Molecular Structures,Vibrational Spectra,Electronic Properties,and Molecular Docking of Two Pyrazoline Derivatives Containing 1-Carboxamide and 1-Carbothioamide: A Comparative Study

Pyrazolines derivatives are nitrogen-containing heterocyclic compound, which exhibit the broad spectrum of biological activities such as antibacterial, antifungal, antiprotozoal, and anti-inflammatory. The optimized geometry, frequency, and intensity of vibrational bands of these compounds are obtained by the density function theory (DFT) using 6–31+G(d,p) basis set. The scaled harmonic vibrational frequencies have been compared with experimental Fourier transform infrared spectroscopy (FTIR) values and found to be in good agreement. The electronic properties of these molecules are discussed with the help of highest occupied molecular orbital (HOMO), lowest unoccupied molecular orbital (LUMO), and molecular electrostatic potential (MESP) surfaces, and a number of electronic and thermodynamic parameters are calculated, which are closely related to their chemical reactivity and reaction path. We also notice that pyrazoline derivatives show biological activity for preventing dyskinesia. This study may provide a further investigation on pyrazolines derivatives for pharmacological importance.     

Substrate Recognition Properties from an Intermediate Structural State of the UreA Transporter

Through a combination of comparative modeling, site-directed and classical random mutagenesis approaches, we previously identified critical residues for binding, recognition, and translocation of urea, and its inhibition by 2-thiourea and acetamide in the Aspergillus nidulans urea transporter, UreA. To deepen the structural characterization of UreA, we employed the artificial intelligence (AI) based AlphaFold2 (AF2) program. In this analysis, the resulting AF2 models lacked inward- and outward-facing cavities, suggesting a structural intermediate state of UreA. Moreover, the orientation of the W82, W84, N279, and T282 side chains showed a large variability, which in the case of W82 and W84, may operate as a gating mechanism in the ligand pathway. To test this hypothesis non-conservative and conservative substitutions of these amino acids were introduced, and binding and transport assessed for urea and its toxic analogue 2-thiourea, as well as binding of the structural analogue acetamide. As a result, residues W82, W84, N279, and T282 were implicated in substrate identification, selection, and translocation. Using molecular docking with Autodock Vina with flexible side chains, we corroborated the AF2 theoretical intermediate model, showing a remarkable correlation between docking scores and experimental affinities determined in wild-type and UreA mutants. The combination of AI-based modeling with classical docking, validated by comprehensive mutational analysis at the binding region, would suggest an unforeseen option to determine structural level details on a challenging family of proteins.     

人血红蛋白分子印迹聚合物的制备及分子识别性能

以丙烯酰胺和甲基丙烯酸为功能单体制备得到了具有选择性识别能力的人血红蛋白分子印迹聚合物. 该印迹聚合物的分子识别特性来自聚合物的结合基团与印迹分子的功能基团之间的氢键和静电作用,以及印迹孔穴的空间几何选择性. 研究表明,聚合单体中甲基丙烯酸的含量会影响聚合物对血红蛋白的特异结合能力及分子印迹聚合物的吸附容量和洗脱性能.     

Molecular Recognition of the HPLC Whelk-O1 Selector towards the Conformational Enantiomers of Nevirapine and Oxcarbazepine

The presence of stereogenic elements is a common feature in pharmaceutical compounds, and affording optically pure stereoisomers is a frequent issue in drug design. In this context, the study of the chiral molecular recognition mechanism fundamentally supports the understanding and optimization of chromatographic separations with chiral stationary phases. We investigated, with molecular docking, the interactions between the chiral HPLC selector Whelk-O1 and the stereoisomers of two bioactive compounds, the antiviral Nevirapine and the anticonvulsant Oxcarbazepine, both characterized by two stereolabile conformational enantiomers. The presence of fast-exchange enantiomers and the rate of the interconversion process were studied using low temperature enantioselective HPLC and VT-NMR with Whelk-O1 applied as chiral solvating agent. The values of the energetic barriers of interconversion indicate, for the single enantiomers of both compounds, half-lives sufficiently long enough to allow their separation only at critically sub-ambient temperatures. The chiral selector Whelk-O1 performed as a strongly selective discriminating agent both when applied as a chiral stationary phase (CSP) in HPLC and as CSA in NMR spectroscopy.     

Combining Molecular Docking and Molecular Dynamics to Predict the Binding Modes of Flavonoid Derivatives with the Neuraminidase of the 2009 H1N1 Influenza A Virus

Control of flavonoid derivatives inhibitors release through the inhibition of neuraminidase has been identified as a potential target for the treatment of H1N1 influenza disease. We have employed molecular dynamics simulation techniques to optimize the 2009 H1N1 influenza neuraminidase X-ray crystal structure. Molecular docking of the compounds revealed the possible binding mode. Our molecular dynamics simulations combined with the solvated interaction energies technique was applied to predict the docking models of the inhibitors in the binding pocket of the H1N1 influenza neuraminidase. In the simulations, the correlation of the predicted and experimental binding free energies of all 20 flavonoid derivatives inhibitors is satisfactory, as indicated by R(2) = 0.75.     

Molecular Mechanism of Food-Derived Polyphenols on PD-L1 Dimerization: A Molecular Dynamics Simulation Study

In cancer immunotherapy, an emerging approach is to block the interactions of programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) using small-molecule inhibitors. The food-derived polyphenols curcumin (CC), resveratrol (RSV) and epigallocatechin gallate (EGCG) have anticancer immunologic functions, which, recently, have been proposed to act via the downregulation of PD-L1 expression. However, it remains unclear whether they can directly target PD-L1 dimerization and, thus, interrupt the PD-1/PD-L1 pathway. To elucidate the molecular mechanism of such compounds on PD-L1 dimerization, molecular docking and nanosecond molecular dynamics simulations were performed. Binding free energy calculations show that the affinities of CC, RSV and EGCG to the PD-L1 dimer follow a trend of CC > RSV > EGCG. Hence, CC is the most effective inhibitor of the PD-1/PD-L1 pathway. Analysis on contact numbers, nonbonded interactions and residue energy decomposition indicate that such compounds mainly interact with the C-, F- and G-sheet fragments of the PD-L1 dimer, which are involved in interactions with PD-1. More importantly, nonpolar interactions between these compounds and the key residues Ile54, Tyr56, Met115, Ala121 and Tyr123 play a dominant role in binding. Free energy landscape and secondary structure analyses further demonstrate that such compounds can stably interact with the binding domain of the PD-L1 dimer. The results provide evidence that CC, RSV and EGCG can inhibit PD-1/PD-L1 interactions by directly targeting PD-L1 dimerization. This provides a novel approach to discovering food-derived small-molecule inhibitors of the PD-1/PD-L1 pathway with potential applications in cancer immunotherapy.     

Molecular Docking and Molecular Dynamics Simulation Studies of Triterpenes from Vernonia patula with the Cannabinoid Type 1 Receptor

A molecular docking approach was employed to evaluate the binding affinity of six triterpenes, namely epifriedelanol, friedelin, α-amyrin, α-amyrin acetate, β-amyrin acetate, and bauerenyl acetate, towards the cannabinoid type 1 receptor (CB1). Molecular docking studies showed that friedelin, α-amyrin, and epifriedelanol had the strongest binding affinity towards CB1. Molecular dynamics simulation studies revealed that friedelin and α-amyrin engaged in stable non-bonding interactions by binding to a pocket close to the active site on the surface of the CB1 target protein. The studied triterpenes showed a good capacity to penetrate the blood–brain barrier. These results help to provide some evidence to justify, at least in part, the previously reported antinociceptive and sedative properties of Vernonia patula.     

Critical Assessment of a Structure-Based Screening Campaign for IDO1 Inhibitors: Tips and Pitfalls

Over the last two decades, indoleamine 2,3-dioxygenase 1 (IDO1) has attracted wide interest as a key player in immune regulation, fostering the design and development of small molecule inhibitors to restore immune response in tumor immunity. In this framework, biochemical, structural, and pharmacological studies have unveiled peculiar structural plasticity of IDO1, with different conformations and functional states that are coupled to fine regulation of its catalytic activity and non-enzymic functions. The large plasticity of IDO1 may affect its ligand recognition process, generating bias in structure-based drug design campaigns. In this work, we report a screening campaign of a fragment library of compounds, grounding on the use of three distinct conformations of IDO1 that recapitulate its structural plasticity to some extent. Results are instrumental to discuss tips and pitfalls that, due to the large plasticity of the enzyme, may influence the identification of novel and differentiated chemical scaffolds of IDO1 ligands in structure-based screening campaigns.     

Acyclic Cucurbit[n]uril-type Receptors: Preparation,Molecular Recognition Properties and Biological Applications

This article traces the development of acyclic cucurbit[n]uril-type receptors with a focus on work from the Isaacs group. First, we describe the synthesis of methylene bridged glycoluril dimers capped with aromatic sidewalls which allowed us to probe the interconversion of the S- and C-shaped dimers which is a fundamental step in CB[n] formation. The C-shaped compounds were found to undergo discrete self-assembly (dimerization) in both water and organic solvents which lead us to investigate multicomponent self-sorting systems. We supressed the self-association of 8 by electrostatic repulsion in the putative dimer which allowed expression of its innate molecular recognition properties toward methylene blue and related planar cationic dyes. Longer glycoluril oligomers (trimer-hexamer, acyclic decamer) were prepared by starving the CB[n]-forming reaction of formaldehyde. The longer oligomers (e. g. 15 and 16 ) bind to alkylammonium ions in water ≈100-fold weaker than macrocyclic CB[n] highlighting the high preorganization of the acyclic but polycyclic framework. We prepared a wide variety of acyclic CB[n] compounds (wall variants, solubilizing group variants, linker variants) based on glycoluril trimer and tetramer. In particular, 26 and 27 have been shown to possess a wide variety of chemically and biologically interesting functions. For example, 26 was used to formulate the insoluble drug Albendazole and treat mice bearing SK-OV-3 xenograft tumors. Compound 27 binds tightly to the neuromuscular blocking agents rocuronium, vecuronium, and cisatracurium and acts as an in vivo reversal agent for these compounds in anesthetized rats. Container 27 was also found to modulate the hyperlocomotive effect of rats that had been treated with methamphetamine. Finally, 38 has been used as a cross reactive component of sensor arrays that are capable of classifying and quantifying cancer related nitroamine and a range of over the counter drugs. Overall, the work demonstrates that acyclic CB[n]-type compounds are nicely pre-organized and therefore retain the essential aspects of the recognition properties of macrocyclic CB[n] but allow for more straightforward tailoring of structure and solubility that enables a variety of chemically and biologically important applications.     

Conformational Insights into the Control of CNF1 Toxin Activity by Peptidyl-Prolyl Isomerization: A Molecular Dynamics Perspective

The cytotoxic necrotizing factor 1 (CNF1) toxin from uropathogenic Escherichia coli constitutively activates Rho GTPases by catalyzing the deamidation of a critical glutamine residue located in the switch II (SWII). In crystallographic structures of the CNF1 catalytic domain (CNF1^CD), surface-exposed P768 and P968 peptidyl-prolyl imide bonds (X-Pro) adopt an unusual cis conformation. Here, we show that mutation of each proline residue into glycine abrogates CNF1^CD in vitro deamidase activity, while mutant forms of CNF1 remain functional on RhoA in cells. Using molecular dynamics simulations coupled to protein-peptide docking, we highlight the long-distance impact of peptidyl-prolyl cis-trans isomerization on the network of interactions between the loops bordering the entrance of the catalytic cleft. The energetically favorable isomerization of P768 compared with P968, induces an enlargement of loop L1 that fosters the invasion of CNF1^CD catalytic cleft by a peptide encompassing SWII of RhoA. The connection of the P968 cis isomer to the catalytic cysteine C866 via a ladder of stacking interactions is alleviated along the cis-trans isomerization. Finally, the cis-trans conversion of P768 favors a switch of the thiol side chain of C866 from a resting to an active orientation. The long-distance impact of peptidyl-prolyl cis-trans isomerizations is expected to have implications for target modification.     

Insights from NMR Spectroscopy into the Conformational Properties of Man‐9 and Its Recognition by Two HIV Binding Proteins

Man₉GlcNAc₂ (Man‐9) present at the surface of HIV makes up the binding sites of several HIV‐neutralizing agents and the mammalian lectin DC‐SIGN, which is involved in cellular immunity and trans‐infections. We describe the conformational properties of Man‐9 in its free state and when bound by the HIV entry‐inhibitor protein microvirin (MVN), and define the minimum epitopes of both MVN and DC‐SIGN by using NMR spectroscopy. To facilitate the implementation of 3D ¹³C‐edited spectra to deconvolute spectral overlap and to determine the solution structure of Man‐9, we developed a robust expression system for the production of ¹³C,¹⁵N‐labeled glycans in mammalian cells. The studies reveal that Man‐9 interacts with HIV‐binding proteins through distinct epitopes and adopts diverse conformations in the bound state. In combination with molecular dynamics simulations we observed receptor‐bound conformations to be sampled by Man‐9 in the free state, thus suggesting a conformational selection mechanism for diverse recognition.     

Evaluation of the Molecular Landscape in PD-L1 Positive Metastatic NSCLC: Data from Campania,Italy

Background: Immune-checkpoint inhibitors (ICIs) have increased and improved the treatment options for patients with non-oncogene-addicted advanced stage non-small cell lung cancer (NSCLC). However, the role of ICIs in oncogene-addicted advanced stage NSCLC patients is still debated. In this study, in an attempt to fill in the informational gap on the effect of ICIs on other driver mutations, we set out to provide a molecular landscape of clinically relevant oncogenic drivers in programmed death-ligand 1 (PD-L1) positive NSCLC patients. Methods: We retrospectively reviewed data on 167 advanced stage NSCLC PD-L1 positive patients (≥1%) who were referred to our clinic for molecular evaluation of five driver oncogenes, namely, EGFR, KRAS, BRAF, ALK and ROS1. Results: Interestingly, n = 93 (55.7%) patients showed at least one genomic alteration within the tested genes. Furthermore, analyzing a subset of patients with PD-L1 tumor proportion score (TPS) ≥ 50% and concomitant gene alterations (n = 8), we found that n = 3 (37.5%) of these patients feature clinical benefit with ICIs administration, despite the presence of a concomitant KRAS gene alteration. Conclusions: In this study, we provide a molecular landscape of clinically relevant biomarkers in NSCLC PD-L1 positive patients, along with data evidencing the clinical benefit of ICIs in patient NSCLC PD-L1 positive alterations.     

Augmenting Structure-Based Design with Experimental Protein-Ligand Interaction Data: Molecular Recognition,Interactive Visualization,and Rescoring

The previously introduced ratio of frequencies (R_F) framework provides statistically sound information on the relative interaction preferences of atoms in crystal structures. By applying the methodology to protein-ligand complexes, we can investigate the significance of interactions that are employed in structure-based drug design. Here, we revisit three aspects of molecular recognition in the light of the R_F framework, namely stacking interactions of heteroaromatic rings with protein amide groups, interactions of acidified C−H groups, and interaction differences between syn and anti lone pairs of carboxylate groups. In addition, we introduce a highly interactive visualization tool that facilitates design idea generation in structure-enabled drug discovery projects. Finally, we show that applying the R_F analysis as a simple rescoring tool after docking improves enrichment factors for the DUD−E diverse targets subset supporting the relevance of our approach.     

A Deep Dive into VDAC1 Conformational Diversity Using All-Atom Simulations Provides New Insights into the Structural Origin of the Closed States

The voltage-dependent anion channel 1 (VDAC1) is a crucial mitochondrial transporter that controls the flow of ions and respiratory metabolites entering or exiting mitochondria. As a voltage-gated channel, VDAC1 can switch between a high-conducting “open” state and a low-conducting “closed” state emerging at high transmembrane (TM) potentials. Although cell homeostasis depends on channel gating to regulate the transport of ions and metabolites, structural hallmarks characterizing the closed states remain unknown. Here, we performed microsecond accelerated molecular dynamics to highlight a vast region of VDAC1 conformational landscape accessible at typical voltages known to promote closure. Conformers exhibiting durable subconducting properties inherent to closed states were identified. In all cases, the low conductance was due to the particular positioning of an unfolded part of the N-terminus, which obstructed the channel pore. While the N-terminal tail was found to be sensitive to voltage orientation, our models suggest that stable low-conducting states of VDAC1 predominantly take place from disordered events and do not result from the displacement of a voltage sensor or a significant change in the pore. In addition, our results were consistent with conductance jumps observed experimentally and corroborated a recent study describing entropy as a key factor for VDAC gating.     

Molecular Recognition in C-Type Lectins: The Cases of DC-SIGN,Langerin, MGL,and L-Sectin

Carbohydrates play a pivotal role in intercellular communication processes. In particular, glycan antigens are key for sustaining homeostasis, helping leukocytes to distinguish damaged tissues and invading pathogens from healthy tissues. From a structural perspective, this cross-talk is fairly complex, and multiple membrane proteins guide these recognition processes, including lectins and Toll-like receptors. Since the beginning of this century, lectins have become potential targets for therapeutics for controlling and/or avoiding the progression of pathologies derived from an incorrect immune outcome, including infectious processes, cancer, or autoimmune diseases. Therefore, a detailed knowledge of these receptors is mandatory for the development of specific treatments. In this review, we summarize the current knowledge about four key C-type lectins whose importance has been steadily growing in recent years, focusing in particular on how glycan recognition takes place at the molecular level, but also looking at recent progresses in the quest for therapeutics.     

Structural Investigation and Molecular Modeling Studies of Strobilurin-Based Fungicides Active against the Rice Blast Pathogen Pyricularia oryzae

The increasing emergence of fungicide-resistant pathogens requires urgent solutions for crop disease management. Here, we describe a structural investigation of new fungicides obtained by combining strobilurin and succinate dehydrogenase inhibitor pharmacophores. We identified compounds endowed with very good activity against wild-type Pyricularia oryzae, combined in some cases with promising activity against strobilurin-resistant strains. The first three-dimensional model of P. oryzae cytochrome bc1 complex containing azoxystrobin as a ligand was developed. The model was validated with a set of commercially available strobilurins, and it well explains both the resistance mechanism to strobilurins mediated by the mutation G143A and the activity of metyltetraprole against strobilurin-resistant strains. The obtained results shed light on the key recognition determinants of strobilurin-like derivatives in the cytochrome bc1 active site and will guide the further rational design of new fungicides able to overcome resistance caused by G143A mutation in the rice blast pathogen.     

Molecular Dynamics Simulation of Tryptophan Hydroxylase-1: Binding Modes and Free Energy Analysis to Phenylalanine Derivative Inhibitors

Serotonin is a neurotransmitter that modulates many central and peripheral functions. Tryptophan hydroxylase-1 (TPH1) is a key enzyme of serotonin synthesis. In the current study, the interaction mechanism of phenylalanine derivative TPH1 inhibitors was investigated using molecular dynamics (MD) simulations, free energy calculations, free energy decomposition analysis and computational alanine scanning. The predicted binding free energies of these complexes are consistent with the experimental data. The analysis of the individual energy terms indicates that although the van der Waals and electrostatics interaction contributions are important in distinguishing the binding affinities of these inhibitors, the electrostatic contribution plays a more crucial role in that. Moreover, it is observed that different configurations of the naphthalene substituent could form different binding patterns with protein, yet lead to similar inhibitory potency. The combination of different molecular modeling techniques is an efficient way to interpret the interaction mechanism of inhibitors and our work could provide valuable information for the TPH1 inhibitor design in the future.     

Structural Studies Provide New Insights into the Role of Lysine Acetylation on Substrate Recognition by CARM1 and Inform the Design of Potent Peptidomimetic Inhibitors

The dynamic interplay of post-translational modifications (PTMs) in chromatin provides a communication system for the regulation of gene expression. An increasing number of studies have highlighted the role that such crosstalk between PTMs plays in chromatin recognition. In this study, (bio)chemical and structural approaches were applied to specifically probe the impact of acetylation of Lys¹⁸ in the histone H3 tail peptide on peptide recognition by the protein methyltransferase coactivator-associated arginine methyltransferase 1 (CARM1). Peptidomimetics that recapitulate the transition state of protein arginine N-methyltransferases, were designed based on the H3 peptide wherein the target Arg¹⁷ was flanked by either a free or an acetylated lysine. Structural studies with these peptidomimetics and the catalytic domain of CARM1 provide new insights into the binding of the H3 peptide within the enzyme active site. While the co-crystal structures reveal that lysine acetylation results in minor conformational differences for both CARM1 and the H3 peptide, acetylation of Lys¹⁸ does lead to additional interactions (Van der Waals and hydrogen bonding) and likely reduces the cost of desolvation upon binding, resulting in increased affinity. Informed by these findings a series of smaller peptidomimetics were also prepared and found to maintain potent and selective CARM1 inhibition. These findings provide new insights both into the mechanism of crosstalk between arginine methylation and lysine acetylation as well as towards the development of peptidomimetic CARM1 inhibitors.     

New Molecules of Diterpene Origin with Inhibitory Properties toward α-Glucosidase

The incidence of diabetes mellitus (DM), one of the most common chronic metabolic disorders, has increased dramatically over the past decade and has resulted in higher rates of morbidity and mortality worldwide. The enzyme, α-Glucosidase (α-GLy), is considered a therapeutic target for the treatment of type 2 DM. Herein, we synthesized arylidene, heterocyclic, cyanoetoxy- and propargylated derivatives of quinopimaric acid (levopimaric acid diene adduct with p-benzoquinone) 1–50 and, first, evaluated their ability to inhibit α-GLy. Among the tested compounds, quinopimaric acid 1, 2,3-dihydroquinopimaric acid 8 and its amide and heterocyclic derivatives 9, 30, 33, 39, 44, with IC₅₀ values of 35.57–65.98 μM, emerged as being good inhibitors of α-GLy. Arylidene 1β-hydroxy and 1β,13α-epoxy methyl dihydroquinopimarate derivatives 6, 7, 26–29, thiadiazole 32, 1a,4a-dehydroquinopimaric acid 40 and its indole, nitrile and propargyl hybrids 35–38, 42, 45, 48, and 50 showed excellent inhibitory activities. The most active compounds 38, 45, 48, and 50 displayed IC₅₀ values of 0.15 to 0.68 μM, being 1206 to 266 more active than acarbose (IC₅₀ of 181.02 μM). Kinetic analysis revealed the most active diterpene indole with an alkyne substituent 45 as a competitive inhibitor with K_i of 50.45 μM. Molecular modeling supported this finding and suggested that the indole core plays a key role in the binding. Compound 45 also has favorable pharmacokinetic and safety properties, according to the computational ADMET profiling. The results suggested that quinopimaric acid derivatives should be considered as potential candidates for novel alternative therapies in the treatment of type 2 diabetes.