Activity-Directed Synthesis of Inhibitors of the p53/hDM2 Protein–Protein Interaction

Protein–protein interactions (PPIs) provide a rich source of potential targets for drug discovery and biomedical science research. However, the identification of structural-diverse starting points for discovery of PPI inhibitors remains a significant challenge. Activity-directed synthesis (ADS), a function-driven discovery approach, was harnessed in the discovery of the p53/hDM2 PPI. Over two rounds of ADS, 346 microscale reactions were performed, with prioritisation on the basis of the activity of the resulting product mixtures. Four distinct and novel series of PPI inhibitors were discovered that, through biophysical characterisation, were shown to have promising ligand efficiencies. It was thus shown that ADS can facilitate ligand discovery for a target that does not have a defined small-molecule binding site, and can provide distinctive starting points for the discovery of PPI inhibitors.     

Ugi–Smiles and Ullmann reactions catalyzed by Schiff base derived from Tröger’s base and BINOL

Research on Chemical Intermediates - A Schiff base catalyst (1a) combining a Tröger’s base-NH2 and the (R)-BINOL-(CHO)2 was used to promote the Ugi-Smiles reaction. By using isocyanide,...     

Isolation and Identification of α-Glucosidase and Protein Glycation Inhibitors from Stereospermum colais

Stereospermum colais (family Bignoniaceae) is a well-known pharmacologically potent medicinal plant reported in traditional systems of medicine. Phytochemical investigation of the roots of S. colais resulted in the isolation of seven compounds, and the metabolites were screened for its α-glucosidase enzyme inhibition and anti-glycation property. The compounds identified were β-sitosterol (1), 2-(4′-hydroxyphenyl) ethyl undecanoate (2), 2-(4′-hydroxyphenyl)ethyl pentadecanoate (3), 5α-ergosta-7,22-dien-3β-ol (4), ursolic acid (5), lapachol (6), and pinoresinol (7). Ursolic acid, lapachol, and pinoresinol possessed IC₅₀ values of 119.01, 130.29, and 125.62 nM, respectively, compared to standard ascorbic acid with an IC₅₀ value of 201.01 nM. The other compounds failed to show the activity. Results of the current study showcased the possible exploration of this medicinal plant for the treatment of type 2 diabetes in line with the development of phytopharmaceutical industry.     

Interaction between a Decahydro-closo-Decaborate(2–) Anion and Aliphatic Carboxylic Acids

Interaction between a closo-decaborate anion B₁₀H^2– ₁₀and carboxylic acids RCOOH (R = H, CH₃, C₂H₅, iso-C₃H₇, C₄H₉) is studied. The mono-, di- tri- and tetrasubstituted products B₁₀H_{10 – n} (OCOR)^2– _n are formed in sequence with the temperature growth. The reaction follows an essentially regioselective mechanism: only one of all possible isomers forms at every stage of the process. The respective hydroxy-closo-decaborates B₁₀H_{10 – n} (OH)^2– _n were prepared by alkaline hydrolysis in aqueous and nonaqueous solutions. All the compounds were identified by chemical analysis and ¹¹B NMR and IR spectroscopy. The crystal structure of [Pb(Bipy)(DMF)(B₁₀H₉OH)] · DMF was determined by X-ray diffraction.     

Interaction between polydeoxyadenylic acid and β-glucan from Lentinus edodes

The interaction between polydeoxyadenylic acid (poly(dA)) and single chains of Lentinan (s-LNT) was investigated by circular dichroism spectra (CD), UV–Vis spectra, nano-differential scanning calorimetry (nano-DSC), dynamic light scattering (DLS), and atomic force microscopy (AFM). All the experimental results indicated that poly(dA) really interacted with s-LNT having molecular weight of 5.3 × 10⁵ to form a novel composite with stiff conformation through hydrogen bonding, whereas the triple helical Lentinan (t-LNT) could not, implying that it was the single chain but not the triple helical chain interacted with poly(dA). Meanwhile, the interaction strongly depended on the concentration of s-LNT and pH. When the poly(dA) concentration was fixed at 5.3 μg/ml, the interaction between poly(dA) and s-LNT increased with an increase in s-LNT concentration, then reached the maximum at ～60 μg/ml, finally decreased with further increase of s-LNT concentration. This was due to self-renaturation of s-LNT into triple helix or incomplete species confirmed by nano-DSC. The poly(dA)/s-LNT complex could exist in solutions with pH 5.5–11.5, and pH 7–10 was the optimal condition for the complex, showing higher stability against pH than the positive control of poly(dA)/s-SPG. The dynamic behaviors of the complex demonstrated that the interaction between s-LNT and poly(dA) occurred rapidly, and reached stable within 3–7 h. Moreover, the thermal stability of poly(dA) was enhanced by complexation with s-LNT. The topograph of the poly(dA)/s-LNT complex was shown as rod-like stiff architecture. This work enlarges the application of LNT in the biomedical field.     

Antibody–Prodrug Conjugates with KSP Inhibitors and Legumain-Mediated Metabolite Formation

Many antibody–drug conjugates (ADCs) have failed to achieve a sufficient therapeutic window in clinical studies either due to target-mediated or off-target toxicities. To achieve an additional safety level, a new class of antibody–prodrug conjugates (APDCs) directed against different targets in solid tumors is here described. The tumor-associated lysosomal endopeptidase legumain with a unique cleavage sequence was utilized for APDC metabolism. Legumain-activatable APDCs were as potent as their cathepsin B-activatable analogues. The peptide sequence susceptible to legumain cleavage was optimized for further discrimination of the formation of active metabolites within tumor cells versus healthy tissues, leveraging different tissue-specific legumain activities. Optimized APDCs with slow legumain-mediated conversion reduced preclinically the levels of active metabolite in healthy organs while retaining high activity against different TWEAKR- and B7H3-expressing tumors.     

Discovery of spirooxindole–ferrocene hybrids as novel MDM2 inhibitors

A series of spirooxindole-ferrocene hybrids bearing five or four contiguous chiral centers were designed and synthesized via organocatalysis. In vitro protein binding and cellular proliferation assays suggested that compound 5 d was the most potent mouse double minute 2 homolog(MDM2) inhibitor. In addition,mechanistic studies indicated that compound 5 d suppressed MDM2-mediated p53 degradation, induced apoptosis and promoted oxidative damage. Molecular docking studies have suggested that 5 d binds to MDM2 by mimicking the Trp23 and Leu26 residues of p53. This work can provide a basis for the development of novel multifunctional MDM2 inhibitors. The further exploration of more derivatives from this library and additional investigation of organocatalysis application in the development of new molecules may generate new potential lead compounds for cancer-targeted therapy.     

Interaction between zero-charged catanionic vesicles and PEO–PPO–PEO triblock copolymers

We investigate the interaction between zero-charged catanionic vesicles and PEO–PPO–PEO (poly(ethylene oxide–poly(propylene oxide)–poly(ethylene oxide)) triblock copolymers. The 25-mg mL^?1 aqueous solution of tetradecyltrimethylammonium laurate (TTAL) contains closely packed uni- and multi-lamellar vesicles and shows viscoelastic properties with a dominant elastic modulus (G′) over a viscous modulus (G″). When a small amount of F127 ((EO)₉₇(PO)₆₉(EO)₉₇) or F68 ((EO)₇₆(PO)₂₉(EO)₇₆) was added, an improvement of the viscoelasticity was observed at suitable polymer concentrations. Freeze–fracture transmission electron microscopy (FF-TEM) observations on an F68-containing system revealed interesting aggregate transition from vesicles to flexible tubules and back to vesicles. The improvement of the viscoelasticity of the vesicular solution containing F68 or F127 can be explained by the formation of tubule and polymer–vesicle associates, while no such phenomenon was noticed for P123 ((EO)₁₉(PO)₆₉(EO)₁₉) which has the highest propylene oxide (PO) content and the strongest ability to self-associate in aqueous solution. In all the cases, vesicles will be destroyed and phase separation can be observed at high polymer contents (>5-mg mL^?1).     

Parikh–Doering oxidation–dehydration–Ugi cyclization cascade in the development of lactams from formidoalkanols (3>chain length>7)

De novo incorporation of N-formylated aminols as strategic substrates in legendary Ugi cyclization. Lactamization of N-formido alkanols is accomplished by a novel Parikh–Doering oxidation–dehydration–Ugi cyclization cascade leading to the formation of lactams up to eight carbons (ring size; n=1–4) in moderate yields using formidoaminol, substituted aniline, and aliphatic/aromatic carboxylic acid derivatives as starting materials.     

Synthesis of sulfonyl chlorides and thiosulfonates from H2O2–TiCl4

A new method is described for the oxidative chlorination of thiols to sulfonyl chlorides using titanium tetrachloride in combination with the oxidant hydrogen peroxide. Direct conversion of thiols into their corresponding thiosulfonates is also reported. Good to excellent yields, short reaction times, high efficiencies, cost-effectiveness, and, facile isolation of the desired products make the present methodology a practical alternative.     

Synthesis and Evaluation of Coumarin–Resveratrol Hybrids as 15-Lipoxygenaze Inhibitors

A series of coumarin–resveratrol hybrids, 3-arylcoumarin derivatives 3a–u, were synthesized through the intermolecular condensation reaction of various salicylaldehydes and phenylacetic acids in the presence of 1,4-diazabicyclo[2.2.2]octane under solvent-free conditions. All the synthesized compounds were screened for their inhibitory potency against soybean 15-lipoxygenase. Among them, three compounds (3c, 3j, and 3q) showed good enzyme-inhibitory activities.     

Synthesis and Evaluation of 2-Deoxy-2-amino-β-cellobiosides as Cellulase Inhibitors

The cellulase mixture of Hypocrea jecorina (formerly Trichoderma reesei) contains a variety of exo- and endoglucanases that belong to different structural families. As such, these enzymes form an interesting model system to study the enzyme-ligand interactions in glycoside hydrolases. The nucleophilic carboxylate of retaining β-glycosidases is believed to form a hydrogen bond with the 2-hydroxyl group of their substrate. Consequently, replacing this hydroxyl group with an amino group should result in a stronger electrostatic interaction and thus an increased affinity for the ligand. In this study, several modified cellobiosides were synthesized and evaluated as cellulase inhibitors. The introduction of an amino group was found to have an unpredictable effect on the inhibitory power of the ligands. However, the enzymes display a very high affinity for the corresponding 2-azido compounds, precursors in the synthetic route. The new ligand m-iodobenzyl 2-deoxy-2-azido-β-cellobioside even is the strongest inhibitor of cellobiohydrolase I known to date (K_I = 1 μM).     

Interaction parameters and (solid + liquid) equilibria calculation for KCl–H2O–HCl–C2H5OH,K2SO4–H2O–H2SO4 and K2SO4–H2O–C2H5OH mixed solvent-electrolyte systems

This work deals with the prediction and experimental measurements of the (solid + liquid) equilibrium (SLE) in acid medium for industrial purposes. Specific systems including KCl–ethanol–water–HCl and K₂SO₄–water–H₂SO₄ were analyzed. At first, a critical discussion of SLE calculations was given, based on the well-known UNIQUAC extended and LIQUAC models. Two new proposals were derived, considering the explicit necessity of a new reference state for SLE calculations for the studied (solvents + acid) mixtures. The solubility of KCl in water–ethanol–HCl mixed solvents was measured in the temperature range of 300.15 to 315.15 K using an analytical gravimetric method. These results combined with some other experimental data reported in the open literature let us to propose a set of parameters for the new models. They included the interaction parameters between ethanol and the H⁺ ion. The prediction capability of the new models, for calculations in acid medium, was illustrated. Experimentally, it was observed that the (K₂SO₄ + water + H₂SO₄) system presented the unusual behavior of increasing K₂SO₄ solubility with an increase in the sulfuric acid concentration. This was accurately predicted by the newly proposed models.     

Anionic–Nonionic Mixed Surfactant Systems: Micellar Interaction and Thermodynamic Behavior

The interactions between an anionic surfactant, viz., sodium dodecylbenzenesulfonate and nonionic surfactants with different secondary ethoxylated chain length, viz., Tergitol 15-S-12, Tergitol 15-S-9, and Tergitol 15-S-7 have been studied in the present article. An attempt has also been made to investigate the effect of ethoxylated chain length on the micellar and the thermodynamic properties of the mixed surfactant systems. The micellar properties like critical micelle concentration (CMC), micellar composition (X_A), interaction parameter (β), and the activity coefficients (f_A and f_NI) have been evaluated using Rubingh's regular solution theory. In addition to micellar studies, thermodynamic parameters like the surface pressure (Π_CMC), surface excess values (Γ_CMC), average area of the monomers at the air–water interface (A_avg), free energy of micellization (ΔG_m), minimum energy at the air–water interface (G_min), etc., have also been calculated. It has been found that in mixtures of anionic and nonionic secondary ethoxylated surfactants, a surfactant containing a smaller ethoxylated chain is favored thermodynamically. Additionally, the adsorption of nonionic species on air/water interface and micelle increases with decreasing secondary ethoxylated chain length. Dynamic light scattering and viscometric studies have also been performed to study the interactions between anionic and nonionic surfactants used.     

Protein–protein recognition: a computational mutagenesis study of the MDM2–P53 complex

Protein P53 is involved in more than 50% of the human cancers and the P53–MDM2 complex is a target for anticancer drug design. It is possible to engineer small P53 mimics that would be expected to disrupt the P53–MDM2 complex, and release P53 to initiate cell-cycle arrest or apoptosis. These small peptides should bind to the functional epitopes of the protein–protein interface, and prevent the interaction between P53 and MDM2. Here, we apply an improved computational alanine scanning mutagenesis method, which allows the determination of the hot spots present in both monomers, P53 and MDM2, of three protein complexes (the P53-binding domain of human MDM2, its analogue from Xenopus laevis, and the structure of human MDM2 in complex with an optimized P53 peptide). The importance of the hydrogen bonds formed by the protein backbone has been neglected due to the difficulty of measuring experimentally their contribution to the binding free energy. In this study we present a computational approach that allows the estimation of the contribution to the binding free energy of the C=O and N–H groups in the backbone of the P53 and MDM2 proteins. We have noticed that the hydrogen bond between the HE1 atom of the hot spot Trp23 and the O atom of the residue Leu54, as well as the NH-pi hydrogen bond between the Ile57 and Met58 were observed in the Molecular dynamics simulation, and their contribution to the binding free energy measured. This study not only shows the reliability of the computational mutagenesis method to detect hot spots but also demonstrates an excellent correlation between the quantitative calculated binding free energy contribution of the C=O and N–H backbone groups of the interfacial residues and the qualitative values expected for this kind of interaction. The study also increases our understanding of the P53–MDM2 interaction.     

Solution and Gas-Phase H/D Exchange of Protein–Small-Molecule Complexes: Cex and Its Inhibitors

The properties of noncovalent complexes of the enzyme exo-1,4-β-D-glycanase (“Cex”) with three aza-sugar inhibitors, deoxynojirimycin (X₂DNJ), isofagomine lactam (X₂IL), and isofagomine (X₂IF), have been studied with solution and gas-phase hydrogen deuterium exchange (H/Dx) and measurements of collision cross sections of gas-phase ions. In solution, complexes have lower H/Dx levels than free Cex because binding the inhibitors blocks some sites from H/Dx and reduces fluctuations of the protein. In mass spectra of complexes, abundant Cex ions are seen, which mostly are formed by dissociation of complexes in the ion sampling interface. Both complex ions and Cex ions formed from a solution containing complexes have lower cross sections than Cex ions from a solution of Cex alone. This suggests the Cex ions formed by dissociation “remember” their solution conformations. For a given charge, ions of the complexes have greater gas-phase H/Dx levels than ions of Cex. Unlike cross sections, H/Dx levels of the complexes do not correlate with the relative gas-phase binding strengths measured by MS/MS. Cex ions from solutions with or without inhibitors, which have different cross sections, show the same H/Dx level after 15 s, indicating the ions may fold or unfold on the seconds time scale of the H/Dx experiment. Thus, cross sections show that complexes have more compact conformations than free protein ions on the time scale of ca. 1 ms. The gas-phase H/Dx measurements show that at least some complexes retain different conformations from the Cex ions on a time scale of seconds.     

Development and Validation of an HPTLC–Densitometric Method for Determination of ACE Inhibitors

A high-performance thin layer chromatographic method coupled with densitometric analysis has been developed for measurement of benazepril and cilazapril, both pure and in their commercial dosage forms. The active substances were extracted from tablets with methanol (mean recovery 102%) and chromatographed on silica gel 60 F₂₅₄ HPTLC plates in horizontal chambers with ethyl acetate–acetone–acetic acid–water, 8:2:0.5:0.5 (v/v), as mobile phase. Chromatographic separation of these ACE inhibitors was followed by UV densitometric quantitation at 215 nm. Calibration plots were constructed in the range 0.4 to 2.0 g L^–1 for benazepril (2.0–10.0 g spot^–1) and from 0.5 to 1.5 g L^–1 for cilazapril (4.0–12.0 g spot^–1) with good correlation coefficients (r 0.990). The method was used to determine benazepril and cilazapril in pharmaceutical preparations with satisfactory precision (1.4% < RSD < 5.6%) and accuracy (1.7 < RE < 5.1).