Structural Basis of Non-Latent Signaling by the Anti-Müllerian Hormone Procomplex.

Most TGFß family ligands exist as procomplexes consisting of a prodomain noncovalently bound to a growth factor (GF); Whereas some prodomains confer latency, the Anti-Müllerian Hormone (AMH) prodomain maintains a remarkably high affinity for the GF yet remains active. Using single particle EM methods, we show the AMH prodomain consists of two subdomains: a vestigial TGFß prodomain-like fold and a novel, helical bundle GF-binding domain, the result of an exon insertion 450 million years ago, that engages both receptor epitopes. When associated with the prodomain, the AMH GF is distorted into a strained, open conformation whose closure upon bivalent binding of AMHR2 displaces the prodomain through a conformational shift mechanism to allow for signaling.

Characterization of the molecular mechanisms that govern anti-Müllerian hormone synthesis and activity.

The roles of anti-Müllerian hormone (AMH) continue to expand, from its discovery as a critical factor in sex determination, through its identification as a regulator of ovarian folliculogenesis, its use in fertility clinics as a measure of ovarian reserve, and its emerging role in hypothalamic-pituitary function. In light of these actions, AMH is considered an attractive therapeutic target to address diverse reproductive needs, including fertility preservation. Here, we set out to characterize the molecular mechanisms that govern AMH synthesis and activity. First, we enhanced the processing of the AMH precursor to >90% by introducing more efficient proprotein convertase cleavage sites (RKKR or ISSRKKRSVSS [SCUT]). Importantly, enhanced processing corresponded with a dramatic increase in secreted AMH activity. Next, based on species differences across the AMH type II receptor-binding interface, we generated a series of human AMH variants and assessed bioactivity. AMHSCUT potency (EC50 4 ng/mL) was increased 5- or 10-fold by incorporating Gln484 Met/Leu535 Thr (EC50 0.8 ng/mL) or Gln484 Met/Gly533 Ser (EC50 0.4 ng/mL) mutations, respectively. Furthermore, the Gln484 Met/Leu535 Thr double mutant displayed enhanced efficacy, relative to AMHSCUT . Finally, we identified residues within the wrist pre-helix of AMH (Trp494 , Gln496 , Ser497 , and Asp498 ) that likely mediate type I receptor binding. Mutagenesis of these residues generated gain- (Trp494 Phe or Gln496 Leu) or loss- (Ser497 Ala) of function AMH variants. Surprisingly, combining activating type I and type II receptor mutations only led to modest additive increases in AMH potency/efficacy. Our study is the first to characterize AMH residues involved in type I receptor binding and suggests a step-wise receptor-complex assembly mechanism, in which enhancement in the affinity of the ligand for either receptor can increase AMH activity beyond the natural level.

Utilization of a kinetic isotope effect to decrease decomposition of ceftriaxone in a mixture of D2O/H2O.

The discovery of cephalosporin and demonstration of its improved stability in aqueous solution, as well as enhanced in vitro activity against penicillin-resistant organisms, were major breakthroughs in the development of ß-lactam antibiotics. Although cephalosporins are more stable with respect to hydrolytic degradation than penicillins, they still experience a variety of chemical transformations. The present study offers an insight into the rates and mechanisms of ceftriaxone degradation at the therapeutic concentration in water, a mixture of water and deuterium oxide, and deuterium oxide itself at the neutral pH. Specific ceftriaxone degradation products were observed in aged samples (including a previously unreported dimer-type species), and by comparing the degradation rates in H2O and D2O, the observation of a kinetic isotope effect provided some valuable insight as to the nature of the initial ceftriaxone degradation. The effect of protium to deuterium isotope change on the degradation kinetics of ceftriaxone was evaluated using the method of initial rates based on HPLC analysis as well as by quantitative 1H NMR spectroscopy. Moreover, computational analysis was utilized to get a molecular insight into chemical processes governing the ceftriaxone degradation and to rationalize the stabilizing effect of replacing H2O with D2O.

Selective Deuteration Improves the Affinity of Adenosine A2A Receptor Ligands: A Computational Case Study with Istradefylline and Caffeine.

We used a range of computational techniques to assess the effect of selective C-H deuteration on the antagonist istradefylline affinity for the adenosine A2A receptor, which was discussed relative to its structural analogue caffeine, a well-known and likely the most widely used stimulant. The obtained results revealed that smaller caffeine shows high receptor flexibility and exchanges between two distinct poses, which agrees with crystallographic data. In contrast, the additional C8-trans-styryl fragment in istradefylline locks the ligand within a uniform binding pose, while contributing to the affinity through the C-H···π and π···π contacts with surface residues, which, together with its much lower hydration prior to binding, enhances the affinity over caffeine. In addition, the aromatic C8-unit shows a higher deuteration sensitivity over the xanthine part, so when both of its methoxy groups are d6-deuterated, the affinity improvement is -0.4 kcal mol-1, which surpasses the overall affinity gain of -0.3 kcal mol-1 in the perdeuterated d9-caffeine. Yet, the latter predicts around 1.7-fold potency increase, being relevant for its pharmaceutical implementations, and also those within the coffee and energy drink production industries. Still, the full potential of our strategy is achieved in polydeuterated d19-istradefylline, whose A2A affinity improves by -0.6 kcal mol-1, signifying a 2.8-fold potency increase that strongly promotes it as a potential synthetic target. This knowledge supports deuterium application in drug design, and while the literature already reports about over 20 deuterated drugs currently in the clinical development, it is easily foreseen that more examples will hit the market in the years to come. With this in mind, we propose that the devised computational methodology, involving the ONIOM division of the QM region for the ligand and the MM region for its environment, with an implicit quantization of nuclear motions relevant for the H/D exchange, allows fast and efficient estimates of the binding isotope effects in any biological system.

Imidazo[4,5-b]pyridine derived tubulin polymerization inhibitors: Design, synthesis, biological activity in vitro and computational analysis.

Imidazo[4,5-b]pyridine derived acrylonitriles were synthesized and explored for their in vitro antiproliferative effect on a diverse human cancer cell line panel. Three compounds, 20, 21 and 33, showed strong activity in the submicromolar range (IC50 0.2-0.6 µM), and were chosen for further biological experiments. Immunofluorescence staining and tubulin polymerization assays confirmed tubulin as the main target, but excluded its colchicine-binding site as a potential interacting unit. This was supported by the computational analysis, which revealed that the most potent ligands act on the extended colchicine site on the surface between interacting tubulin subunits, where they interfere with their polymerization and reveal pronounced antitumor properties. In addition, lead molecule 21 potently inhibited cancer cell migration, while it did not affect the viability of normal cells even at the highest concentration tested (100 µM).

COVID-19 infection and neurodegeneration: Computational evidence for interactions between the SARS-CoV-2 spike protein and monoamine oxidase enzymes.

Although COVID-19 has been primarily associated with pneumonia, recent data show that its causative agent, the SARS-CoV-2 coronavirus, can infect many vital organs beyond the lungs, including the heart, kidneys and the brain. The literature agrees that COVID-19 is likely to have long-term mental health effects on infected individuals, which signifies a need to understand the role of the virus in the pathophysiology of brain disorders that is currently unknown and widely debated. Our docking and molecular dynamics simulations show that the affinity of the spike protein from the wild type (WT) and the South African B.1.351 (SA) variant towards MAO enzymes is comparable to that for its ACE2 receptor. This allows for the WT/SA⋅⋅⋅MAO complex formation, which changes MAO affinities for their neurotransmitter substrates, thereby impacting their metabolic conversion and misbalancing their levels. Knowing that this fine regulation is strongly linked with the etiology of various brain pathologies, these results are the first to highlight the possibility that the interference with the brain MAO catalytic activity is responsible for the increased neurodegenerative illnesses following a COVID-19 infection, thus placing a neurobiological link between these two conditions in the spotlight. Since the obtained insight suggests that a more contagious SA variant causes even larger disturbances, and with new and more problematic strains likely emerging in the near future, we firmly advise that the presented prospect of the SARS-CoV-2 induced neurological complications should not be ignored, but rather requires further clinical investigations to achieve an early diagnosis and timely therapeutic interventions.

Synthesis, Computational Analysis, and Antiproliferative Activity of Novel Benzimidazole Acrylonitriles as Tubulin Polymerization Inhibitors: Part 2.

We used classical linear and microwave-assisted synthesis methods to prepare novel N-substituted, benzimidazole-derived acrylonitriles with antiproliferative activity against several cancer cells in vitro. The most potent systems showed pronounced activity against all tested hematological cancer cell lines, with favorable selectivity towards normal cells. The selection of lead compounds was also tested in vitro for tubulin polymerization inhibition as a possible mechanism of biological action. A combination of docking and molecular dynamics simulations confirmed the suitability of the employed organic skeleton for the design of antitumor drugs and demonstrated that their biological activity relies on binding to the colchicine binding site in tubulin. In addition, it also underlined that higher tubulin affinities are linked with (i) bulkier alkyl and aryl moieties on the benzimidazole nitrogen and (ii) electron-donating substituents on the phenyl group that allow deeper entrance into the hydrophobic pocket within the tubulin's ß-subunit, consisting of Leu255, Leu248, Met259, Ala354, and Ile378 residues.

Potentiometric Surfactant Sensor Based on 1,3-Dihexadecyl-1H-benzo[d]imidazol-3-ium for Anionic Surfactants in Detergents and Household Care Products.

A 1,3-dihexadecyl-1H-benzo[d]imidazol-3-ium-tetraphenylborate (DHBI-TPB) ion-pair implemented in DHBI-TPB surfactant sensor was used for the potentiometric quantification of anionic surfactants in detergents and commercial household care products. The DHBI-TPB ion-pair was characterized by FTIR spectroscopy and computational analysis which revealed a crucial contribution of the C-H∙∙∙π contacts for the optimal complex formation. The DHBI-TPB sensor potentiometric response showed excellent analytical properties and Nernstian slope for SDS (60.1 mV/decade) with LOD 3.2 × 10-7 M; and DBS (58.4 mV/decade) with LOD 6.1 × 10-7 M was obtained. The sensor possesses exceptional resistance to different organic and inorganic interferences in broad pH (2-10) range. DMIC used as a titrant demonstrated superior analytical performances for potentiometric titrations of SDS, compared to other tested cationic surfactants (DMIC > CTAB > CPC > Hyamine 1622). The combination of DHBI-TPB sensor and DMIC was successfully employed to perform titrations of the highly soluble alkane sulfonate homologues. Nonionic surfactants (increased concentration and number of EO groups) had a negative impact on anionic surfactant titration curves and a signal change. The DHBI-TPB sensor was effectively employed for the determination of technical grade anionic surfactants presenting the recoveries from 99.5 to 101.3%. The sensor was applied on twelve powered samples as well as liquid-gel and handwashing home care detergents containing anionic surfactants. The obtained results showed good agreement compared to the outcomes measured by ISE surfactant sensor and a two-phase titration method. The developed DHBI-TPB surfactant sensor could be used for quality control in industry and has great potential in environmental monitoring.

Direct Metal-Free Transformation of Alkynes to Nitriles: Computational Evidence for the Precise Reaction Mechanism.

Density functional theory calculations elucidated the precise reaction mechanism for the conversion of diphenylacetylenes into benzonitriles involving the cleavage of the triple C≡C bond, with N-iodosuccinimide (NIS) as an oxidant and trimethylsilyl azide (TMSN3) as a nitrogen donor. The reaction requires six steps with the activation barrier ΔG‡ = 33.5 kcal mol-1 and a highly exergonic reaction free-energy ΔGR = -191.9 kcal mol-1 in MeCN. Reaction profiles agree with several experimental observations, offering evidence for the formation of molecular I2, interpreting the necessity to increase the temperature to finalize the reaction, and revealing thermodynamic aspects allowing higher yields for alkynes with para-electron-donating groups. In addition, the proposed mechanism indicates usefulness of this concept for both internal and terminal alkynes, eliminates the option to replace NIS by its Cl- or Br-analogues, and strongly promotes NaN3 as an alternative to TMSN3. Lastly, our results advise increasing the solvent polarity as another route to advance this metal-free strategy towards more efficient processes.

Amidino substituted 2-aminophenols: biologically important building blocks for the amidino-functionalization of 2-substituted benzoxazoles.

Unlike the closely related and widely investigated amidino-substituted benzimidazoles and benzothiazoles with a range of demonstrated biological activities, the matching benzoxazole analogues still remain a largely understudied and not systematically evaluated class of compounds. To address this challenge, we utilized the Pinner reaction to convert isomeric cyano-substituted 2-aminophenols into their amidine derivatives, which were isolated as hydrochlorides and/or zwitterions, and whose structure was confirmed by single crystal X-ray diffraction. The key step during the Pinner synthesis of the crucial carboximidate intermediates was characterized through mechanistic DFT calculations, with the obtained kinetic and thermodynamic parameters indicating full agreement with the experimental observations. The obtained amidines were subjected to a condensation reaction with aryl carboxylic acids that allowed the synthesis of a new library of 5- and 6-amidino substituted 2-arylbenzoxazoles. Their antiproliferative features against four human tumour cell lines (SW620, HepG2, CFPAC-1, HeLa) revealed sub-micromolar activities on SW620 for several cyclic amidino 2-naphthyl benzoxazoles, thus demonstrating the usefulness of the proposed synthetic strategy and promoting amidino substituted 2-aminophenols as important building blocks towards biologically active systems.

The Effect of Deuteration on the H2 Receptor Histamine Binding Profile: A Computational Insight into Modified Hydrogen Bonding Interactions.

We used a range of computational techniques to reveal an increased histamine affinity for its H2 receptor upon deuteration, which was interpreted through altered hydrogen bonding interactions within the receptor and the aqueous environment preceding the binding. Molecular docking identified the area between third and fifth transmembrane α-helices as the likely binding pocket for several histamine poses, with the most favorable binding energy of -7.4 kcal mol-1 closely matching the experimental value of -5.9 kcal mol-1. The subsequent molecular dynamics simulation and MM-GBSA analysis recognized Asp98 as the most dominant residue, accounting for 40% of the total binding energy, established through a persistent hydrogen bonding with the histamine -NH3+ group, the latter further held in place through the N-H∙∙∙O hydrogen bonding with Tyr250. Unlike earlier literature proposals, the important role of Thr190 is not evident in hydrogen bonds through its -OH group, but rather in the C-H∙∙∙π contacts with the imidazole ring, while its former moiety is constantly engaged in the hydrogen bonding with Asp186. Lastly, quantum-chemical calculations within the receptor cluster model and utilizing the empirical quantization of the ionizable X-H bonds (X = N, O, S), supported the deuteration-induced affinity increase, with the calculated difference in the binding free energy of -0.85 kcal mol-1, being in excellent agreement with an experimental value of -0.75 kcal mol-1, thus confirming the relevance of hydrogen bonding for the H2 receptor activation.

Experimental and Computational Study of the Antioxidative Potential of Novel Nitro and Amino Substituted Benzimidazole/Benzothiazole-2-Carboxamides with Antiproliferative Activity.

We present the synthesis of a range of benzimidazole/benzothiazole-2-carboxamides with a variable number of methoxy and hydroxy groups, substituted with nitro, amino, or amino protonated moieties, which were evaluated for their antiproliferative activity in vitro and the antioxidant capacity. Antiproliferative features were tested on three human cancer cells, while the antioxidative activity was measured using 1,1-diphenyl-picrylhydrazyl (DPPH) free radical scavenging and ferric reducing antioxidant power (FRAP) assays. Trimethoxy substituted benzimidazole-2-carboxamide 8 showed the most promising antiproliferative activity (IC50 = 0.6-2.0 µM), while trihydroxy substituted benzothiazole-2-carboxamide 29 was identified as the most promising antioxidant, being significantly more potent than the reference butylated hydroxytoluene BHT in both assays. Moreover, the latter also displays antioxidative activity in tumor cells. The measured antioxidative capacities were rationalized through density functional theory (DFT) calculations, showing that 29 owes its activity to the formation of two [O•∙∙∙H-O] hydrogen bonds in the formed radical. Systems 8 and 29 were both chosen as lead compounds for further optimization of the benzazole-2-carboxamide scaffold in order to develop more efficient antioxidants and/or systems with the antiproliferative activity.

Racemization of oxazepam and chiral 1,4-benzodiazepines. DFT study of the reaction mechanism in aqueous solution.

The tranquilizer and hypnotic drug oxazepam undergoes the racemization process in aqueous medium, which is relevant for its pharmacological profile. The experimental barrier value (ΔG‡298 ≈ 91 kJ mol-1) was determined earlier, but the exact mechanism of enantiomerization is not known. Four different mechanisms have been proposed in the literature: C3-H/H exchange reaction, keto-enol tautomerization, solvolytic identity reaction, and ring-chain tautomerization. However, none of the reported reactions has been confirmed as the main pathway for racemization. In this work, all these mechanisms were subjected to comprehensive analysis performed by high-level quantum-chemical models. Two density functionals (B3LYP and M062X) were employed for geometry optimization of all stationary points at the corresponding potential surfaces, and the double-hybrid model (B2PLYP) was used for improved energy calculations. Out of all the tested mechanisms, only the ring-chain tautomerism fits the two experimental targets: the measured energy barrier and the pH-rate profile of racemization. The latter reveals that no acid/base catalysis is required for racemization to occur. The ring-chain tautomerism is initiated by intramolecular proton transfer from the C3-hydroxyl group to the imine nitrogen, which triggers the benzodiazepine ring opening and the formation of the achiral aldehyde intermediate. The latter undergoes ring closure which results in the inverted configuration at the C3-chiral atom of oxazepam. Our computational results suggest that the same mechanism is operative in the fast racemization of different 1,4-benzodiazepines, which posses the hydroxyl group at the stereogenic C3-centre (e.g. lorazepam or temazepam). In other benzodiazepine members (e.g. cinazepam or camazepam) the keto-enol tautomerization and/or the C3-H/H exchange mechanism may become relevant for their much slower racemization. This computational study is not only revealing in terms of mechanistic details, but also has predictive power for optical stability estimates in the family of benzodiazepines and similar heterocycles.

Chlorination of 5-fluorouracil: Reaction mechanism and ecotoxicity assessment of chlorinated products.

What happens to drugs in the chlorinating environment? Degradation products may vary in pharmacological profiles and in ecotoxicity potentials compared to the parent compound. This study combines synthesis, NMR spectroscopy, quantum chemical calculations, and toxicity experiments on Daphnia magna to investigate chemical fate of antineoplastic drug 5-fluorouracil (5-FU) in chlorinated environment, which is common in waste-water treatment procedures, but also endogenous in activated neutrophils. A reduction of toxicity (EC50 after 48 h is 50% higher than for the parent 5-FU) was observed after the first chlorination step, in which a chlorohydrin 5-chloro-5-fluoro-6-hydroxy-5,6-dihydrouracil was formed. Further chlorination leads to N-chlorinated intermediate, that undergoes the pyrimidine ring opening reaction. The final product, 2-chloro-2-fluoro-3,3-dihydroxypropanoic acid was obtained after the loss of the chlorinated urea fragment. This is the most potent compound in the reaction sequence, with toxicity parameter EC50, after 48 h, more than twice lower compared to the parent 5-FU. Clearly, the contact time between chlorinating species and degradation products provide different ecotoxicological properties of reaction mixtures. Interplay between experimental and theoretical procedures, to properly describe reaction pathways and provide more information on toxicity profiles, is a way forward in environmental science research.