Discovery of pyrazolo[3,4-d]pyrimidines as novel mitogen-activated protein kinase kinase 3 (MKK3) inhibitors.

The dual-specificity protein kinase MKK3 has been implicated in tumor cell proliferation and survival, yet its precise role in cancer remains inconclusive. A critical step in elucidating the kinase's involvement in disease biology is the identification of potent, cell-permeable kinase inhibitors. Presently, MKK3 lacks a dedicated tool compound for these purposes, along with validated methods for the facile screening, identification, and optimization of inhibitors. In this study, we have developed a TR-FRET-based enzymatic assay for the detection of MKK3 activity in vitro and a BRET-based assay to assess ligand binding to this enzyme within intact human cells. These assays were instrumental in identifying hit compounds against MKK3 that share a common chemical scaffold, sourced from a library of bioactive kinase inhibitors. Initial hits were subsequently expanded through the synthesis of novel analogs. The resulting structure-activity relationship (SAR) was rationalized using molecular dynamics simulations against a homology model of MKK3. We expect our findings to expedite the development of novel, potent, selective, and bioactive inhibitors, thus facilitating investigations into MKK3's role in various cancers.

Synthesis and evaluation of hybrid sulfonamide-chalcones with potential antileishmanial activity.

Leishmaniasis is an emerging tropical infectious disease caused by a protozoan parasite of the genus Leishmania. In this work, the molecular hybridization between a trimethoxy chalcone and a sulfonamide group was used to generate a series of sulfonamide-chalcones. A series of eight sulfonamide-chalcone hybrids were made with good yields (up to 95%). These sulfonamide-chalcones were tested against promastigotes of Leishmania amazonensis and cytotoxicity against mouse macrophages, which showed good antileishmanial activity with IC50 = 1.72-3.19 µM. Three of them (10c, 10g, and 10h) were also highly active against intracellular amastigotes and had a good selectivity index (SI > 9). Thus, those three compounds were docked in the cytosolic tryparedoxin peroxidase (cTXNPx) enzyme of the parasite, and molecular dynamics simulations were carried out. This enzyme was selected as a target protein for the sulfonamide-chalcones due to the fact of the anterior report, which identified a strong and stable interaction between the chalcone NAT22 (6) and the cTXNPx. In addition, a prediction of the drug-likeness, and the pharmacokinetic profile of all compounds were made, demonstrating a good profile of those chalcones.

Identification of potential inhibitors of casein kinase 2 alpha of Plasmodium falciparum with potent in vitro activity.

Drug resistance to commercially available antimalarials is a major obstacle in malaria control and elimination, creating the need to find new antiparasitic compounds with novel mechanisms of action. The success of kinase inhibitors for oncological treatments has paved the way for the exploitation of protein kinases as drug targets in various diseases, including malaria. Casein kinases are ubiquitous serine/threonine kinases involved in a wide range of cellular processes such as mitotic checkpoint signaling, DNA damage response, and circadian rhythm. In Plasmodium, it is suggested that these protein kinases are essential for both asexual and sexual blood-stage parasites, reinforcing their potential as targets for multi-stage antimalarials. To identify new putative PfCK2α inhibitors, we utilized an in silico chemogenomic strategy involving virtual screening with docking simulations and quantitative structure-activity relationship predictions. Our investigation resulted in the discovery of a new quinazoline molecule (542), which exhibited potent activity against asexual blood stages and a high selectivity index (>100). Subsequently, we conducted chemical-genetic interaction analysis on yeasts with mutations in casein kinases. Our chemical-genetic interaction results are consistent with the hypothesis that 542 inhibits yeast Cka1, which has a hinge region with high similarity to PfCK2α. This finding is in agreement with our in silico results suggesting that 542 inhibits PfCK2α via hinge region interaction.

Aurones: A Promising Scaffold to Inhibit SARS-CoV-2 Replication.

Aurones are a small subgroup of flavonoids in which the basic C6-C3-C6 skeleton is arranged as (Z)-2-benzylidenebenzofuran-3(2H)-one. These compounds are structural isomers of flavones and flavonols, natural products reported as potent inhibitors of SARS-CoV-2 replication. Herein, we report the design, synthesis, and anti-SARS-CoV-2 activity of a series of 25 aurones bearing different oxygenated groups (OH, OCH3, OCH2OCH3, OCH2O, OCF2H, and OCH2C6H4R) at the A- and/or B-rings using cell-based screening assays. We observed that 12 of the 25 compounds exhibit EC50 < 3 µM (8e, 8h, 8j, 8k, 8l, 8m, 8p, 8q, 8r, 8w, 8x, and 8y), of which five presented EC50 < 1 µM (8h, 8m, 8p, 8q, and 8w) without evident cytotoxic effect in Calu-3 cells. The substitution of the A- and/or B-ring with OCH3, OCH2OCH3, and OCF2H groups seems beneficial for the antiviral activity, while the corresponding phenolic derivatives showed a significant decrease in the anti-SARS-CoV-2 activity. The most potent compound of the series, aurone 8q (EC50 = 0.4 µM, SI = 2441.3), is 2 to 3 times more effective than the polyphenolic flavonoids myricetin (2) and baicalein (1), respectively. Investigation of the five more active compounds as inhibitors of SARS-CoV-2 3CLpro based on molecular dynamic calculations suggested that these aurones should detach from the active site of 3CLpro, and, probably, they could bind to another SARS-CoV-2 protein target (either receptor or enzyme).

Differences in Gluco and Galacto Substrate-Binding Interactions in a Dual 6Pß-Glucosidase/6Pß-Galactosidase Glycoside Hydrolase 1 Enzyme from Bacillus licheniformis.

Bacterial glycoside hydrolase 1 (GH1) enzymes with 6-phospho-ß-galactosidase and 6-phospho-ß-glucosidase activities have the important task of releasing phosphorylated and nonphosphorylated monosaccharides into the cytoplasm. Curiously, dual 6-phospho-ß-galactosidase/6-phospho-ß-glucosidase (dual-phospho) enzymes have broad specificity and are able to hydrolyze galacto- and gluco-derived substrates. This study investigates the structure and substrate specificity of a GH family 1 enzyme from Bacillus licheniformis, hereafter known as BlBglC. The enzyme structure has been solved, and sequence analysis, molecular dynamics simulations, and binding free energy calculations offered evidence of dual-phospho activity. Both test ligands p-nitrophenyl-ß-d-galactoside-6-phosphate (PNP6Pgal) and p-nitrophenyl-ß-d-glucoside-6-phosphate (PNP6Pglc) demonstrated strong binding to BlBglC although the pose and interactions of the PNP6Pglc triplicates were slightly more consistent. Interestingly, known specificity-inducing residues, Gln23 and Trp433, bind strongly to the ligand O3 hydroxyl group in the PNP6Pgal-BlBglC complex and to the ligand O4 hydroxyl group in the PNP6Pglc-BlBglC complex. Additionally, the BlBglC-His124 residue is a major contributor of hydrogen bonds to the PNP6Pgal O3 hydroxyl group but does not form any hydrogen bonds with PNP6Pglc. On the other hand, BlBglC residues Tyr173, Tyr301, Gln302, and Thr321 form hydrogen bonds with PNP6Pglc but not PNP6Pgal. These findings provide important details of the broad specificity of dual-phospho activity GH1 enzymes.

X-ray Structure, Bioinformatics Analysis, and Substrate Specificity of a 6-Phospho-ß-glucosidase Glycoside Hydrolase 1 Enzyme from Bacillus licheniformis.

In bacteria, mono- and disaccharides are phosphorylated during the uptake processes through the vastly spread transport system phosphoenolpyruvate-dependent phosphotransferase. As an initial step in the phosphorylated disaccharide metabolism pathway, 6-phospho-ß-glucosidases and 6-phospho-ß-galactosidases play a crucial role by releasing phosphorylated and nonphosphorylated monosaccharides. However, structural determinants for the specificity of these enzymes still need to be clarified. Here, an X-ray structure of a glycoside hydrolase family 1 enzyme from Bacillus licheniformis, hereafter known as BlBglH, was determined at 2.2 Å resolution, and its substrate specificity was investigated. The sequence of BlBglH was compared to the sequences of 58 other GH1 enzymes using sequence alignments, sequence identity calculations, phylogenetic analysis, and motif discovery. Through these various analyses, BlBglH was found to have sequence features characteristic of the 6-phospho-ß-glucosidase activity enzymes. Motif and structural observations highlighted the importance of loop L8 in 6-phospho-ß-glucosidase activity enzymes. To further affirm enzyme specificity, molecular docking and molecular dynamics simulations were performed using the crystallographic structure of BlBglH. Docking was carried out with a 6-phospho-ß-glucosidase enzyme activity positive and negative control ligand, followed by 400 ns of MD simulations. The positive and negative control ligands were PNP6Pglc and PNP6Pgal, respectively. PNP6Pglc maintained favorable interactions within the active site until the end of the MD simulation, while PNP6Pgal exhibited instability. The favorable binding of substrate stabilized the loops that surround the active site. Binding free energy calculations showed that the PNP6Pglc complex had a substantially lower binding energy compared to the PNP6Pgal complex. Altogether, the findings of this study suggest that BlBglH possesses 6-phospho-ß-glucosidase enzymatic activity and revealed sequence and structural differences between bacterial GH1 enzymes of various activities.

Novel Dihydropteridinone Derivatives As Potent Inhibitors of the Understudied Human Kinases Vaccinia-Related Kinase 1 and Casein Kinase 1δ/ε.

Vaccinia-related kinase 1 (VRK1) and the δ and ε isoforms of casein kinase 1 (CK1) are linked to various disease-relevant pathways. However, the lack of tool compounds for these kinases has significantly hampered our understanding of their cellular functions and therapeutic potential. Here, we describe the structure-based development of potent inhibitors of VRK1, a kinase highly expressed in various tumor types and crucial for cell proliferation and genome integrity. Kinome-wide profiling revealed that our compounds also inhibit CK1δ and CK1ε. We demonstrate that dihydropteridinones 35 and 36 mimic the cellular outcomes of VRK1 depletion. Complementary studies with existing CK1δ and CK1ε inhibitors suggest that these kinases may play overlapping roles in cell proliferation and genome instability. Together, our findings highlight the potential of VRK1 inhibition in treating p53-deficient tumors and possibly enhancing the efficacy of existing cancer therapies that target DNA stability or cell division.

A simple method to determine changes in the affinity between HisF and HisH in the Imidazole Glycerol Phosphate Synthase heterodimer.

The bi-enzyme HisF-HisH heterodimer is part of the pathway that produces histidine and purines in bacteria and lower eukaryotes, but it is absent in mammals. This heterodimer has been largely studied probing the basis of the allosteric effects and the structural stability in proteins. It is also a potential target for antibacterial drugs. In this work, we developed a simple method to evaluate changes in the affinity between HisF and HisH in the heterodimer of the bacteria Thermotoga maritima. HisH contains a single tryptophan residue, which is exposed in the free protein, but buried in the heterodimer interface. Hence, the intrinsic fluorescence maximum of this residue changes to shorter wavelengths upon dimerization. Thus, we used the fluorescence intensity at this shorter wavelength to monitor heterodimer accumulation when HisH was combined with sub-stoichiometric HisF. Under conditions where the HisF-HisH heterodimer is in equilibrium with the free states of these enzymes, when [HisH] > [HisF], we deduced a linear function connecting [HisF-HisH] to [HisF], in which the slope depends on the heterodimer dissociation constant (Kd). Based on this equation, taking fluorescence intensities as proxies of the heterodimer and HisF concentrations, we experimentally determined the Kd at four different temperatures. These Kd values were compared to those evaluated using ITC. Both methods revealed an increase in the HisF and HisH binding affinity as the temperature increases. In spite of differences in their absolute values, the Kd determined using these methods presented an evident linear correlation. To demonstrate the effectiveness of the fluorescence method we determined the effect on the Kd caused by 12 single mutations in HisF. Coherently, this test singled out the only mutation in the binding interface. In brief, the method described here effectively probes qualitative effects on the Kd, can be carried out using common laboratory equipment and is scalable.

Homodimerization of a glycoside hydrolase family GH1 ß-glucosidase suggests distinct activity of enzyme different states.

In this work, we investigated how activity and oligomeric state are related in a purified GH1 ß-glucosidase from Spodoptera frugiperda (Sfßgly). Gel filtration chromatography coupled to a multiple angle light scattering detector allowed separation of the homodimer and monomer states and determination of the dimer dissociation constant (KD ), which was in the micromolar range. Enzyme kinetic parameters showed that the dimer is on average 2.5-fold more active. Later, we evaluated the kinetics of homodimerization, scanning the changes in the Sfßgly intrinsic fluorescence over time when the dimer dissociates into the monomer after a large dilution. We described how the rate constant of monomerization (koff ) is affected by temperature, revealing the enthalpic and entropic contributions to the process. We also evaluated how the rate constant (kobs ) by which equilibrium is reached after dimer dilution behaves when varying the initial Sfßgly concentration. These data indicated that Sfßgly dimerizes through the conformational selection mechanism, in which the monomer undergoes a conformational exchange and then binds to a similar monomer, forming a more active homodimer. Finally, we noted that conformational selection reports and experiments usually rely on a ligand whose concentration is in excess, but for homodimerization, this approach does not hold. Hence, since our approach overcomes this limitation, this study not only is a new contribution to the comprehension of GH1 ß-glucosidases, but it can also help to elucidate protein interaction pathways.

Optimum temperature may be a misleading parameter in enzyme characterization and application.

The optimum temperature is commonly determined in enzyme characterization. A search in the PubMed database for "optimum temperature" and "enzymes" yielded more than 1,700 manuscripts reporting this parameter over the last five years. Here, we show that the optimum temperature is not a constant. The catalytic activity of the mesophylic ß-glucosidase Sfßgly was determined at different temperatures using different assay times and enzyme concentrations. We observed that the optimum temperature for Sfßgly changed by 5°C simply by modifying the assay length, and it was inversely correlated with enzyme concentration. These observations rely on the fact that close to the melting temperature, thermal denaturation continuously decreases the active enzyme concentration as the assay progresses. Thus, as the denaturation rate increases with increasing temperature, the bell-shaped curves observed in "activity versus temperature plots" occur only if the enzyme is denatured at and above the optimum temperature, which was confirmed using the thermostable ß-glucosidase bglTm. Thus, the optimum temperature hardly reflects an intrinsic enzyme property and is actually a mere consequence of the assay condition. Thus, adoption of the "optimum temperature" determined under bench conditions for large-scale uses, which differ in assay length and enzyme concentration, may result in lower yields and financial losses.

Search for independent (ß/α)4 subdomains in a (ß/α)8 barrel ß-glucosidase.

Proteins that fold as (ß/α)8 barrels are thought to have evolved from half-barrels that underwent duplication and fusion events. The evidence is particularly clear for small barrels, which have almost identical halves. Additionally, computational calculations of the thermodynamic stability of these structures in the presence of denaturants have revealed that (ß/α)8 barrels contain two subunits or domains corresponding to half-barrels. Hence, within (ß/α)8 barrels, half-barrels are self-contained units. Here, we tested this hypothesis using ß-glucosidase from the bacterium Thermotoga maritima (bglTm), which has a (ß/α)8 barrel structure. Mutations were introduced to disrupt the noncovalent contacts between its halves and reveal the presence of two domains within bglTm, thus resulting in the creation of mutants T1 (containing W12A and I217A mutations) and T2 (containing W12A, H195A, I217A and F404A mutations). Mutants T1 and T2 were properly folded, as indicated by their fluorescence spectra and enzyme kinetic parameters. T1 and wild-type bglTm were equally stable, as shown by the results of thermal inactivation, differential scanning fluorimetry and guanidine hydrochloride denaturation experiments. However, T2 showed a first-order inactivation at 80°C, a single melting temperature of 82°C and only one transition concentration (c50) in 2.4 M guanidine hydrochloride. Additionally, T1 and T2 exhibited a cooperative denaturation process that followed a two-state model (m-values equal to 1.4 and 1.6 kcal/mol/M, respectively), similar to that of wild-type bglTm (1.2 kcal/mol/M). Hence, T1 and T2 each denatured as a single unit, although they contained different degrees of disruption between their halves. In conclusion, bglTm halves are equivalent in terms of their thermal and chemical stability; thus, their separate contributions to (ß/α)8 barrel unfolding cannot be disentangled.

Anticancer drug screening: standardization of in vitro wound healing assay / Triagem de drogas anticâncer: padronização do ensaio de ranhura in vitro

ABSTRACT Introduction: Gliomas are characterized by rapid proliferation and aggressive invasion into normal surrounding brain tissue. In medical laboratories, the in vitro wound healing assay stands out as a simple, easy, inexpensive and affordable method to evaluate cell migration and proliferation. Objective: To standardize the in vitro wound healing assay using antimicrotubule drugs as positive controls. Methods: U87MG glioma cells were seeded at different densities and, after 24 h, the monolayer was scratched using different micropipette tip size to create a gap with no cells. The cells were then treated with colchicine and paclitaxel in culture medium with the presence or absence of fetal bovine serum. The wound was photographed with the aid of an inverted microscope and the wound area was measured using the Image J software. Results: Better defined edges scratches and monolayer with approximately 90% confluence were obtained at 1.5 and 2 x 105 cells/well density. The width and area of the scratch were, respectively, 948 pm/2.193221 mm2; 964 pm/2.266 mm2 and 1448 pm/3.221 mm2 to 10. 200 and 1000 pl micropipette tips. Colchicine inhibited wound closure by 12.6% or 3.4%, both in the presence or absence of serum; paclitaxel 2.4 and 6.7% respectively. Conclusion: Under standardized conditions, colchicine and paclitaxel proved to be efficient positive controls into the in vitro wound healing assay.

RESUMEN Introducción: Gliomas se caracterizan por rápida proliferación e invasion agresiva del tejido cerebral normal circundante. En laboratorios médicos, el ensayo de cierre de herida - una prueba in vitro - se destaca por ser un método simple, fácil, de bajo costo y accesiblepara evaluar la migración y la proliferación celular. Objetivo: Estandarizar el ensayo de cierre de herida usando agentes anti-microtúbulos como control positivo. Métodos: Las células de glioma U87MG fueron sembradas en diferentes concentracionesy, después de 24 horas, la monocamadafue rayada con punteras de diferentes tamanos para crear una hendidura sin células. Las células fueron entonces tratadas con colchicina y paclitaxel, en medio con o sin suero fetal bovino. La ranura fue fotografiada con la ayuda de un microscopio invertido, y el área de la ranura fue medida mediante el programa Image J. Resultados: Ranuras con bordes más bien-definidos y monocamada con alrededor de 90% de confluencia se obtuvieron con 1,5 y 2 x 105 células/pozo. La anchura y el área de las ranuras obtenidas fueron, respectivamente, 948 p.m/2,193 mm2; 964p.m/2,266mm2; y 1448p.m/3,221 mm2 para las punteras de 10,200y 1000pl. La colchicina inhibió el cierre de las ranuras en 12,6% o 2,4%, tanto en presencia como en ausencia de suero; el paclitaxel, 3,4% y 6,7%, respectivamente. Conclusión: En condiciones estandarizadas, colchicina y paclitaxel pueden ser usados como control positivo en el ensayo de cierre de herida in vitro.

RESUMO Introdução: Gliomas são caracterizados por terem rápida proliferação e invasão agressiva no tecido cerebral circundante normal. Em laboratórios médicos, o ensaio de ranhura - um teste in vitro - destaca-se por ser um método simples, fácil, barato e acessível para avaliar a migração e a proliferação celular. Objetivo: Padronizar o ensaio de ranhura, utilizando drogas antimicrotúbulos como controles positivos. Métodos: As células de glioma U87MG foram semeadas em diferentes concentrações e, após 24 horas, a monocamada foi arranhada usando ponteiras de diferentes tamanhos para criar uma fenda sem células. As células foram então tratadas com colchicina e paclitaxel, com meio em ausência ou presença de soro fetal bovino. A ranhura foi fotografada com auxílio de microscópio invertido, e a área da ranhura foi medida por meio do programa Image J. Resultados: Ranhuras com bordas mais bem definidas e monocamada com aproximadamente 90% de confluência foram obtidas com 1,5 e 2 x 105 células/poço. A largura e a área das ranhuras obtidasforam, respectivamente, 948pm/2,193 mm2;964pm/2,266mm2; e 1448 pm/3,221 mm2 para as ponteiras de 10, 200 e 1000 pl. A colchicina inibiu o fechamento das ranhuras em 12,6% ou 2,4%, tanto na presença quanto na ausência de soro; o paclitaxel, em 3,4% e 6,7%, respectivamente. Conclusão: Em condições padronizadas, colchicina e paclitaxel podem ser usados como controles positivos eficientes no teste in vitro de ranhura.