Structure-Based Drug Design of ADRA2A Antagonists Derived from Yohimbine.

Yohimbine, a natural indole alkaloid and a nonselective adrenoceptor antagonist, possesses potential benefits in treating inflammatory disorders and sepsis. Nevertheless, its broader clinical use faces challenges due to its low receptor selectivity. A structure-activity relationship study of novel yohimbine analogues identified amino esters of yohimbic acid as potent and selective ADRA2A antagonists. Specifically, amino ester 4n, in comparison to yohimbine, showed a 6-fold higher ADRA1A/ADRA2A selectivity index (SI > 556 for 4n) and a 25-fold higher ADRA2B/ADRA2A selectivity index. Compound 4n also demonstrated high plasma and microsomal stability, moderate-to-low membrane permeability determining its limited ability to cross the blood-brain barrier, and negligible toxicity on nontumor normal human dermal fibroblasts. Compound 4n represents an important complementary pharmacological tool to study the involvement of adrenoceptor subtypes in pathophysiologic conditions such as inflammation and sepsis and a novel candidate for further preclinical development to treat ADRA2A-mediated pathologies.

Synthesis and unexpected binding of monofluorinated N,N'-diacetylchitobiose and LacdiNAc to wheat germ agglutinin.

Fluorination of carbohydrate ligands of lectins is a useful approach to examine their binding profile, improve their metabolic stability and lipophilicity, and convert them into 19F NMR-active probes. However, monofluorination of monovalent carbohydrate ligands often leads to a decreased or completely lost affinity. By chemical glycosylation, we synthesized the full series of methyl ß-glycosides of N,N'-diacetylchitobiose (GlcNAcß(1-4)GlcNAcß1-OMe) and LacdiNAc (GalNAcß(1-4)GlcNAcß1-OMe) systematically monofluorinated at all hydroxyl positions. A competitive enzyme-linked lectin assay revealed that the fluorination at the 6'-position of chitobioside resulted in an unprecedented increase in affinity to wheat germ agglutinin (WGA) by one order of magnitude. For the first time, we have characterized the binding profile of a previously underexplored WGA ligand LacdiNAc. Surprisingly, 4'-fluoro-LacdiNAc bound WGA even stronger than unmodified LacdiNAc. These observations were interpreted using molecular dynamic calculations along with STD and transferred NOESY NMR techniques, which gave evidence for the strengthening of CH/π interactions after deoxyfluorination of the side chain of the non-reducing GlcNAc. These results highlight the potential of fluorinated glycomimetics as high-affinity ligands of lectins and 19F NMR-active probes.

Exploring long-range fluorine-carbon J-coupling for conformational analysis of deoxyfluorinated disaccharides: A combined computational and NMR study.

In this study, we investigated the potential of long-range fluorine-carbon J-coupling for determining the structures of deoxyfluorinated disaccharides. Three disaccharides, previously synthesized as potential galectin inhibitors, exhibited through-space fluorine-carbon J-couplings. In our independent conformational analysis of these disaccharide derivatives, we employed a combination of density functional theory (DFT) calculations and nuclear magnetic resonance (NMR) experiments. By comparing the calculated nuclear shieldings with the experimental carbon chemical shifts, we were able to identify the most probable conformers for each compound. A model comprising fluoromethane and methane molecules was used to study the relationship between molecular arrangements and intermolecular through-space J-coupling. Our study demonstrates the important effect of internuclear distance and molecular orientation on the magnitude of fluorine-carbon coupling. The experimental values for the fluorine-carbon through-space couplings (TSCs) of the disaccharides corresponded with values calculated for the most probable conformers identified by the conformational analysis. These results unlock the broader application of fluorine-carbon TSCs as powerful tools for conformational analysis of flexible molecules, offering valuable insights for future structural investigations.

The Stability of Hydrogen-Bonded Ion-Pair Complex Unexpectedly Increases with Increasing Solvent Polarity.

The generally observed decrease of the electrostatic energy in the complex with increasing solvent polarity has led to the assumption that the stability of the complexes with ion-pair hydrogen bonds decreases with increasing solvent polarity. Besides, the smaller solvent-accessible surface area (SASA) of the complex in comparison with the isolated subsystems results in a smaller solvation energy of the latter, leading to a destabilization of the complex in the solvent compared to the gas phase. In our study, which combines Nuclear Magnetic Resonance, Infrared Spectroscopy experiments, quantum chemical calculations, and molecular dynamics (MD) simulations, we question the general validity of this statement. We demonstrate that the binding free energy of the ion-pair hydrogen-bonded complex between 2-fluoropropionic acid and n-butylamine (CH3CHFCOO-…NH3But+) increases with increased solvent polarity. This phenomenon is rationalized by a substantial charge transfer between the subsystems that constitute the ion-pair hydrogen-bonded complex. This unexpected finding introduces a new perspective to our understanding of solvation dynamics, emphasizing the interplay between solvent polarity and molecular stability within hydrogen-bonded systems.

NMR crystallography of amino acids.

The development of NMR crystallography methods requires a reliable database of chemical shifts measured for systems with known crystal structure. We measured and assigned carbon and hydrogen chemical shifts of twenty solid natural amino acids of known polymorphic structure, meticulously determined using powder X-ray diffraction. We then correlated the experimental data with DFT-calculated isotropic shieldings. The small size of the unit cell of most amino acids allowed for advanced computations using various families of DFT functionals, including generalized gradient approximation (GGA), meta-GGA and hybrid DFT functionals. We tested several combinations of functionals for geometry optimizations and NMR calculations. For carbon shieldings, the widely used GGA functional PBE performed very well, although an improvement could be achieved by adding shielding corrections calculated for isolated molecules using a hybrid functional. For hydrogen nuclei, we observed the best performance for NMR calculations carried out with structures optimized at the hybrid DFT level. The high fidelity of the calculations made it possible to assign additional signals that could not be assigned based on experiments alone, for example signals of two non-equivalent molecules in the unit cell of some of the amino acids.

What are the minimal folding seeds in proteins? Experimental and theoretical assessment of secondary structure propensities of small peptide fragments.

Certain peptide sequences, some of them as short as amino acid triplets, are significantly overpopulated in specific secondary structure motifs in folded protein structures. For example, 74% of the EAM triplet is found in α-helices, and only 3% occurs in the extended parts of proteins (typically ß-sheets). In contrast, other triplets (such as VIV and IYI) appear almost exclusively in extended parts (79% and 69%, respectively). In order to determine whether such preferences are structurally encoded in a particular peptide fragment or appear only at the level of a complex protein structure, NMR, VCD, and ECD experiments were carried out on selected tripeptides: EAM (denoted as pro-'α-helical' in proteins), KAM(α), ALA(α), DIC(α), EKF(α), IYI(pro-ß-sheet or more generally, pro-extended), and VIV(ß), and the reference α-helical CATWEAMEKCK undecapeptide. The experimental data were in very good agreement with extensive quantum mechanical conformational sampling. Altogether, we clearly showed that the pro-helical vs. pro-extended propensities start to emerge already at the level of tripeptides and can be fully developed at longer sequences. We postulate that certain short peptide sequences can be considered minimal "folding seeds". Admittedly, the inherent secondary structure propensity can be overruled by the large intramolecular interaction energies within the folded and compact protein structures. Still, the correlation of experimental and computational data presented herein suggests that the secondary structure propensity should be considered as one of the key factors that may lead to understanding the underlying physico-chemical principles of protein structure and folding from the first principles.

Photocatalytic Generation of Trifluoromethyl Nitrene for Alkene Aziridination.

N-Trifluoromethylated organics may be applied in drug design, agrochemical synthesis, and materials science, among other areas. Yet, despite recent advances in the synthesis of aliphatic, cyclic and heterocyclic N-trifluoromethyl compounds, no strategy based on trifluoromethyl nitrene has hitherto been explored. Here we describe the formation of triplet trifluoromethyl nitrene from azidotrifluoromethane, a stable and safe-to-use precursor, by visible light photocatalysis. The addition of CF3 N to alkenes via biradical intermediates afforded previously unknown aziridines substituted with trifluoromethyl group on the nitrogen atom. The obtained aziridines were converted into either N-trifluoromethylimidazolines, via formal [3+2] cycloaddition with nitriles, mediated by a Lewis acid, or into N-trifluoromethylaldimines, via ring opening and aryl group migration mediated by a strong Brønsted acid. Our findings open new opportunities for the development of novel classes of N-CF3 compounds with possible applications in the life sciences.

Regular arrays of C60-based molecular rotors mounted on the surface of tris(o-phenylenedioxy)cyclotriphosphazene nanocrystals.

Dielectric spectroscopy has been used to determine the barriers of rotation of surface-mounted fullerenes (2.3 ± 0.1 and 4.3 ± 0.1 kcal mol-1). In order to achieve this, a C60 derivative equipped with an anchoring group designed to form a surface inclusion with the hexagonal form of tris(o-phenylenedioxy)cyclotriphosphazene (TPP) has been synthesized. Solid-state NMR analysis revealed that approximately 50% of the surface-mounted molecules have a chemical environment different from the others suggesting two distinct insertion modes. These observations correlate with results of DFT calculations.

Regular Arrays of Rod-Shaped Molecular Photoswitches: Synthesis, Preparation, Characterization, and Selective Photoswitching within Mono- and Bilayer Systems.

We assembled photoresponsive mono- and bilayer systems with well-defined properties from rod-shaped molecules equipped with different photoswitches. Using properly chosen chromophores (diarylethene-based switch and unidirectional light-driven molecular motor), we then selectively targeted layers made of the same types of photoswitches using appropriate monochromatic light. UV-vis analysis confirmed smooth and unrestricted photoisomerization. To achieve this, we synthesized a new class of triptycene-based molecular pedestals adept at forming sturdy Langmuir-Blodgett films on a water-air interface. The films were smoothly transferred to gold and quartz surfaces. Repeated deposition afforded bilayer systems: one layer containing diarylethene-based photoswitches and the other a unidirectional light-driven molecular motor. Structural analysis of both mono- and bilayer systems revealed the molecules to be tilted with carboxylic functions pointing to the surface. At least two different polymorphs differing in monolayer thickness and tilt angle (~40° and ~60°) were identified on the gold surface.

Solvent-controlled formation of alkali and alkali-earth-secured cucurbituril/guest trimers.

Cucurbit[7]uril (CB[7]) encapsulates adamantyl and trimethylsilyl substituents of positively charged guests in dimethyl sulfoxide (DMSO). Unlike in water or deuterium oxide, addition of a selection of alkali and alkali-earth cations with van der Waals radii between 1.0 and 1.4 Å (Na+, K+, Ca2+, Sr2+, Ba2+ and Eu3+) to the CB[7]/guest complexes triggers their cation-mediated trimerization, a process that is very slow on the nuclear magnetic resonance (NMR) time scale. Smaller (Li+, Mg2+) or larger cations (Rb+, Cs+ or NH4+) are inert. The trimers display extensive CH-O interactions between the equatorial and pseudo-equatorial hydrogens of CB[7] and the carbonyl rim of the neighboring CB[7] unit in the trimer, and a deeply nested cation between the three interacting carbonylated CB[7] rims; a counteranion is likely perched in the shallow cavity formed by the three outer walls of CB[7] in the trimer. Remarkably, a guest must occupy the cavity of CB[7] for trimerization to take place. Using a combination of semi-empirical and density functional theory techniques in conjunction with continuum solvation models, we showed that trimerization is favored in DMSO, and not in water, because the penalty for the partial desolvation of three of the six CB[7] portals upon aggregation into a trimer is less unfavorable in DMSO compared to water.

Vinylphosphonate-based cyclic dinucleotides enhance STING-mediated cancer immunotherapy.

Cyclic dinucleotides (CDNs) trigger the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, which plays a key role in cytosolic DNA sensing and thus in immunomodulation against infections, cell damage and cancer. However, cancer immunotherapy trials with CDNs have shown immune activation, but not complete tumor regression. Nevertheless, we designed a novel class of CDNs containing vinylphosphonate based on a STING-affinity screening assay. In vitro, acyloxymethyl phosphate/phosphonate prodrugs of these vinylphosphonate CDNs were up to 1000-fold more potent than the clinical candidate ADU-S100. In vivo, the lead prodrug induced tumor-specific T cell priming and facilitated tumor regression in the 4T1 syngeneic mouse model of breast cancer. Moreover, we solved the crystal structure of this ligand bound to the STING protein. Therefore, our findings not only validate the therapeutic potential of vinylphosphonate CDNs but also open up opportunities for drug development in cancer immunotherapy bridging innate and adaptive immunity.

Inhibition of FLT3-ITD Kinase in Acute Myeloid Leukemia by New Imidazo[1,2-b]pyridazine Derivatives Identified by Scaffold Hopping.

FLT3 kinase is a potential drug target in acute myeloid leukemia (AML). Patients with FLT3 mutations typically have higher relapse rates and worse outcomes than patients without FLT3 mutations. In this study, we investigated the suitability of various heterocycles as central cores of FLT3 inhibitors, including thieno[3,2-d]pyrimidine, pyrazolo[1,5-a]pyrimidine, imidazo[4,5-b]pyridine, pyrido[4,3-d]pyrimidine, and imidazo[1,2-b]pyridazine. Our assays revealed a series of imidazo[1,2-b]pyridazines with high potency against FLT3. Compound 34f showed nanomolar inhibitory activity against recombinant FLT3-ITD and FLT3-D835Y (IC50 values 4 and 1 nM, respectively) as well as in the FLT3-ITD-positive AML cell lines MV4-11, MOLM-13, and MOLM-13 expressing the FLT3-ITD-D835Y mutant (GI50 values of 7, 9, and 4 nM, respectively). In contrast, FLT3-independent cell lines were much less sensitive. In vitro experiments confirmed suppression of FLT3 downstream signaling pathways. Finally, the treatment of MV4-11 xenograft-bearing mice with 34f at doses of 5 and 10 mg/kg markedly blocked tumor growth without any adverse effects.

Hydrogen-Bonding Interactions of 8-Substituted Purine Derivatives.

Hydrogen bonding between nucleobases is a crucial noncovalent interaction for life on Earth. Canonical nucleobases form base pairs according to two main geometries: Watson-Crick pairing, which enables the static functions of nucleic acids, such as the storing of genetic information; and Hoogsteen pairing, which facilitates the dynamic functions of these biomacromolecules. This precisely tuned system can be affected by oxidation or substitution of nucleobases, leading to changes in their hydrogen-bonding patterns. This paper presents an investigation into the intermolecular interactions of various 8-substituted purine derivatives with their hydrogen-bonding partners. The systems were analyzed using nuclear magnetic resonance spectroscopy and density functional theory calculations. Our results demonstrate that the stability of hydrogen-bonded complexes, or base pairs, depends primarily on the number of intermolecular H-bonds and their donor-acceptor alternation. No strong preferences for a particular geometry, either Watson-Crick or Hoogsteen, were found.

Triazinium Ligation: Bioorthogonal Reaction of N1-Alkyl 1,2,4-Triazinium Salts.

The development of reagents that can selectively react in complex biological media is an important challenge. Here we show that N1-alkylation of 1,2,4-triazines yields the corresponding triazinium salts, which are three orders of magnitude more reactive in reactions with strained alkynes than the parent 1,2,4-triazines. This powerful bioorthogonal ligation enables efficient modification of peptides and proteins. The positively charged N1-alkyl triazinium salts exhibit favorable cell permeability, which makes them superior for intracellular fluorescent labeling applications when compared to analogous 1,2,4,5-tetrazines. Due to their high reactivity, stability, synthetic accessibility and improved water solubility, the new ionic heterodienes represent a valuable addition to the repertoire of existing modern bioorthogonal reagents.

Design and Synthesis of Novel HIV-1 NNRTIs with Bicyclic Cores and with Improved Physicochemical Properties.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) represent cornerstones of current regimens for treatment of human immunodeficiency virus type 1 (HIV-1) infections. However, NNRTIs usually suffer from low aqueous solubility and the emergence of resistant viral strains. In the present work, novel bicyclic NNRTIs derived from etravirine (ETV) and rilpivirine (RPV), bearing modified purine, tetrahydropteridine, and pyrimidodiazepine cores, were designed and prepared. Compounds 2, 4, and 6 carrying the acrylonitrile moiety displayed single-digit nanomolar activities against the wild-type (WT) virus (EC50 = 2.5, 2.7, and 3.0 nM, respectively), where the low nanomolar activity was retained against HXB2 (EC50 = 2.2-2.8 nM) and the K103N and Y181C mutated strains (fold change, 1.2-6.7×). Most importantly, compound 2 exhibited significantly improved phosphate-buffered saline solubility (10.4 µM) compared to ETV and RPV (≪1 µM). Additionally, the binding modes of compounds 2, 4, and 6 to the reverse transcriptase were studied by X-ray crystallography.

A Crystalline Dimeric Steroidal Diboronate with Electronically Impeded Rotation.

The dimeric steroid SMR-3, featuring a 1,4-phenyldiboronic ester flanked by two pregnan-triol frameworks, was synthesized to explore the intramolecular dynamics of its central component. The structural data from single-crystal X-ray diffraction studies and the Hirshfeld analyses indicate small steric effects around the aromatic ring that should favor the intended motion. However, solid-state NMR data obtained through VT 13C{1H} CPMAS and 2H spin-echo experiments, using the deuterated analogue SMR-3D4, revealed that this component is rigid even at temperatures where other reported steroidal molecular rotors experience fast rotation (85 °C). A combination of classical molecular dynamics, molecular mechanics, and correlated ab initio calculations allowed us to distinguish the steric and electronic factors that restrict the potential motion in this compound. The experimental and computational data reveal that electronic components dominate the behavior and are responsible for the high rotational barrier in the SMR-3 crystal.

The hydrogen bond continuum in solid isonicotinic acid.

The understanding and correct description of intermolecular hydrogen bonds are crucial in the field of multicomponent pharmaceutical solids, such as salts and cocrystals. Solid isonicotinic acid can serve as a suitable model for the development of methods that can accurately characterize these hydrogen bonds. Experimental solid-state NMR has revealed a remarkable temperature dependence and deuterium-isotope-induced changes of the chemical shifts of the atoms involved in the intermolecular hydrogen bond; these NMR data are related to changes of the average position of the hydrogen atom. These changes of NMR parameters were interpreted using periodic DFT path-integral molecular dynamics (PIMD) simulations. The small size of the unit cell of isonicotinic acid allowed for PIMD simulations with the computationally demanding hybrid DFT functional. Calculations of NMR parameters based on the hybrid-functional PIMD simulations are in excellent agreement with experiment. It is thus demonstrated that an accurate characterization of intermolecular hydrogen bonds can be achieved by a combination of NMR experiments and advanced computations.

Cosolvent Exclusion Drives Protein Stability in Trimethylamine N-Oxide and Betaine Solutions.

Using a combination of molecular dynamics simulation, dialysis experiments, and electronic circular dichroism measurements, we studied the solvation thermodynamics of proteins in two osmolyte solutions, trimethylamine N-oxide (TMAO) and betaine. We showed that existing force fields are unable to capture the solvation properties of the proteins lysozyme and ribonuclease T1 and that the inaccurate parametrization of protein-osmolyte interactions in these force fields promoted an unphysical strong thermal denaturation of the trpcage protein. We developed a novel force field for betaine (the KBB force field) which reproduces the experimental solution Kirkwood-Buff integrals and density. We further introduced appropriate scaling to protein-osmolyte interactions in both the betaine and TMAO force fields which led to successful reproduction of experimental protein-osmolyte preferential binding coefficients for lysozyme and ribonuclease T1 and prevention of the unphysical denaturation of trpcage in osmolyte solutions. Correct parametrization of protein-TMAO interactions also led to the stabilization of the collapsed conformations of a disordered elastin-like peptide, while the uncorrected parameters destabilized the collapsed structures. Our results establish that the thermodynamic stability of proteins in both betaine and TMAO solutions is governed by osmolyte exclusion from proteins.

Structure of Zinc and Nickel Histidine Complexes in Solution Revealed by Molecular Dynamics and Raman Optical Activity.

The histidine residue has an exceptional affinity for metals, but solution structure of its complexes are difficult to study. For zinc and nickel complexes, Raman and Raman optical activity (ROA) spectroscopy methods to investigate the link between spectral shapes and the geometry were used. The spectra were recorded and interpreted on the basis of ionic equilibria, molecular dynamics, ab initio molecular dynamics, and density functional theory. For zwitterionic histidine the dominant tautomer was determined by the decomposition of experimental spectra into calculated subspectra. An octahedral structure was found to prevail for the ZnHis2 complex in solution, in contrast to a tetrahedral arrangement in the crystal phase. The solution geometry of NiHis2 is more similar to the octahedral structure found by X-ray. The Raman and ROA structural determinations of metal complexes are dependent on extensive computations, but reveal unique information about the studied systems.

Diketo-Ketoenol Tautomers in Curcuminoids: Synthesis, Separation of Tautomers, and Kinetic and Structural Studies.

Curcumin and its congeners exist in an equilibrium between diketo and ketoenol tautomers, which have different potencies to bind biomolecules. This work describes procedures for the preparation of 4-alkylated curcumin derivatives and the separation of their two tautomeric forms. Comprehensive NMR studies of the tautomer equilibria in various solvents have been accomplished. Additionally, a pure ketoenol tautomeric form of the active pharmaceutical ingredient (API) ASC-JM17 has been unequivocally determined by X-ray crystallography. Two different polymorphs of this API have been microscopically identified in the X-ray sample and manually separated, and a solid-state NMR study of the two polymorphs has also been performed. This work reports on the slow kinetics of diketo-ketoenol tautomerization in particular solvents that allow the separation and full characterization of both curcuminoids' tautomers.