In silico and in vitro approaches to develop Dimethylarginine dimethylaminohydrolase-1 inhibitors

Introduction: Dimethylarginine dimethylaminohydrolases (DDAH) metabolise the endogenous nitric oxide synthase (NOS) inhibitors: asymmetric dimethylarginine (ADMA) and monomethylarginine1. In sepsis excessive nitric oxide partially contributes to acute circulatory failure, and pharmacological DDAH1 inhibition has been proposed in order to increase methylarginines and reduce NO levels 2. The SR257 arginine analogue, with NG-methoxyethyl substituent, inhibits DDAH1 with an IC50 22 µM without directly inhibiting NOSs1,3. Methods: Acyclic and cyclic NG,NG-disubstituted arginines were made as previously described4 using Katritzky’s synthesis preparing trisubstituted guanidines from di-(benzotriazol-1-yl)methanimine5. Molecular docking was employed to explore interactions of these NG,NG-disubstituted arginines with human DDAH1 (PDB 2JAJ) using Glide (Schroedinger6) and Autodock47. The published SR257 ligand was used to define the binding site with both software tools. Recombinant human DDAH1 activity was measured using colorometric citrulline assay8 containing ADMA (100 µM), sodium phosphate (10 mM pH7.4); with symmetric dimethylarginine (100 µM), not a substrate for DDAH1, as blank. Experiments were carried out in duplicate, and repeated on at least 3 separate occasions. Results: Recombinant DDAH1 activity was reduced to less than 25% of control (ADMA substrate, 100 µM) in the presence of 100 µM piperidinyl, methoxyethyl/methyl, N-methylpiperazinyl, with morpholinyl and pyrrolidinyl substituents reducing activity to less than 10% of control. The in silico Glide docking score and predicted Autodock4 binding energy for human DDAH1 (PDB, 2JAJ) for the known SR257 DDAH1 inhibitor and NG,NG-disubstituted arginines are shown in the table: Conclusion: Both Autodock4 and Glide docking predicted higher binding energies for morpholinyl, pyrrolidinyl and piperinyl than the known SR257 compound. In vitro assays confirmed these NG,NG-disubstituted arginines reduced DDAH1 activity. There was variation between Glide and Autodock4 in the docking predictions for methoxyethyl/methyl and N-methylpiperazinyl. In silico prediction of DDAH1-ligand interactions may assist in the future design and development of novel NG,NG-disubstituted arginines. References: 1 Leiper, J. et al. (2007) Nat Med. 13:198-203. 2 Wang, Z et al. (2014) Biochem J. 460:309 3 Rossiter, S. et al. (2005) J Med Chem. 48:4670-4678. 4 Morfill, C et al. (2012) http://www.pA2online.org/abstracts/Vol10Issue4abst197P.pdf 5 Katritzky, A et al. (2000) J. Org. Chem. 65: 8080-8082. 6 Friesner, RA et al. (2006) J Med Chem. 49:6177-6196. 7 Morris, GM et al. (2009) J. Comp. Chem. 16:2785-91. 8 Knipp, M & Vasak, M (2000) Anal Biochem 286:257-64

Deep learning for novel antimicrobial peptide design

Antimicrobial resistance is an increasing issue in healthcare as the overuse of antibacterial agents rises during the COVID-19 pandemic. The need for new antibiotics is high, while the arsenal of available agents is decreasing, especially for the treatment of infections by Gram-negative bacteria like Escherichia coli. Antimicrobial peptides (AMPs) are offering a promising route for novel antibiotic development and deep learning techniques can be utilised for successful AMP design. In this study, a long short-term memory (LSTM) generative model and a bidirectional LSTM classification model were constructed to design short novel AMP sequences with potential antibacterial activity against E. coli. Two versions of the generative model and six versions of the classification model were trained and optimised using Bayesian hyperparameter optimisation. These models were used to generate sets of short novel sequences that were classified as antimicrobial or non-antimicrobial. The validation accuracies of the classification models were 81.6–88.9% and the novel AMPs were classified as antimicrobial with accuracies of 70.6–91.7%. Predicted three-dimensional conformations of selected short AMPs exhibited the alpha-helical structure with amphipathic surfaces. This demonstrates that LSTMs are effective tools for generating novel AMPs against targeted bacteria and could be utilised in the search for new antibiotics leads

Novel C-3-(N-alkyl-aryl)-aminomethyl rifamycin SV derivatives exhibit activity against rifampicin-resistant Mycobacterium tuberculosis RpoBS522L strain and display a different binding mode at the RNAP β-subunit site compared to rifampicin

Antimicrobial resistance is a main concern in tuberculosis treatment and is often associated with the emergence of Mycobacterium tuberculosis strains resistant to rifampicin (RIF), which is one of the cornerstones of tuberculosis chemotherapy. In this study, aminoalkyl-aromatic ring tails were appended to the C3 position of rifamycin core to assess the role of C3 substitutions to the anti-mycobacterial activity of the rifamycin antibiotics. The typical hydrazone unit of RIF was replaced by an amino-alkyl linkage to connect the aromatic ring tails with the rifamycin naphthoquinone core. Eight novel C3-(N-alkyl-aryl)-aminoalkyl analogues of rifamycin SV were synthesised and screened in vitro against wild-type HR37Rv and “hypervirulent” HN-878 strains, and a panel of rifampicin-resistant M. tuberculosis clinical isolates carrying mutations at the 522, 531 and 455 positions of the rpoB gene (RpoBS522L, RpoBS531L and RpoBH455D strains). The analogues exhibited anti-tubercular activity against H37Rv and HN-878 at submicromolar or nanomolar concentrations, and against clinical H37Rv isolates bearing the S522L mutations at low micromolar concentration. Benzylamine moiety-including analogue 8 was as active as rifampicin against HN-878 with a MIC90 value of 0.02 μM, whereas 14 and 15, which included tryptamine and para-methyl-sulfonylbenzylamine C3-substituents, respectively, showed higher anti-tubercular activity (MIC90 = 3 μM) compared to rifampicin against the S522L mutated H37Rv strain. Detailed in silico analysis of different RNAP molecular systems predicted a distinct, possibly novel, binding mode for the new rifamycin analogues. These were found to occupy a different space in the binding pockets of both wild type and mutated RNAP proteins compared to that of rifampicin. Moreover, the molecular modelling experiments investigated the ability of the novel analogues aromatic tails to establish key interactions at the RNAP binding site. These interesting findings might pave the way for generating rifamycin analogues that can overcome anti-microbial resistance in M. tuberculosis

DFT study of the radical scavenging activity of isoxanthohumol, humulones (α-acids), and iso-α-acids from beer

Humulones and iso-humulones are potent natural antioxidants found in beer. In this study, density functional theory (DFT) method was applied for elucidating the structure-antioxidant activity relationship and molecular mechanism of antioxidant activity of eight bioactive humulones previously identified in different beer samples: isoxanthohumol, (R)- and (S)-adhumulone, cis- and trans-iso-adhumulone, cis- and trans-iso-n-humulone, and desdimethyl-octahydro-iso-cohumulone. The calculated bond dissociation enthalpies (BDEs) suggest that desdimethyl-octahydro-iso-cohumulone was the most potent compound with BDEs 5.1 and 23.9 kJ/mol lower compared to the values for resveratrol in gas phase and water, respectively. The enolic –OH is the most reactive site for hydrogen atom transfer (HAT). The presence of β-keto group with respect to enolic –OH diminishes the HAT potency via the formation of a strong intramolecular hydrogen bond. Another common antioxidant mechanism, single electron transfer followed by proton transfer (SET-PT), is only feasible for isoxanthohumol. The results of this study indicate a strong correlation between the increased antioxidant activity of beer products and the higher content of reduced iso-α-acids

An Evaluation of the Potential of NMR Spectroscopy and Computational Modelling Methods to Inform Biopharmaceutical Formulations

Protein-based therapeutics are considered to be one of the most important classes of pharmaceuticals on the market. The growing need to prolong stability of high protein concentrations in liquid form has proven to be challenging. Therefore, significant effort is being made to design formulations which can enable the storage of these highly concentrated protein therapies for up to 2 years. Currently, the excipient selection approach involves empirical high-throughput screening, but does not reveal details on aggregation mechanisms or the molecular-level effects of the formulations under storage conditions. Computational modelling approaches have the potential to elucidate such mechanisms, and rapidly screen in silico prior to experimental testing. Nuclear Magnetic Resonance (NMR) spectroscopy can also provide complementary insights into excipient–protein interactions. This review will highlight the underpinning principles of molecular modelling and NMR spectroscopy. It will also discuss the advancements in the applications of computational and NMR approaches in investigating excipient–protein interactions

Synthesis and antitubercular activity of novel 4-arylalkyl substituted thio-, oxy- and sulfoxy-quinoline analogues targeting the cytochrome bc1 complex

A library of 4-substituted quinolines was synthesised based on the structural features of the privileged 4-(benzylthio)-6-methoxy-2-methylquinoline scaffold. Quinoline-based chemical probes have proven to be effective anti-tuberculosis agents with the ability of inhibiting components of Mycobacterium tuberculosis (MTB) respiratory chain including the b subunit of the cytochrome bc1 complex. Novel 4-(arylalkyl)-thio, -oxy and sulfoxy-quinoline analogues were tested for their ability to inhibit the growth of MTB H37Rv and QcrB mutant strains, and the compounds mode of action was investigated. Members of the 4-subtituted thio- and sulfoxyquinoline series exhibited significant growth inhibitory activity in the high nanomolar range against wild-type MTB and induced depletion of intracellular ATP. These probes also showed reduced potency in the QcrB T313I mutant strain, thus indicating the cytochrome bc1 oxidase complex as the molecular target. Interestingly, new 4-(quinolin-2-yl)oxy-quinoline 4i was more selective for the QcrB T313I strain compared to the wild-type strain

An Evaluation of the Potential of NMR Spectroscopy and Computational Modelling Methods to Inform Biopharmaceutical Formulations

Protein-based therapeutics are considered to be one of the most important classes of pharmaceuticals on the market. The growing need to prolong stability of high protein concentrations in liquid form has proven to be challenging. Therefore, significant effort is being made to design formulations which can enable the storage of these highly concentrated protein therapies for up to 2 years. Currently, the excipient selection approach involves empirical high-throughput screening, but does not reveal details on aggregation mechanisms or the molecular-level effects of the formulations under storage conditions. Computational modelling approaches have the potential to elucidate such mechanisms, and rapidly screen in silico prior to experimental testing. Nuclear Magnetic Resonance (NMR) spectroscopy can also provide complementary insights into excipient⁻protein interactions. This review will highlight the underpinning principles of molecular modelling and NMR spectroscopy. It will also discuss the advancements in the applications of computational and NMR approaches in investigating excipient⁻protein interactions

VEGFA, B, C: Implications of the C-Terminal Sequence Variations for the Interaction with Neuropilins

Vascular endothelial growth factors (VEGFs) are the key regulators of blood and lymphatic vessels’ formation and function. Each of the proteins from the homologous family VEGFA, VEGFB, VEGFC and VEGFD employs a core cysteine-knot structural domain for the specific interaction with one or more of the cognate tyrosine kinase receptors. Additional diversity is exhibited by the involvement of neuropilins–transmembrane co-receptors, whose b1 domain contains the binding site for the C-terminal sequence of VEGFs. Although all relevant isoforms of VEGFs that interact with neuropilins contain the required C-terminal Arg residue, there is selectivity of neuropilins and VEGF receptors for the VEGF proteins, which is reflected in the physiological roles that they mediate. To decipher the contribution made by the C-terminal sequences of the individual VEGF proteins to that functional differentiation, we determined structures of molecular complexes of neuropilins and VEGFderived peptides and examined binding interactions for all neuropilin-VEGF pairs experimentally and computationally. While X-ray crystal structures and ligand-binding experiments highlighted similarities between the ligands, the molecular dynamics simulations uncovered conformational preferences of VEGF-derived peptides beyond the C-terminal arginine that contribute to the ligand selectivity of neuropilins. The implications for the design of the selective antagonists of neuropilins’ functions are discussed

A phytochemical comparison of saw palmetto products using gas chromatography and H nuclear magnetic resonance spectroscopy metabolomic profiling

Objectives: Preparations containing saw palmetto berries are used in the treatment of benign prostatic hyperplasia (BPH). There are many products on the market, and relatively little is known about their chemical variability and specifically the composition and quality of different saw palmetto products notwithstanding that in 2000, an international consultation paper from the major urological associations from the five continents on treatments for BPH demanded further research on this topic. Here, we compare two analytical approaches and characterise 57 different saw palmetto products. Methods: An established method - gas chromatography - was used for the quantification of nine fatty acids, while a novel approach of metabolomic profiling using H nuclear magnetic resonance (NMR) spectroscopy was used as a fingerprinting tool to assess the overall composition of the extracts. Key findings: The phytochemical analysis determining the fatty acids showed a high level of heterogeneity of the different products in the total amount and of nine single fatty acids. A robust and reproducible H NMR spectroscopy method was established, and the results showed that it was possible to statistically differentiate between saw palmetto products that had been extracted under different conditions but not between products that used a similar extraction method. Principal component analysis was able to determine those products that had significantly different metabolites. Conclusions: The metabolomic approach developed offers novel opportunities for quality control along the value chain of saw palmetto and needs to be followed further, as with this method, the complexity of a herbal extract can be better assessed than with the analysis of a single group of constituents. © 2014 The Authors

Poly-glutamic dendrimer-based conjugates for cancer vaccination - a computational design for targeted delivery of antigens

© 2017 Taylor & Francis. This is an accepted manuscript of an article published by Taylor & Francis in Journal of Drug Targeting on 10 Aug 2017, available online: https://doi.org/10.1080/1061186X.2017.1363213.Computational techniques are useful to predict interaction models and molecular properties for the design of drug delivery systems, such as dendrimers. Dendrimers are hyperbranched macromolecules with repetitive building blocks, defined architecture and functionality. This work evaluated the impact of surface modifications of mannosamine-conjugated multifunctional poly (glutamic acid) (PG)-dendrimers as nanocarriers of the tumor associated antigens (TAA) MART-1, gp100:44 and gp100:209. Their potential to target antigen presenting cells through molecular interactions with mannose receptor (MR1) to promote an efficient and selective antitumor immunotherapeutic effect was also evaluated. Molecular dynamics (MD) simulations and docking studies were performed. Results showed that nitrobenzoxadiazole (NBD)-PG-G4-dendrimer displayed 64 carboxylic groups, however the frontier molecular orbital theory study has indicated that only 32 of those carboxylic groups present on the backbone were available to form covalent bonds with either mannosamine or TAA. No differences in the gap energy of HOMO of conjugated NBD-PG-G4-dendrimer and LUMO of conjugating agents were observed while increasing conjugation loading. When the number of mannosamines conjugated to dendrimer was increased from 16 to 32, the conjugated dendrimer interacted with the receptor with higher affinity. However, due to absence of available carboxylic end groups of backbone chain for further conjugation in a dendrimer with 32 mannosamine, the 16 mannosamines-NBD-PG-G4-dendrimer was chosen to conjugate TAA for added functionality. Docking results showed that the majority of TAA-conjugated NBD-PG-G4-dendrimer demonstrated a favorable interaction with mannosamine binding site on MR1, thus constituting a promising tool for the targeted delivery of TAA. Our in silico approach effectively narrows down the selection of the best candidates for the synthesis of functionalized PG-dendrimers with desired functionalities. The results of this study will significantly reduce the time and efforts required to experimentally obtain modified dendrimers for optimal controlled delivery of antigens to targeted DC.Peer reviewedFinal Accepted Versio