Michela I. Simone

A compendium of cyclic sugar amino acids and their carbocyclic and heterocyclic nitrogen analogues

This compendium focuses on functionalised sugar amino acids (SAAs) and their 3- to 6-membered nitrogen heterocyclic and carbocyclic analogues. The main benefit of using SAAs and their related nitrogen and carbon congeners in the production of peptidomimetics and glycomimetics is that their properties can be readily altered via modification of their ring size, chemical manipulation of their numerous functional groups and fine-tuning of the stereochemical arrangement of their ring substituents. These building blocks provide access to hydrophilic and hydrophobic peptide isosteres whose physical properties allow entry to a region of chemotherapeutic space which is still under-explored by medicinal chemists. These building blocks are also important in providing amino acids whose inherent conformational bias leads to predisposition to secondary structure upon oligomerisation in relatively short sequences. These foldamers, particularly those containing ω-amino acids, provide an additional opportunity to expand access to the control of structures by artificial peptides. The synthesis and biological evaluation of these building blocks in glycomimetics and peptidomimetics systems keep expanding the reach of the glycosciences to the medical sciences, provide a greater outlook onto the wide range of cellular functions of saccharides and their derivatives involved and greater insight into the nature of oligosaccharide and protein folding.

Endothelial C-type natriuretic peptide maintains vascular homeostasis

The endothelium plays a fundamental role in maintaining vascular homeostasis by releasing factors that regulate local blood flow, systemic blood pressure, and the reactivity of leukocytes and platelets. Accordingly, endothelial dysfunction underpins many cardiovascular diseases, including hypertension, myocardial infarction, and stroke. Herein, we evaluated mice with endothelial-specific deletion of Nppc, which encodes C-type natriuretic peptide (CNP), and determined that this mediator is essential for multiple aspects of vascular regulation. Specifically, disruption of CNP leads to endothelial dysfunction, hypertension, atherogenesis, and aneurysm. Moreover, we identified natriuretic peptide receptor-C (NPR-C) as the cognate receptor that primarily underlies CNP-dependent vasoprotective functions and developed small-molecule NPR-C agonists to target this pathway. Administration of NPR-C agonists promotes a vasorelaxation of isolated resistance arteries and a reduction in blood pressure in wild-type animals that is diminished in mice lacking NPR-C. This work provides a mechanistic explanation for genome-wide association studies that have linked the NPR-C (Npr3) locus with hypertension by demonstrating the importance of CNP/NPR-C signaling in preserving vascular homoeostasis. Furthermore, these results suggest that the CNP/NPR-C pathway has potential as a disease-modifying therapeutic target for cardiovascular disorders.

Selective Inhibition of the Mitochondrial Permeability Transition Pore Protects against Neurodegeneration in Experimental Multiple Sclerosis

The mitochondrial permeability transition pore is a recognized drug target for neurodegenerative conditions such as multiple sclerosis and for ischemia-reperfusion injury in the brain and heart. The peptidylprolyl isomerase, cyclophilin D (CypD, PPIF), is a positive regulator of the pore, and genetic down-regulation or knock-out improves outcomes in disease models. Current inhibitors of peptidylprolyl isomerases show no selectivity between the tightly conserved cyclophilin paralogs and exhibit significant off-target effects, immunosuppression, and toxicity. We therefore designed and synthesized a new mitochondrially targeted CypD inhibitor, JW47, using a quinolinium cation tethered to cyclosporine. X-ray analysis was used to validate the design concept, and biological evaluation revealed selective cellular inhibition of CypD and the permeability transition pore with reduced cellular toxicity compared with cyclosporine. In an experimental autoimmune encephalomyelitis disease model of neurodegeneration in multiple sclerosis, JW47 demonstrated significant protection of axons and improved motor assessments with minimal immunosuppression. These findings suggest that selective CypD inhibition may represent a viable therapeutic strategy for MS and identify quinolinium as a mitochondrial targeting group for in vivo use.

A mitochondrial-targeted cyclosporin A with high binding affinity for cyclophilin D yields improved cytoprotection of cardiomyocytes

Mitochondrial CyP-D (cyclophilin-D) catalyses formation of the PT (permeability transition) pore, a key lesion in the pathogenesis of I/R (ischaemia/reperfusion) injury. There is evidence [Malouitre, Dube, Selwood and Crompton (2010) Biochem. J. 425, 137–148] that cytoprotection by the CyP inhibitor CsA (cyclosporin A) is improved by selective targeting to mitochondria. To investigate this further, we have developed an improved mtCsA (mitochondrial-targeted CsA) by modifying the spacer linking the CsA to the TPP+ (triphenylphosphonium) (mitochondrial-targeting) cation. The new mtCsA exhibits an 18-fold increase in binding affinity for CyP-D over the prototype and a 12-fold increase in potency of inhibition of the PT in isolated mitochondria, owing to a marked decrease in non-specific binding. The cytoprotective capacity was assessed in isolated rat cardiomyocytes subjected to transient glucose and oxygen deprivation (pseudo-I/R). The new mtCsA was maximally effective at lower concentrations than CsA (3–15 nM compared with 50–100 nM) and yielded improved cytoprotection for up to 3 h following the pseudo-ischaemic insult (near complete compared with 40%). These data indicate the potential value of selective CyP-D inhibition in cytoprotection.

C-3 branched δ-3,5-cis- and trans-THF sugar amino acids: synthesis of the first generation of branched homooligomers

This article describes the efficient synthesis of the first generation of branched sugar amino acid (SAA) oligomers in solution phase via two main routes: by the use of a standard coupling reagent and via the use of active ester intermediates. Benzyl-protected dimeric carbopeptoid and methyl-protected dimeric and tetrameric, hexameric and octameric carbopeptoids were obtained from a branched δ-3,5-trans-tetrahydrofuran (THF) SAA and methyl-protected dimeric and tetrameric carbopeptoids were synthesised from a branched δ-3,5-cis-THF SAA. These systems are of interest because of their potential to display foldameric properties reminiscent of those observed in α-peptides and proteins. Amongst their many uses, foldamers provide simpler models in the study of the factors which induce the folding and unfolding of proteins and, ultimately, potential insights into their functioning.

2- and 3-substituted imidazo[1,2-a]pyrazines as inhibitors of bacterial type IV secretion

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Exploring the interaction between siRNA and the SMoC biomolecule transporters: Implications for small molecule-mediated delivery of siRNA

The small molecule carrier class of biomolecule transporters, modeled on the third helix of the Antennapedia homeodomain, has previously been shown to transport active proteins into cells. Here, we show an improved synthetic route to small molecule carriers, including Molander chemistry using trifluoroborate salts to improve the yield of the Suzuki–Miyaura coupling step for the formation of the biphenyl backbone. The required boronic acids could be formed by the reaction of a 2‐(dimethylamino)ethyl ether‐modified aryl Grignard reagent with triisopropyl borate. The potential for the use of small molecule carriers as oligonucleotide‐transporting agents was also explored by characterizing the interactions between small molecule carriers and siRNA. Molecular dynamics and NMR analysis indicated that the small molecule carrier guanidines are stabilized by π‐cation interactions with the biphenyl system, thus not only increasing the basicity or pKa but also shielding the charge. The binding affinities of various small molecule carriers for siRNA were investigated using isothermal calorimetry and gel shift assays. Small molecule carrier‐mediated siRNA delivery to cultured fibroblasts is demonstrated, showing that small molecule carriers possess the ability to transport functional siRNA into cells. Knockdown of Cdc7 kinase, a target for cancer, is achieved.

Improved fluorescence assays to measure the defects associated with F508del-CFTR allow identification of new active compounds

Cystic fibrosis (CF) is a debilitating disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which codes for a Clˉ/HCO3ˉ channel. F508del, the most common CF‐associated mutation, causes both gating and biogenesis defects in the CFTR protein. This paper describes the optimization of two fluorescence assays, capable of measuring CFTR function and cellular localization, and their use in a pilot drug screen.

An integrated flow and microwave approach to a broad spectrum protein kinase inhibitor

The protein kinase inhibitor CTx-0152960 (6, 2-((5-chloro-2-((4-morpholinophenyl)amino)pyrimidin-4-yl)amino)-N-methylbenzamide), and the piperazinyl analogue, CTx-0294885 (7, 2-((5-chloro-2-((4-piperazin-1-ylphenyl)amino)pyrimidin-4-yl)amino)-N-methylbenzamide), were prepared using a hybrid flow and microwave approach. The use of flow chemistry approaches avoided the need for Boc-protection of piperidine in the key SNAr coupling with 1-fluoro-4-nitrobenzene. Microwave coupling of 4-morphilinoaniline 8 and 4-(piperazine-1-yl)aniline 9 with 2-(2,5-dichloropyrimidine-4-ylamino)-N-methylbenzamide 10, proved to be the most efficacious route to the target analogues 6 and 7. This hybrid methodology reduced the number of synthetic steps, gave enhanced overall yields and increased atom economy through step reduction and minimal requirement for chromatographic purification, relative to the original batch synthesis approach.

Penta­fluoro­phenyl (3R,4R,5S)-5-{[(3R,4R,5S)-5-azido­methyl-3,4-dimeth­oxy-2,3,4,5-tetra­hydro­furan-3-carboxamido]­meth­yl}-3,4-dimeth­oxy-2,3,4,5-tetra­hydro­furan-3-carboxyl­ate

The title compound, C22H25F5N4O9, is a stable pentafluorophenyl ester intermediate in the synthesis of novel homo-oligomeric structures containing branched carbon chains. The structure is epimeric to the previously characterized dimeric pentafluorophenyl ester with stereochemistry (3R,4R,5R), which was synthesized using d-ribose as starting material. The crystal structure of the title molecule removes any ambiguities arising from the relative stereochemistries of the six chiral centres. Two hydrogen bonds, bifurcating from the NH group, stabilize the crystal: one intramolecular and one intermolecular, both involving O atoms of the methoxy groups. The asymmetric unit contains two independent molecules not related by any pseudo-symmetry operators. The major conformational differences are localized, leading to one molecule being extended compared to the other. The collected crystal was twinned (twin ratio is 0.939:0.061), and the azide group is positionally disordered over two positions in one molecule [occupancy ratio 0.511 (18):0.489 (18)].