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.

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.

Phosphopantetheinyl transferase binding and inhibition by amidino-urea and hydroxypyrimidinethione compounds.

Owing to their role in activating enzymes essential for bacterial viability and pathogenicity, phosphopantetheinyl transferases represent novel and attractive drug targets. In this work, we examined the inhibitory effect of the aminido-urea 8918 compound against the phosphopantetheinyl transferases PptAb from Mycobacterium abscessus and PcpS from Pseudomonas aeruginosa, two pathogenic bacteria associated with cystic fibrosis and bronchiectasis, respectively. Compound 8918 exhibits inhibitory activity against PptAb but displays no activity against PcpS in vitro, while no antimicrobial activity against Mycobacterium abscessus or Pseudomonas aeruginosa could be detected. X-ray crystallographic analysis of 8918 bound to PptAb-CoA alone and in complex with an acyl carrier protein domain in addition to the crystal structure of PcpS in complex with CoA revealed the structural basis for the inhibition mechanism of PptAb by 8918 and its ineffectiveness against PcpS. Finally, in crystallo screening of potent inhibitors from the National Cancer Institute library identified a hydroxypyrimidinethione derivative that binds PptAb. Both compounds could serve as scaffolds for the future development of phosphopantetheinyl transferases inhibitors.

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.

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.

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.

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.

Development of a CERT START Domain-Ceramide HTRF Binding Assay and Application to Pharmacological Studies and Screening.

Sphingomyelin (SM) metabolism deregulation was recently associated with cell metastasis and chemoresistance, and several pharmacological strategies targeting SM metabolism have emerged. The ceramide (Cer) generated in the endoplasmic reticulum (ER) is transferred to the Golgi apparatus to be transformed into SM. CERamide Transfer (CERT) protein is responsible for the nonvesicular trafficking of Cer to Golgi. Blocking the CERT-mediated ER-to-Golgi Cer transfer is an interesting antioncogenic therapeutic approach. Here, we developed a protein-lipid interaction assay for the identification of new CERT-Cer interaction inhibitors. Frequently used for protein-protein interaction by enzymatic and analyte dosage assays, homogeneous time-resolved fluorescence technology was adapted for the first time to a lipid-protein binding assay. This test was developed for high-throughput screening, and a library of 672 molecules was screened. Seven hits were identified, and their inhibitory effect quantified by EC50 measurements showed binding inhibition three orders of magnitude more potent than that of HPA12, the unique known CERT antagonist to date. Each compound was tested on an independent test, confirming its high affinity and pharmacological potential.

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.

Alteration of ceramide 1-O-functionalization as a promising approach for cancer therapy.

Sphingolipids, which are complex lipidic components of the cell membranes, lie in a key position to modulate the pathways of trans-membrane signaling and allow the cell to adapt to environmental stresses. In malignancies, reduced production of some sphingolipid species able to induce apoptosis such as ceramide and conversely, increased levels of some other metabolites involved in tumor progression and drug resistance of cancer cells, are often described. In this context, the discovery of new chemical entities able to specifically modify ceramide metabolism should offer novel pharmacological tools in cancer therapy. The review dedicates particular attention to the enzymes that modify ceramide at the C1-OH position generating other biologically important sphingolipids in cancer, such as sphingomyelin, ceramide-1-phosphate or glucosylceramide. Findings reported in the literature leading to the development of new chemical entities specifically designed to achieve the above goals have been collected and are discussed. The effects of enzyme inhibitors of sphingomyelin synthase, ceramide kinase and glucosylceramide synthase on cancer cell proliferation, sensitivity to chemotherapeutics, induction of apoptosis or growth of xenografts are presented.

Flexible and enantioselective access to jaspine B and biologically active chain-modified analogues thereof.

Whereas the all-cis tetrahydrofuran framework of the cytotoxic anhydrophytosphingosine jaspine B is considered as a relevant pharmacophore, little is known about the influence of the aliphatic chain of this amphiphilic molecule on its activity. We developed a synthetic strategy allowing flexible introduction of various lipophilic fragments in the jaspine's skeleton. The route was validated with two distinct approaches to jaspine B. Five chain-modified analogues were also prepared. Biological evaluation of these derivatives demonstrated a good correlation between their cytotoxicity and their capacity to inhibit conversion of ceramide into sphingomyelin in melanoma cells. A series of potent and selective inhibitors of sphingomyelin production was thus identified. Furthermore, the good overall potency of an omega-aminated analogue allowed us to dissociate of the pharmacological action of jaspine B from its amphiphilic nature.