Self-assembling dendrimer nanosystems for specific fluorine magnetic resonance imaging and effective theranostic treatment of tumors.

Fluorine magnetic resonance imaging (19F-MRI) is particularly promising for biomedical applications owing to the absence of fluorine in most biological systems. However, its use has been limited by the lack of safe and water-soluble imaging agents with high fluorine contents and suitable relaxation properties. We report innovative 19F-MRI agents based on supramolecular dendrimers self-assembled by an amphiphilic dendrimer composed of a hydrophobic alkyl chain and a hydrophilic dendron. Specifically, this amphiphilic dendrimer bears multiple negatively charged terminals with high fluorine content, which effectively prevented intra- and intermolecular aggregation of fluorinated entities via electrostatic repulsion. This permitted high fluorine nuclei mobility alongside good water solubility with favorable relaxation properties for use in 19F-MRI. Importantly, the self-assembling 19F-MRI agent was able to encapsulate the near-infrared fluorescence (NIRF) agent DiR and the anticancer drug paclitaxel for multimodal 19F-MRI and NIRF imaging of and theranostics for pancreatic cancer, a deadly disease for which there remains no adequate early detection method or efficacious treatment. The 19F-MRI and multimodal 19F-MRI and NIRF imaging studies on human pancreatic cancer xenografts in mice confirmed the capability of both imaging modalities to specifically image the tumors and demonstrated the efficacy of the theranostic agent in cancer treatment, largely outperforming the clinical anticancer drug paclitaxel. Consequently, these dendrimer nanosystems constitute promising 19F-MRI agents for effective cancer management. This study offers a broad avenue to the construction of 19F-MRI agents and theranostics, exploiting self-assembling supramolecular dendrimer chemistry.

Molecular Ballet: Investigating the Complex Interaction between Self-Assembling Dendrimers and Human Serum Albumin via Computational and Experimental Methods.

Dendrimers, intricate macromolecules with highly branched nanostructures, offer unique attributes including precise control over size, shape, and functionality, making them promising candidates for a wide range of biomedical applications. The exploration of their interaction with biological environments, particularly human serum albumin (HSA), holds significant importance for biomedical utilization. In this study, the interaction between HSA and a recently developed self-assembling amphiphilic dendrimer (AD) was investigated using various experimental techniques. Fluorescence spectroscopy and isothermal titration calorimetry revealed moderate interactions between the protein and the AD nanomicelles (NMs), primarily attributed to favorable enthalpic contributions arising from electrostatic interactions and hydrogen bonding. Structural analysis indicated minimal changes in HSA upon complexation with the AD NMs, which was further supported by computational simulations demonstrating stable interactions at the atomistic level. These findings provide valuable insights into the binding mechanisms and thermodynamic parameters governing HSA/AD NM interactions, thereby contributing to the understanding of their potential biomedical applications.

[18F]Fluspidine-A PET Tracer for Imaging of σ1 Receptors in the Central Nervous System.

σ1 receptors play a crucial role in various neurological and neurodegenerative diseases including pain, psychosis, Alzheimer's disease, and depression. Spirocyclic piperidines represent a promising class of potent σ1 receptor ligands. The relationship between structural modifications and σ1 receptor affinity and selectivity over σ2 receptors led to the 2-fluoroethyl derivative fluspidine (2, Ki = 0.59 nM). Enantiomerically pure (S)-configured fluspidine ((S)-2) was prepared by the enantioselective reduction of the α,ß-unsaturated ester 23 with NaBH4 and the enantiomerically pure co-catalyst (S,S)-24. The pharmacokinetic properties of both fluspidine enantiomers (R)-2 and (S)-2 were analyzed in vitro. Molecular dynamics simulations revealed very similar interactions of both fluspidine enantiomers with the σ1 receptor protein, with a strong ionic interaction between the protonated amino moiety of the piperidine ring and the COO- moiety of glutamate 172. The 18F-labeled radiotracers (S)-[18F]2 and (R)-[18F]2 were synthesized in automated syntheses using a TRACERlab FX FN synthesis module. High radiochemical yields and radiochemical purity were achieved. Radiometabolites were not found in the brains of mice, piglets, and rhesus monkeys. While both enantiomers revealed similar initial brain uptake, the slow washout of (R)-[18F]2 indicated a kind of irreversible binding. In the first clinical trial, (S)-[18F]2 was used to visualize σ1 receptors in the brains of patients with major depressive disorder (MDD). This study revealed an increased density of σ1 receptors in cortico-striato-(para)limbic brain regions of MDD patients. The increased density of σ1 receptors correlated with the severity of the depressive symptoms. In an occupancy study with the PET tracer (S)-[18F]2, the selective binding of pridopidine at σ1 receptors in the brain of healthy volunteers and HD patients was shown.

Biophysical and docking study on the interaction of anticancer drugs encorafenib and binimetinib with human serum albumin.

The utilization of BRAF and MEK inhibitors in combination therapy has demonstrated superior outcomes in the treatment of melanoma as compared to monotherapy. In the present scenario, the combination therapy of Encorafenib (ENC), a BRAF inhibitor, and Binimetinib (BINI), a MEK inhibitor, has been identified as one of the most efficacious treatment modalities for this malignancy. Investigations of protein binding, particularly with human serum albumin (HSA), are essential to understand drug performance and enhance therapeutic outcomes. The investigation of the interplay between small molecule drugs and HSA is of paramount importance, given that such interactions can exert a substantial influence on the pharmacokinetics of these therapeutic agents. The present study aims to bridge these lacunae by implementing a comprehensive approach that integrates fluorescence spectroscopy (FS), isothermal titration calorimetry (ITC), far-ultraviolet circular dichroism (far-UV CD), and molecular simulations. Through analysis of the fluorescence quenching of HSA at three distinct temperatures, it was ascertained that the association constants for the complexes formed between drugs and HSA were of the magnitude of 104 M-1. This suggests that the interactions between the compounds and albumin were moderate and comparable. Simultaneously, the investigation of fluorescence indicated a contrasting binding mechanism for the two inhibitors: ENC predominantly binds to HSA through enthalpic interaction, while BINI/HSA is stabilized by entropic contributions. The data obtained was confirmed through experimental procedures conducted using the ITC method. The results of ligand-competitive displacement experiments indicate that ENC and BINI can bind to HSA within subdomain IIA, specifically Sudlow site I. However, far-UV CD studies show that there are no notable alterations in the structure of HSA upon binding with either of the two inhibitors. Ultimately, the results were supported by computational molecular analysis, which identified the key interactions that contribute to the stabilization of the two ligand/HSA complexes.

The Spicy Science of Dendrimers in the Realm of Cancer Nanomedicine: A Report from the COST Action CA17140 Nano2Clinic.

COST Action CA17140 Cancer Nanomedicine-from the bench to the bedside (Nano2Clinic,) is the first, pan-European interdisciplinary network of representatives from academic institutions and small and medium enterprises including clinical research organizations (CROs) devoted to the development of nanosystems carrying anticancer drugs from their initial design, preclinical testing of efficacy, pharmacokinetics and toxicity to the preparation of detailed protocols needed for the first phase of their clinical studies. By promoting scientific exchanges, technological implementation, and innovative solutions, the action aims at providing a timely instrument to rationalize and focus research efforts at the European level in dealing with the grand challenge of nanomedicine translation in cancer, one of the major and societal-burdening human pathologies. Within CA17140, dendrimers in all their forms (from covalent to self-assembling dendrons) play a vital role as powerful nanotheranostic agents in oncology; therefore, the purpose of this review work is to gather and summarize the major results in the field stemming from collaborative efforts in the framework of the European Nano2Clinic COST Action.

Cargo-selective and adaptive delivery of nucleic acid therapeutics by bola-amphiphilic dendrimers.

Nucleic acid therapeutics are becoming an important drug modality, offering the unique opportunity to address "undruggable" targets, respond rapidly to evolving pathogens, and treat diseases at the gene level for precision medicine. However, nucleic acid therapeutics have poor bioavailability and are chemolabile and enzymolabile, imposing the need for delivery vectors. Dendrimers, by virtue of their well-defined structure and cooperative multivalence, represent precision delivery systems. We synthesized and studied bola-amphiphilic dendrimers for cargo-selective and on-demand delivery of DNA and small interfering RNA (siRNA), both important nucleic acid therapeutics. Remarkably, superior performances were achieved for siRNA delivery with the second-generation dendrimer, yet for DNA delivery with the third generation. We systematically studied these dendrimers with regard to cargo binding, cellular uptake, endosomal release, and in vivo delivery. Differences in size both of the dendrimers and their nucleic acid cargos impacted the cooperative multivalent interactions for cargo binding and release, leading to cargo-adaptive and selective delivery. Moreover, both dendrimers harnessed the advantages of lipid and polymer vectors, while offering nanotechnology-based tumor targeting and redox-responsive cargo release. Notably, they allowed tumor- and cancer cell-specific delivery of siRNA and DNA therapeutics for effective treatment in different cancer models, including aggressive and metastatic malignancies, outperforming the currently available vectors. This study provides avenues to engineer tailor-made vectors for nucleic acid delivery and precision medicine.

Conformationally Restricted σ1 Receptor Antagonists from (-)-Isopulegol.

Antagonists at σ1 receptors have great potential for the treatment of neuropathic pain. Starting from monoterpene (-)-isopulegol (1), aminodiols 8-11 were obtained and transformed into bicyclic 13-16 and tricyclic ligands 19-22. Aminodiols 8-11 showed higher σ1 affinity than the corresponding bicyclic 13-16 and tricyclic derivatives 19-22. (R)-configuration in the side chain of aminodiols (8 and 10) led to higher σ1 affinity than (S)-configuration (9 and 11). 4-Benzylpiperidines (b-series) revealed higher σ1 affinity than 4-phenylbutylamines (a-series). Aminodiol 8b showed very high σ1 affinity (Ki = 1.2 nM), excellent selectivity over σ2 receptors, and promising logD7.4 (3.05) and lipophilic ligand efficiency (5.87) values. Molecular dynamics simulations were conducted to analyze the σ1 affinity and selectivity on an atomistic level. In the capsaicin assay, 8b exhibited similar antiallodynic activity to the prototypical σ1 antagonist S1RA. The antiallodynic activity of 8b was removed by co-application of the σ1 agonist PRE-084, proving σ1 antagonism being involved in the antiallodynic effect.

The cyclic peptide G4CP2 enables the modulation of galactose metabolism in yeast by interfering with GAL4 transcriptional activity.

Genetically-encoded combinatorial peptide libraries are convenient tools to identify peptides to be used as therapeutics, antimicrobials and functional synthetic biology modules. Here, we report the identification and characterization of a cyclic peptide, G4CP2, that interferes with the GAL4 protein, a transcription factor responsible for the activation of galactose catabolism in yeast and widely exploited in molecular biology. G4CP2 was identified by screening CYCLIC, a Yeast Two-Hybrid-based combinatorial library of cyclic peptides developed in our laboratory. G4CP2 interferes with GAL4-mediated activation of galactose metabolic enzymes both when expressed intracellularly, as a recombinant peptide, and when provided exogenously, as a chemically-synthesized cyclic peptide. Our results support the application of G4CP2 in microbial biotechnology and, additionally, demonstrate that CYCLIC can be used as a tool for the rapid identification of peptides, virtually without any limitations with respect to the target protein. The possible biotechnological applications of cyclic peptides are also discussed.

Thioxanthenone-based derivatives as multitarget therapeutic leads for Alzheimer's disease.

A set of twenty-five thioxanthene-9-one and xanthene-9-one derivatives, that were previously shown to inhibit cholinesterases (ChEs) and amyloid ß (Aß40) aggregation, were evaluated for the inhibition of tau protein aggregation. All compounds exhibited a good activity, and eight of them (5-8, 10, 14, 15 and 20) shared comparable low micromolar inhibitory potency versus Aß40 aggregation and human acetylcholinesterase (AChE), while inhibiting human butyrylcholinesterase (BChE) even at submicromolar concentration. Compound 20 showed outstanding biological data, inhibiting tau protein and Aß40 aggregation with IC50 = 1.8 and 1.3 µM, respectively. Moreover, at 0.1-10 µM it also exhibited neuroprotective activity against tau toxicity induced by okadoic acid in human neuroblastoma SH-SY5Y cells, that was comparable to that of estradiol and PD38. In preliminary toxicity studies, these interesting results for compound 20 are somewhat conflicting with a narrow safety window. However, compound 10, although endowed with a little lower potency for tau and Aß aggregation inhibition additionally demonstrated good inhibition of ChEs and rather low cytotoxicity. Compound 4 is also worth of note for its high potency as hBChE inhibitor (IC50 = 7 nM) and for the three order of magnitude selectivity versus hAChE. Molecular modelling studies were performed to explain the different behavior of compounds 4 and 20 towards hBChE. The observed balance of the inhibitory potencies versus the relevant targets indicates the thioxanthene-9-one derivatives as potential MTDLs for AD therapy, provided that the safety window will be improved by further structural variations, currently under investigation.

Some things old, new and borrowed: Delivery of dabrafenib and vemurafenib to melanoma cells via self-assembled nanomicelles based on an amphiphilic dendrimer.

Two clinically approved anticancer drugs targeting BRAF in melanoma patients - dabrafenib (DAB) and vemurafenib (VEM) - have been successfully encapsulated into nanomicelles formed upon self-assembly of an amphiphilic dendrimer AD based on two C18 aliphatic chains and a G2 PAMAM head. The process resulted in the formation of well-defined (∼10 nm) core-shell nanomicelles (NMs) with excellent encapsulation efficiency (∼70% for DAB and ∼60% for VEM) and good drug loading capacity (∼27% and ∼24% for DAB and VEM, respectively). Dynamic light scattering (DLS), transmission electron microscopy (TEM), small-angle x-ray scattering (SAXS), nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC), and molecular simulation (MS) experiments were used, respectively, to determine the size and structure of the empty and drug-loaded nanomicelles (DLNMs), along with the interactions between the NMs and their cargoes. The in vitro release data revealed profiles governed by Fickian diffusion; moreover, for both anticancer molecules, an acidic environment (pH = 5.0) facilitated drug release with respect to physiological pH conditions (pH = 7.4). Finally, both DAB- and VEM-loaded NMs elicited enhanced response with respect to free drug treatments in 4 different melanoma cell lines.

Novel epigenetic therapeutic strategies and targets in cancer.

The critical role of dysregulated epigenetic pathways in cancer genesis, development, and therapy has typically been established as a result of scientific and technical innovations in next generation sequencing. RNA interference, histone modification, DNA methylation and chromatin remodelling are epigenetic processes that control gene expression without causing mutations in the DNA. Although epigenetic abnormalities are thought to be a symptom of cell tumorigenesis and malignant events that impact tumor growth and drug resistance, physicians believe that related processes might be a key therapeutic target for cancer treatment and prevention due to the reversible nature of these processes. A plethora of novel strategies for addressing epigenetics in cancer therapy for immuno-oncological complications are currently available - ranging from basic treatment to epigenetic editing. - and they will be the subject of this comprehensive review. In this review, we cover most of the advancements made in the field of targeting epigenetics with special emphasis on microbiology, plasma science, biophysics, pharmacology, molecular biology, phytochemistry, and nanoscience.

Transformation of L-DOPA and Dopamine on the Surface of Gold Nanoparticles: An NMR and Computational Study.

Gold nanoparticles (AuNPs) have found applications in biomedicine as diagnostic tools, but extensive research efforts have been also directed toward their development as more efficient drug delivery agents. The high specific surface area of AuNPs may provide dense loading of molecules like catechols (L-DOPA and dopamine) on nanosurfaces, enabling functionalization strategies for advancing conventional therapy and diagnostic approaches of neurodegenerative diseases. Despite numerous well-described procedures in the literature for preparation of different AuNPs, possible transformation and structural changes of surface functionalization agents have not been considered thoroughly. As a case in point, the catechols L-DOPA and dopamine were selected because of their susceptibility to oxidation, cyclization, and polymerization. To assess the fate of coating and functionalization agents during the preparation of AuNPs or interaction at the nano-bio interface, a combination of spectroscopy, light scattering, and microscopy techniques was used while structural information and reaction mechanism were obtained by NMR in combination with computational tools. The results revealed that the final form of catechol on the AuNP nanosurface depends on the molar ratio of Au used for AuNP preparation. A large molar excess of L-DOPA or dopamine is needed to prepare AuNPs funtionalized with fully reduced catechols. In the case of molar excess of Au, the oxidation of catechols to dopamine quinone and dopaquinone was promoted, and dopaquinone underwent intramolecular cyclization in which additional oxidation products, leukodopachrome, dopachrome, or its tautomer, were formed because of the larger intrinsic acidity of the more nucleophilic amino group in dopaquinone. MD simulations showed that, of the oxidation products, dopachrome had the highest affinity for binding to the AuNPs surface. The results highlight how a more versatile methodological approach, combining experimental and in silico techniques, allows more reliable characterization of binding events at the surface of AuNPs for possible applications in biomedicine.

Bola-Amphiphilic Glycodendrimers: New Carbohydrate-Mimicking Scaffolds to Target Carbohydrate-Binding Proteins.

Dendrimers are appealing scaffolds for creating carbohydrate mimics with unique multivalent cooperativity. We report here novel bola-amphiphilic glycodendrimers bearing mannose and glucose terminals, and a hydrophobic thioacetal core responsive to reactive oxygen species. The peculiar bola-amphiphilic feature enabled stronger binding to lectin compared to conventional amphiphiles. In addition, these dendrimers are able to target mannose receptors and glucose transporters expressed at the surface of cells, thus allowing effective and specific cellular uptake. This highlights their great promise for targeted delivery.

Smart IM-MS and NMR study of natural diastereomers: the study case of the essential oil from Senecio transiens.

Unambiguous identification of the components of a natural mixture remains a challenging and meticulous issue. Usually, different analytical techniques and laborious separation protocols are employed; nevertheless, in some cases, delicate and equivocal problems are hardly addressed by traditional methods. In this context, an original methodology for the analysis of natural samples consisting of recent mass spectrometry methods based on ion mobility (MS-IM) is proposed. As an example, a polar fraction obtained by the essential oil prepared from Senecio transiens, an endemic plant harvested on the Corsica Island, was selected for this study to show how IM-MS-based methods easily provide very useful insights suggesting the presence of two diastereomers. To unambiguously confirm this hypothesis and verify reliability of the IM-MS results, the purified compounds were further analysed by means of nuclear magnetic resonance (NMR) methodologies, allowing the structural elucidation and the identification of two new natural compounds, diastereomers of 4-acetoxy-5,9-dimethyl-3-(2-methylpropenyl)-2-oxabicyclo[4.4.0] dec-9-ene, reported here for the first time.

Dynamic self-assembling supramolecular dendrimer nanosystems as potent antibacterial candidates against drug-resistant bacteria and biofilms.

The alarming and prevailing antibiotic resistance crisis urgently calls for innovative "outside of the box" antibacterial agents, which can differ substantially from conventional antibiotics. In this context, we have established antibacterial candidates based on dynamic supramolecular dendrimer nanosystems self-assembled with amphiphilic dendrimers composed of a long hydrophobic alkyl chain and a small hydrophilic poly(amidoamine) dendron bearing distinct terminal functionalities. Remarkably, the amphiphilic dendrimer with amine terminals exhibited strong antibacterial activity against both Gram-positive and Gram-negative as well as drug-resistant bacteria, and prevented biofilm formation. Multidisciplinary studies combining experimental approaches and computer modelling together demonstrate that the dendrimer interacts and binds via electrostatic interactions with the bacterial membrane, where it becomes enriched and then dynamically self-assembles into supramolecular nanoassemblies for stronger and multivalent interactions. These, in turn, rapidly promote the insertion of the hydrophobic dendrimer tail into the bacterial membrane thereby inducing bacterial cell lysis and constituting powerful antibacterial activity. Our study presents a novel concept for creating nanotechnology-based antibacterial candidates via dynamic self-assembly and offers a new perspective for combatting recalcitrant bacterial infection.

Binding of the B-Raf Inhibitors Dabrafenib and Vemurafenib to Human Serum Albumin: A Biophysical and Molecular Simulation Study.

Drug binding to human serum albumin (HSA) significantly affects in vivo drug transport and biological activity. To gain insight into the binding mechanism of the two B-Raf tyrosine kinase inhibitors dabrafenib and vemurafenib to HSA, in this work, we adopted a combined strategy based on fluorescence spectroscopy, isothermal titration calorimetry (ITC), circular dichroism (CD), and molecular simulations. Both anticancer drugs are found to bind spontaneously and with a 1:1 stoichiometry within the same binding pocket, located in Sudlow's site II (subdomain IIIA) of the protein with comparable affinity and without substantially perturbing the protein secondary structure. However, the nature of each drug-protein interactions is distinct: whereas the formation of the dabrafenib/HSA complex is more entropically driven, the formation of the alternative vemurafenib/HSA assembly is prevalently enthalpic in nature. Kinetic analysis also indicates that the association rate is similar for the two drugs, whereas the residence time of vemurafenib within the HSA binding pocket is somewhat higher than that determined for the alternative B-Raf inhibitor.

Synthesis of Aminoethyl-Substituted Piperidine Derivatives as σ1 Receptor Ligands with Antiproliferative Properties.

A series of novel σ1 receptor ligands with a 4-(2-aminoethyl)piperidine scaffold was prepared and biologically evaluated. The underlying concept of our project was the improvement of the lipophilic ligand efficiency of previously synthesized potent σ1 ligands. The key steps of the synthesis comprise the conjugate addition of phenylboronic acid at dihydropyridin-4(1H)-ones 7, homologation of the ketones 8 and introduction of diverse amino moieties and piperidine N-substituents. 1-Methylpiperidines showed particular high σ1 receptor affinity and selectivity over the σ2 subtype, whilst piperidines with a proton, a tosyl moiety or an ethyl moiety exhibited considerably lower σ1 affinity. Molecular dynamics simulations with per-residue binding free energy deconvolution demonstrated that different interactions of the basic piperidine-N-atom and its substituents (or the cyclohexane ring) with the lipophilic binding pocket consisting of Leu105, Thr181, Leu182, Ala185, Leu186, Thr202 and Tyr206 are responsible for the different σ1 receptor affinities. Recorded logD7.4 and calculated clogP values of 4a and 18a indicate low lipophilicity and thus high lipophilic ligand efficiency. Piperidine 4a inhibited the growth of human non-small cell lung cancer cells A427 to a similar extent as the σ1 antagonist haloperidol. 1-Methylpiperidines 20a, 21a and 22a showed stronger antiproliferative effects on androgen negative human prostate cancer cells DU145 than the σ1 ligands NE100 and S1RA.

The fellowship of the RING: BRCA1, its partner BARD1 and their liaison in DNA repair and cancer.

The breast cancer type 1 susceptibility protein (BRCA1) and its partner - the BRCA1-associated RING domain protein 1 (BARD1) - are key players in a plethora of fundamental biological functions including, among others, DNA repair, replication fork protection, cell cycle progression, telomere maintenance, chromatin remodeling, apoptosis and tumor suppression. However, mutations in their encoding genes transform them into dangerous threats, and substantially increase the risk of developing cancer and other malignancies during the lifetime of the affected individuals. Understanding how BRCA1 and BARD1 perform their biological activities therefore not only provides a powerful mean to prevent such fatal occurrences but can also pave the way to the development of new targeted therapeutics. Thus, through this review work we aim at presenting the major efforts focused on the functional characterization and structural insights of BRCA1 and BARD1, per se and in combination with all their principal mediators and regulators, and on the multifaceted roles these proteins play in the maintenance of human genome integrity.

Molecular rationale for SARS-CoV-2 spike circulating mutations able to escape bamlanivimab and etesevimab monoclonal antibodies.

The purpose of this work is to provide an in silico molecular rationale of the role eventually played by currently circulating mutations in the receptor binding domain of the SARS-CoV-2 spike protein (S-RBDCoV­2) in evading the immune surveillance effects elicited by the two Eli Lilly LY-CoV555/bamlanivimab and LY-CoV016/etesevimab monoclonal antibodies. The main findings from this study show that, compared to the wild-type SARS-CoV-2 spike protein, mutations E484A/G/K/Q/R/V, Q493K/L/R, S494A/P/R, L452R and F490S are predicted to be markedly resistant to neutralization by LY-CoV555, while mutations K417E/N/T, D420A/G/N, N460I/K/S/T, T415P, and Y489C/S are predicted to confer LY-CoV016 escaping advantage to the viral protein. A challenge of our global in silico results against relevant experimental data resulted in an overall 90% agreement. Thus, the results presented provide a molecular-based rationale for all relative experimental findings, constitute a fast and reliable tool for identifying and prioritizing all present and newly reported circulating spike SARS-CoV-2 variants with respect to antibody neutralization, and yield substantial structural information for the development of next-generation vaccines and monoclonal antibodies more resilient to viral evolution.

Propellanes as Rigid Scaffolds for the Stereodefined Attachment of σ-Pharmacophoric Structural Elements to Achieve σ Affinity.

Following the concept of conformationally restriction of ligands to achieve high receptor affinity, we exploited the propellane system as rigid scaffold allowing the stereodefined attachment of various substituents. Three types of ligands were designed, synthesized and pharmacologically evaluated as σ1 receptor ligands. Propellanes with (1) a 2-methoxy-5-methylphenylcarbamate group at the "left" five-membered ring and various amino groups on the "right" side; (2) benzylamino or analogous amino moieties on the "right" side and various substituents at the left five-membered ring and (3) various urea derivatives at one five-membered ring were investigated. The benzylamino substituted carbamate syn,syn-4a showed the highest σ1 affinity within the group of four stereoisomers emphasizing the importance of the stereochemistry. The cyclohexylmethylamine 18 without further substituents at the propellane scaffold revealed unexpectedly high σ1 affinity (Ki = 34 nM) confirming the relevance of the bioisosteric replacement of the benzylamino moiety by the cyclohexylmethylamino moiety. Reduction of the distance between the basic amino moiety and the "left" hydrophobic region by incorporation of the amino moiety into the propellane scaffold resulted in azapropellanes with particular high σ1 affinity. As shown for the propellanamine 18, removal of the carbamate moiety increased the σ1 affinity of 9a (Ki = 17 nM) considerably. Replacement of the basic amino moiety by H-bond forming urea did not lead to potent σ ligands. According to molecular dynamics simulations, both azapropellanes anti-5 and 9a as well as propellane 18 adopt binding poses at the σ1 receptor, which result in energetic values correlating well with their different σ1 affinities. The affinity of the ligands is enthalpy driven. The additional interactions of the carbamate moiety of anti-5 with the σ1 receptor protein cannot compensate the suboptimal orientations of the rigid propellane and its N-benzyl moiety within the σ1 receptor-binding pocket, which explains the higher σ1 affinity of the unsubstituted azapropellane 9a.