of iminosugar-based ceramide mimics for the development of new START domain ligands potentially targeting either protein isoforms. Strategic choice of (i) an iminoxylitol core structure and (ii) the positioning of two dodecyl residues led to an extent of protein binding comparable to that of the natural cargo lipid ceramide or the archetypical inhibitor HPA-12. Molecular docking study evidenced a possible mode of protein binding fully coherent with the one observed in crystalline co-structures of known ligands. The present study thus paves the way for cellular CERT inhibition studies en route to the development of pharmacological tools aiming at deciphering the respective function and therapeutic potential of the two CERT/GPBP protein isoforms.

Chemistry and Biology of HPAs: A Family of Ceramide Trafficking Inhibitors.

In 2001, two years before the disclosure of the CERT-associated Cer transfer machinery, N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)alkanamides (HPAs) were described as the first, and to date unique, family of intracellular Cer trafficking inhibitors. The dodecanamide derivative, HPA-12, turned out to be a benchmark as a cellular inhibitor of CERT-mediated de novo sphingomyelin biosynthesis. In only 15 years after its first disclosure, this compound has prompted a growing number of biological and chemical studies. Its initial chemical development closely paralleled the study of the CERT protein. It was only after its structural revision in 2011 that HPA-12 received broad attention from the synthetic chemistry community, leading to novel analogues with enhanced protein binding. This Minireview aims at presenting an exhaustive report of the syntheses of HPA-12 and analogues. Biological activities of this CERT inhibitor and structure-activity relationships are also presented to afford a comprehensive overview of the chemistry and biology of the HPA series.

Asymmetric Synthesis and Binding Study of New Long-Chain HPA-12 Analogues as Potent Ligands of the Ceramide Transfer Protein CERT.

A series of 12 analogues of the Cer transfer protein (CERT) antagonist HPA-12 with long aliphatic chains were prepared as their (1R,3S)-syn and (1R,3R)-anti stereoisomers from pivotal chiral oxoamino acids. The enantioselective access to these intermediates as well as their ensuing transformation relied on a practical crystallization-induced asymmetric transformation (CIAT) process. Sonogashira coupling followed by triple bond reduction and thiophene ring hydrodesulfurization (HDS) into the corresponding alkane moieties was then implemented to complete the synthetic routes delivering the targeted HPA-12 analogues in concise 4- to 6-step reaction sequences. Ten compounds were evaluated regarding their ability to bind to the CERT START domain by using the recently developed time-resolved FRET-based homogeneous (HTR-FRET) binding assay. The introduction of a lipophilic appendage on the phenyl moiety led to an overall 10- to 1000-fold enhancement of the protein binding, with the highest effect being observed for a n-hexyl residue in the meta position. The importance of the phenyl ring for the activity was indicated by the reduced potency of the 3-deoxyphytoceramide aliphatic analogues. The 1,3-syn stereoisomers were systematically more potent than their 1,3-anti analogues. In silico studies were used to rationalized these trends, leading to a model of protein recognition coherent with the stronger binding of (1R,3S)-syn HPAs.

The CERT antagonist HPA-12: first practical synthesis and individual binding evaluation of the four stereoisomers.

The first unified synthetic route to the four enantiopure HPA-12 stereoisomers in multi-gram scale is reported based on Crystallization-Induced Asymmetric Transformation (CIAT) technology. This preparative stereoselective synthesis allowed the unprecedented comparative evaluation of HPA-12 stereoisomers regarding their interaction with the CERT START domain. In vitro binding assay coupled to in silico docking approach indicate a possible interaction for the four derivatives. The first TR-FRET homogeneous-phase assay was developed to quantify their binding to the START domain, allowing complete determination of HPA-12 EC50. Results indicate that not only the (1R,3S) lead to the strongest binding, but that both 1R and 3S stereocenters similarly contribute to extent of recognition This automated homogenous assay further opens up promising prospect for the identification of novel potential CERT antagonist by means of high throughput screening.

A novel jaspine B-ceramide hybrid modulates sphingolipid metabolism.

A new sphingolipid hybrid molecule was designed to assemble, within a tail-to-tail double-chain structure, the ceramide hydrophilic moiety and the tetrahydrofuran pharmacophore of jaspine B, a natural product known to interfere with sphingolipid metabolism. This compound was prepared through acylation of sphingosine with a jaspine B derivative bearing a COOH group in the terminal position of the aliphatic backbone. This new hybrid molecule was evaluated for its capacities to affect melanoma cell viability and sphingolipid metabolism. While retaining the cytotoxicity of ceramide itself, this compound was shown to lower the sphingomyelin cellular levels and significantly enhance the production of sphingosine-1-phosphate, thus representing a novel sphingolipid metabolism modulator.

Identification of novel CERT ligands as potential ceramide trafficking inhibitors.

A highly compartmentalized enzymatic network regulates the pro-apoptotic and proliferative effects of sphingolipids. Over-conversion of ceramide (Cer) correlates with insensitivity to apoptosis signaling (in response to chemotherapy) and to drug resistance of cancer cells. De novo sphingomyelin biosynthesis relies on non-vesicular ceramide trafficking by the CERT (CERamide Transfer) protein. Therefore, blocking CERT transfer, thus leading to increased intracellular ceramide availability, represents a potential anticancer strategy. Our study is based on the implementation of an in vitro binding assay, supported by in silico molecular docking. It constitutes the first attempt to explore at the molecular level for the identification of novel CERT ligands. This approach is the first step toward in silico design and optimization of CERT inhibitor candidates, potentially relevant as innovative ceramide-transfer-targeting therapeutic agents.

Prediction of pulmonary restriction from forced vital capacity in elderly is similar using GLI and ERS equations.

INTRODUCTION: The prevalence of pulmonary restriction increases in the elderly and detection could be challenging due to the difficulty in measuring lung volumes in older patients. The recently published Global Lung Function Initiative (GLI) equations were found to predict better restriction in middle-aged patients compared to ERS'93 norms. However, the ability of the GLI equations to detect pulmonary restriction in older patients has not been investigated. PATIENTS AND METHODS: We extracted spirometric data in patients older than 85 years from the database of our pulmonary function testing laboratory. The population with pulmonary restriction was defined as those having a total lung capacity value (TLC) below the lower limit of normal (LLN) using ERS'93 equations. We then compared the ability of the ERS'93 and GLI equations to detect this when the forced vital capacity (FVC) was below the LLN. RESULTS: We analyzed data from 285 patients. A true restrictive defect was found in 66 patients (23%). Sensitivity to detect a reduced TLC was higher when calculated from the GLI than the ERS'93 equations, (70 vs 45%). By contrast, specificity was lower (74 vs 89%, respectively); there was no difference in the negative predictive value (89 and 84%). Using receiver operating curves, both sets of equations performed similarly to detect spirometric restriction. CONCLUSIONS: In conclusion, both sets of equations similarly predicted a pulmonary restriction in older subjects. The high negative predictive value of the GLI equations thus allows for static lung volume measurement to be avoided in older patients when the FCV exceeds the LLN whatever the predicted equation used.

Phenyl dialkynylcarbinols, a Bioinspired Series of Synthetic Antitumor Acetylenic Lipids.

A series of 25 chiral anti-cancer lipidic alkynylcarbinols (LACs) were devised by introducing an (hetero)aromatic ring between the aliphatic chain and the dialkynylcarbinol warhead. The resulting phenyl-dialkynylcarbinols (PACs) exhibit enhanced stability, while retaining cytotoxicity against HCT116 and U2OS cell lines with IC50 down to 40 nM for resolved eutomers. A clickable probe was used to confirm the PAC prodrug behavior: upon enantiospecific bio-oxidation of the carbinol by the HSD17B11 short-chain dehydrogenase/reductase (SDR), the resulting ynones covalently modify cellular proteins, leading to endoplasmic reticulum stress, ubiquitin-proteasome system inhibition, and apoptosis. Insights into the design of LAC prodrugs specifically bioactivated by HSD17B11 vs its paralogue HSD17B13 were obtained. The HSD17B11/HSD17B13-dependent cytotoxicity of PACs was exploited to develop a cellular assay to identify specific inhibitors of these enzymes. A docking study was performed with the HSD17B11 AlphaFold model, providing a molecular basis of the SDR substrates mimicry by PACs. The safety profile of a representative PAC was established in mice.

The effect of a pressure ventilatory support on quadriceps endurance is maintained after exercise training in severe COPD patients. A longitudinal randomized, cross over study.

Purpose: In severe chronic obstructive pulmonary disease (COPD) patients, the application of an inspiratory pressure support (IPS) during exercise increases exercise tolerance and the benefit of exercise training during pulmonary rehabilitation (PR). Moreover, it improves quadriceps endurance after a session of cycling exercise suggesting a reduced muscle fatigue. We looked for the persistence of this effect after PR and sought an association between the improved quadriceps endurance with IPS and the training load during PR. Patients and methods: We studied 20 patients with severe COPD (6 in stage 3and 14 in stage 4 of GOLD) before and after PR. As part of a PR program, patients completed 16 cycling sessions over 6 weeks with the addition of IPS during exercise. As a surrogate of muscular fatigue, quadriceps endurance was measured at 70% of maximal strength in a control condition, after a constant work rate exercise test (CWR) with IPS (TlimQ IPS) or with a sham ventilation (TlimQsham), in a random order. These tests were repeated similarly at the end of PR. Results: PR was associated with a significant increase in maximal power output, cycling endurance, quadriceps strength and endurance. Session training load (power output x duration of the session) increased by 142% during the course of the program. Before PR, CWR duration increases with IPS compared to sham ventilation (Δtime = +244s, p = 0.001). Compared to control condition, post-exercise TlimQ reduction was lower with IPS at isotime than at the end of CWR or than with sham ventilation (-9 ± 21%, -18 ± 16% and -23 ± 18%, respectively, p = 0.09, p < 0.0001 and p < 0.0001). After PR, the post-exercise decrease of TlimQ was reduced after IPS compared to sham (-9 ± 18% vs. -21 ± 17%, respectively, p = 0.004). No relationship was found between the prevention of quadriceps fatigue and the training load. Conclusion: In severe COPD patients, the beneficial effect of a ventilator support on quadriceps endurance persisted after PR with IPS. However, it was not related to the increase in training load, and could not predict the training response to non-invasive ventilation during exercise.

SDR enzymes oxidize specific lipidic alkynylcarbinols into cytotoxic protein-reactive species.

Hundreds of cytotoxic natural or synthetic lipidic compounds contain chiral alkynylcarbinol motifs, but the mechanism of action of those potential therapeutic agents remains unknown. Using a genetic screen in haploid human cells, we discovered that the enantiospecific cytotoxicity of numerous terminal alkynylcarbinols, including the highly cytotoxic dialkynylcarbinols, involves a bioactivation by HSD17B11, a short-chain dehydrogenase/reductase (SDR) known to oxidize the C-17 carbinol center of androstan-3-alpha,17-beta-diol to the corresponding ketone. A similar oxidation of dialkynylcarbinols generates dialkynylketones, that we characterize as highly protein-reactive electrophiles. We established that, once bioactivated in cells, the dialkynylcarbinols covalently modify several proteins involved in protein-quality control mechanisms, resulting in their lipoxidation on cysteines and lysines through Michael addition. For some proteins, this triggers their association to cellular membranes and results in endoplasmic reticulum stress, unfolded protein response activation, ubiquitin-proteasome system inhibition and cell death by apoptosis. Finally, as a proof-of-concept, we show that generic lipidic alkynylcarbinols can be devised to be bioactivated by other SDRs, including human RDH11 and HPGD/15-PGDH. Given that the SDR superfamily is one of the largest and most ubiquitous, this unique cytotoxic mechanism-of-action could be widely exploited to treat diseases, in particular cancer, through the design of tailored prodrugs.

Fragment-Based Ligand Discovery Applied to the Mycolic Acid Methyltransferase Hma (MmaA4) from Mycobacterium tuberculosis: A Crystallographic and Molecular Modelling Study.

The mycolic acid biosynthetic pathway represents a promising source of pharmacological targets in the fight against tuberculosis. In Mycobacterium tuberculosis, mycolic acids are subject to specific chemical modifications introduced by a set of eight S-adenosylmethionine dependent methyltransferases. Among these, Hma (MmaA4) is responsible for the introduction of oxygenated modifications. Crystallographic screening of a library of fragments allowed the identification of seven ligands of Hma. Two mutually exclusive binding modes were identified, depending on the conformation of residues 147-154. These residues are disordered in apo-Hma but fold upon binding of the S-adenosylmethionine (SAM) cofactor as well as of analogues, resulting in the formation of the short η1-helix. One of the observed conformations would be incompatible with the presence of the cofactor, suggesting that allosteric inhibitors could be designed against Hma. Chimeric compounds were designed by fusing some of the bound fragments, and the relative binding affinities of initial fragments and evolved compounds were investigated using molecular dynamics simulation and generalised Born and Poisson-Boltzmann calculations coupled to the surface area continuum solvation method. Molecular dynamics simulations were also performed on apo-Hma to assess the structural plasticity of the unliganded protein. Our results indicate a significant improvement in the binding properties of the designed compounds, suggesting that they could be further optimised to inhibit Hma activity.