Biofilms can be prevalent in natural, industrial and hospital settings and are associated to ca. 80% of all human infections ¹. The increased antimicrobial resistance and mutation rate of bacteria in biofilms contributes to the development of antibiotic resistance, greatly limiting the therapeutic options to a variety of infections, posing a critical problem to the biomedical sector. Preventing biofilm formation could dramatically reduce the effects of infectious diseases ².

Quorum-sensing (QS) is the cell-to-cell communication in bacteria and contributes to the formation of organized structural communities of bacteria in biofilms ³. Several different microbial-derived signaling molecule types and receptors have been recently identified, offering a very appealing opportunity for rational design of new drugs.

This work reports the creation of a database containing all the available experimental X-ray structures for all the synthases and receptors known to be involved in quorum sensing and includes also structural and biological information on all the known compounds with demonstrated inhibitory activity against each of these protein targets.

This database will provide useful atomic-level information for researchers working on this field with direct application in drug design and development efforts through docking, virtual screening, molecular dynamics and QSAR techniques.

1. Worthington, R. J.; Richards, J. J.; Melander, C., Small molecule control of bacterial biofilms. Organic & biomolecular chemistry 2012, 10 (37), 7457-74.
2. Subhadra, B.; Kim, D. H.; Woo, K.; Surendran, S.; Choi, C. H., Control of Biofilm Formation in Healthcare: Recent Advances Exploiting Quorum-Sensing Interference Strategies and Multidrug Efflux Pump Inhibitors. Materials (Basel, Switzerland) 2018, 11 (9).
3. Banerjee, G.; Ray, A. K., Quorum-sensing network-associated gene regulation in Gram-positive bacteria. Acta microbiologica et immunologica Hungarica 2017, 64 (4), 439-453.

• Background: Quillaja saponaria Mol. bark contains a high concentration of triterpene saponins that have been used for centuries as a cleansing, antiinflammatory and analgesic agent in Chilean folk medicine. In earlier studies we demonstrated, in mice, both the anti-inflammatory as well as the antinociceptive effect of the major sapogenin, quillaic acid (QA).
• Objective: To determine the antihyperalgesic effect of QA one and seven days after itpl administration of complete Freund's adjuvant (CFA) in male mice using the hot plate test in the presence of complete Freund's adjuvant (HP/CFA) as an acute and chronic skeletal muscle pain model.
• Methods: The present study evaluated the antihyperalgesic activity of QA against acute and chronic skeletal muscle pain models in mice using the hot plate test in the presence of complete Freund's adjuvant (HP/CFA), at 24 h (acute assay) and 7 days (chronic assay) , with dexketoprofen (DEX) as the reference drug.
• Results: In acute and chronic skeletal muscle pain assays, QA at 30 mg/kg ip elicited its maximal antihyperalgesic effects (65.0% and 53.4%) at 24 h and 7 days respectively. The maximal effect of DEX (99.0 and 94.1 at 24 h and 7 days respectively) was induced at 100 mg/kg.
• Conclusion: QA and DEX elicit dose-dependent antihyperalgesic effects against acute and chronic skeletal muscle pain, but QA is more potent than DEX in the early and late periods of inflammatory pain induced by CFA.

The technological integration of software development components and languages ​​allows the creation of business management platforms that include metrics and standards in data processing[1]. In this work we propose the generation of a scalable and integrable web management application, through the asynchronous use of events and the MVVM development pattern[2,3].

Phosphorylation is one of the most important post-translational modifications in living cells and the phosphate groups are present in many of its biomolecules. Despite the fact that the phosphate group is ubiquitous, their role and the environment to which is is exposed can change dramatically. Therefore, in computational methods, the phosphate parameters are probably the among the least transferable, e.g. a phosphate group in phospholipids is significantly different from a phosphoryl tyrosine, in terms of electrostatics. In current forcefields, phosphate groups have been parameterized in the context of lipids and in connection with nucleotides, while parameters for phosphorylated amino acids are still scarce. Most attempts made at these parameters, were based on RESP calculations on the ESP generated from QM calculations [1]. The phosphate group is pH-active, with the second a pKa value around 6, which is problematic to be studied using conventional MD simulations. With constant-pH MD (CpHMD) simulations [2], we can capture the coupling between protonation and conformation for the phosphate group in their different environments.

We have devised several strategies to obtain the GROMOS 54A7 charge parameters for phosphoryl tyrosine (pTyr), serine (pSet), and threonine (pThr), namely: RESP calculations performed on the ESP generated either by Hartree-Fock or DFT calculations; directly adapting the charge set from Wojciechowski et al. [1]; and by manually curating the best charge set obtained taking in consideration the experimental data available. With all charge sets it was possible to run CpHMD simulations on simple pentapeptides to calibrate the pKa value against experimental data. We tested the parameters with simple systems like the Gly-Gly-X-Ala tetrapeptides and with more complex systems, like the phosphorylated dodecapeptide, free or complexed with a phospholipase [3]. We observed that for the simple systems, all charge sets, after calibration, are able to predict their unshifted pKa values. However, all recipe-based charge parameterization processes, failed (sometimes even qualitatively) in predicting the pKa shift for the complex, which could only be corrected by manually curating the charges.

[1] Wojciechowski, M., Grycuk, T., Antosiewicz, J.M., Lesyng, B., Biophys. J., 2003, 84, 750-6.

[2] Machuqueiro, M., Baptista, A. M., J. Phys Chem. B, 2006, 110, 2927-2933.

[3] Singer, A.U., Forman-Kay, J.D., Prot. Sci., 1997, 6, 1910-9.

β-lactamases are a primary cause of bacterial resistance to β-lactam antibiotics for many important human pathogens (particularly Gram-negative bacteria) [1]. Inhibitors of β-lactamase have been implemented as a dual therapy with antibiotics, but there are only four inhibitors clinically approved and resistance to these compounds is also rising [2]. For β-lactam inhibitors, after acylation, the opening of five-membered ring leads to the formation of a transient imine intermediate then it rearranges several times to form a trans or cis final enamine inhibition-products. Slow hydrolysis of this product by the enzyme leads to an inhibited β-lactamase [2]. A computational study of reaction mechanism for the first step on the deacylation of the inhibitor clavulanate with TEM-1 (inhibited) and KPC-2 (hydrolyzed) enzymes using QM/MM Umbrella Sampling with DFTB method is presented [3]. 2D free energies surfaces for the reactions were calculated using the weighted histogram analysis method (WHAM) and the minimum energy path (MEP) was identified; where the highest point along the MEP is taken as the transition state giving the activation free energy “ΔG_calc”.Our results show that TEM-1 and KPC-2 have an approximate 5 kcal/mol difference in ΔG_calc. Such results are in good agreement with inhibition experimental data for two enzymes in which KPC-2 is less inhibited by clavulanate than TEM-1. We hope our methodology can assist the design and development of covalent inhibitors through a computational screening of inhibitory activity of other molecules.

References:

[1] Hermann, J. C., Pradon, J., Harvey, J. N., & Mulholland, A. J. J. Phys. Chem. A (2009), 113(43), 11984–11994.

[2] Drawz, S. M., Papp-Wallace, K. M., & Bonomo, R. a. Antimicrob. Agents Chemother (2014), 58(4), 1835–1846.

[3] Chudyk, E. I., Limb, M. a L., Jones, C., Spencer, J., van der Kamp, M. W., & Mulholland, A. J. Chem Commun, (2014), 50, 14736–14739.

Acknowledgements:

R.F thanks the Royal Society of Chemistry (RSC) for financial support through Researcher Mobility Grant (R. Fritz 16 Round 1). JAM and RF thank financial support through project FONDECYT No. 1140618.

The intent of present study is to develop the Isradipine controlled release tablets through compression coating of mini-tablets with the help of hydrophilic and hydrophobic polymers. Isradipine mini-tablets were prepared by direct compression method and compression coated using various concentrations of HPMC K15M, Ethyl cellulose and combination of Ethyl cellulose and HPMC K15M. The prepared tablets were characterized for weight variation, hardness, thickness, friability, drug content and swelling studies. Formulations were evaluated for the release of Isradipine over a period of 12 h using type-II USP XXIV standard dissolution apparatus in 6.8 pH phosphate buffer. From the in vitro drug release studies, F5 tablets showed 99.43±0.72% % drug release in 12 h and it followed zero order drug release. The mean dissolution time of all formulations was found to be 4.48 – 10.52 h and it was higher for formulations with ethyl cellulose when compared to HPMC K 15M due to its hydrophobic nature. Time in hours to take 80% drug release explained the ability of prolonged release and they were found to be 10.2 h for best formulation F5. From the stability study, similarity factor (f2) was found as 80.48, which is more than 50 indicates similarity between the dissolution profile before and after storage. Hence the development of Isradipine compression coated mini-tablets is a promising way to control the drug release as per therapeutic requirement.

The identification of folding and aggregation intermediate states is important, both from a fundamental standpoint and for the design of new therapies for conformational disorders. Here, we use the single point mutant (D76N) of β2m, the causing agent of a hereditary systemic amyloidosis affecting visceral organs, as a model system to study the aggregation mechanism of β2m using molecular simulations. We present our predictions on the early molecular events triggering the amyloid cascade for the D76N mutant. Folding simulations highlight the existence of an aggregation-prone intermediate called I1 which presents an unstructured C-terminus and of an aggregation-prone intermediate featuring two unstructured termini called I2. Additionally, Monte Carlo docking simulations suggest that both intermediates have high aggregation-propensity. These simulations support an essential role of the termini and of the DE and EF-loops in the dimerization of both intermediates. The relevance of the C-terminus is higher at the acidic pH 5.2 while the N-terminus become more important at pH 6.2. At physiological pH, the DE and EF-loops are the most important regions for dimerization. These predictions rationalize experimental results that support the involvement of Lys-19, Phe-56, Trp-60 and Phe-62 in amyloidogenesis in the wild-type and other model systems of β2m.

Human fatty acid synthase (hFAS) is a multidomain enzyme that catalyzes all steps of fatty acid biosynthesis [1], which is deregulated in many varieties of human cancers. Studies have shown that FAS inhibitors exhibit anticancer activity without relevant side-effects over healthy cells [2]. Thus, the molecular characterization of all hFAS domains is an important goal for the development of novel anticancer therapies. The malonyl-acetyl transferase (MAT) domain loads acetyl and malonyl moieties to the phosphopantetheine arm of the acyl-carrier protein (ACP) domain, a carrier for reaction intermediates [3]. In this study, we have employed computational hybrid QM/MM methods at the DLPNO−CCSD(T)/CBS:AMBER level of theory [4] to study the MAT catalytic mechanism. The results indicate that the transfer of acyl moieties from CoA to MAT occurs in two catalytic steps: (1) concerted nucleophilic attack on the thioester carbonyl group of the substrate, centered on a Ser-His dyad and (2) tetrahedral intermediate breakdown. The Gibbs activation barrier of the first step is 13.0 kcal·mol−1 for the MAT-acetyl-CoA complex, and 10.9 kcal·mol−1 for the MAT-malonyl-CoA system. As for the second catalytic step, Gibbs energy barriers of 6.4 kcal·mol−1 and 8.0 kcal·mol−1 were obtained for the MAT-acetyl-CoA and MAT-malonyl-CoA complexes, respectively. Two hydrophobic residues, Phe553 and Phe682, are responsible for the positioning of the acetyl moiety in the active site. Additionally, persistent ionic interactions between Arg606 and the carboxylate anion of the malonyl moiety maintain the substrate in a catalysis-favorable position, which corroborates previous experimental findings [5]. The backbone amines of Met499 and Leu582 form an oxyanion hole that accommodates the negative charge of the substrate carbonyl, reducing the activation barrier of the first step for both substrates. The results from this work instigate future studies that aim for the full understanding of MAT’s catalytic machinery and that explore the therapeutic potential of hFAS.

REFERENCES:
[1] Smith, S.; Tsai, S. C. The Type I Fatty Acid and Polyketide Synthases: A Tale of Two Megasynthases. Nat. Prod. Rep. 2007, 24, 1041−72.
[2] Pandey, P. R.; Liu, W.; Xing, F.; Fukuda, K.; Watabe, K. AntiCancer Drugs Targeting Fatty Acid Synthase (Fas). Recent Pat. AntiCancer Drug Discovery 2012, 7, 185−197.
[3] Maier, T.; Leibundgut, M.; Ban, N. The Crystal Structure of a Mammalian Fatty Acid Synthase. Science 2008, 321, 1315−22.
[4] Riplinger, C.; Neese, F. An Efficient and near Linear Scaling Pair Natural Orbital Based Local Coupled Cluster Method. J. Chem. Phys. 2013, 138, 034106.
[5] Rangan, V. S.; Smith, S., Alteration of the Substrate Specificity of the Malonyl-Coa/Acetyl-Coa:Acyl Carrier Protein S-Acyltransferase Domain of the Multifunctional Fatty Acid Synthase by Mutation of a Single Arginine Residue. J. Biol. Chem. 1997, 272, 11975-8.

It is known that fossil fuel combustion is one of the main environmental problems of the modern era, and the sulfur content of crude oil [1] contributes heavily to this. One of the main sulphurous compounds present in crude oil is dibenzothiophene (DBT). Due to its harmfulness, several governments around the world have been imposing stricter restrictions regarding the sulfur content in fossil fuels. The desulfurization of crude oil is currently carried out using costly chemical processes. One alternative to these costly chemical processes involves the use of specific microorganisms, such as Rhodococcus erythropolis, capable of utilizing DBT as a sole source of sulfur. The process carried out by R. erytrhopolis is called the 4S pathway and is conducted by the action of four enzymes of the dibenzothiophene desulfurization enzymes (dsz) family. DszA, DszB, DszC and DszD. The major limitation of this pathway is the slow catalytic rates of the four enzymes, which limits its application in industry. The enhancement of the catalytic power of enzymes is a subject of enormous interest both for science and for industry. The latter, in particular, due to the vast applications enzymes can have in industrial processes. In this work, we sought to enhance the turnover rate of DszD from Rhodococcus erythropolis, a NADH-FMN oxidoreductase responsible to supply FMNH₂ to DszA and DszC in the biodesulfurization process of crude oil, the 4S pathway. For that purpose, we replaced the wild type spectator residue of the rate-limiting step of the reduction of FMN to FMNH₂ catalysed by DszD, known to have an important role in its energetic profile, with all the natural occurring amino acids, one at a time, using computational methodologies, and repeated the above-mentioned reaction with each mutant. To calculate the different free energy profiles, one for each mutated model, we applied quantum mechanical methods (namely DFT) within an ONIOM scheme. The free energy barriers obtained for the different mutated models varied between 15.1 kcal.mol^-1 and 29.9 kcal.mol^-1. Multiple factors contributed to the different ΔGs. The most relevant were electrostatic interactions and the induction of a favourable alignment between substrate and cofactor. These results confirm the great potential that chirurgic mutations have to increase the catalytic power of DszD in relation to the wt enzyme.

[1] N. Kamali, M. Tavallaie, B. Bambai, A. A. Karkhane and M. Miri, Biotechnol Lett, 2010, 32, 921-927.

Tryptophan Synthase (TSase) is a bifunctional enzyme that catalyzes the last two steps in the synthesis of tryptophan (TRP). Each reaction is catalyzed in different active sites that are located in separate α and β subunits. The active site of the α-subunit catalyzes the formation of indole and gliceraldeyde-3-phosphate (G3P) from indole 3-glycerolphosphate (IGP). Indole is then transported through a 25 ≈ physical tunnel to the active site of the β-subunit where it is added to a molecule of acrylate, derived from serine, to produce TRP, in a PLP dependent reaction¹.

Since TSase is absent in mammals, it is a promising target for the development of new antibiotics and vaccines against infectious bacteria, such as Mycobacterium tuberculosis.

The complex allosteric regulation of the enzyme has turn it difficult to co-crystalize the enzyme in its closed conformation with both substrates correctly placed in the α and β-active sites. In this work, we modulated three enzyme models for the posterior construction of QM/MM models: Model 1 (α-IGP and β-Ain); Model 2 (β-Aex-Ser); Model 3 (β-Q2). All the models were based on the crystallographic structure with PDB ID: 3PR2 and the ligands were either obtained from other crystallographic structures (PDB ID:1QOQ) or modulated from the analogs. Each of the three models were emerged in a box of waters and subjected to a MD simulation of 30 ns for detailed analysis and sampling of the interactions formed. The RMSd analysis of the last 20 ns of the three MD simulations did not evidence any abnormal fluctuation, and the equilibrated region presents a low RMSd average value of respectively 2.90 ± 0.17 Å for MD1; 2.64 ± 0.12 Å for MD2 and 2.37± 0.10 Å for MD3. We concluded that all the models are stable and can be the basis for further studies.

Afterwards four ONIOM QM/MM hybrid model were built for geometry optimization, validation of the initial enzyme-ligand interaction, and posterior study of the catalytic mechanism of both α and β subunits of the enzyme.

Acknowledgments:

This work was supported by national funds from Fundação para a Ciência e a Tecnologia (SFRH/BD/114886/2016, IF/01310/2013, IF/00052/2014, and PTDC/QUI-QFI/31689/2017) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728). We acknowledge the use of HPC facilities (QTREX) provided by REQUIMTE where these calculations were performed.

Bibliography:

1 Dunn, M. F., Niks, D., Ngo, H., Barends, T. R. & Schlichting, I. Tryptophan synthase: the workings of a channeling nanomachine. Trends in biochemical sciences 33, 254-264, doi:10.1016/j.tibs.2008.04.008 (2008).