KNeMAP: a network mapping approach for knowledge-driven comparison of transcriptomic profiles.

MOTIVATION: Transcriptomic data can be used to describe the mechanism of action (MOA) of a chemical compound. However, omics data tend to be complex and prone to noise, making the comparison of different datasets challenging. Often, transcriptomic profiles are compared at the level of individual gene expression values, or sets of differentially expressed genes. Such approaches can suffer from underlying technical and biological variance, such as the biological system exposed on or the machine/method used to measure gene expression data, technical errors and further neglect the relationships between the genes. We propose a network mapping approach for knowledge-driven comparison of transcriptomic profiles (KNeMAP), which combines genes into similarity groups based on multiple levels of prior information, hence adding a higher-level view onto the individual gene view. When comparing KNeMAP with fold change (expression) based and deregulated gene set-based methods, KNeMAP was able to group compounds with higher accuracy with respect to prior information as well as is less prone to noise corrupted data. RESULT: We applied KNeMAP to analyze the Connectivity Map dataset, where the gene expression changes of three cell lines were analyzed after treatment with 676 drugs as well as the Fortino et al. dataset where two cell lines with 31 nanomaterials were analyzed. Although the expression profiles across the biological systems are highly different, KNeMAP was able to identify sets of compounds that induce similar molecular responses when exposed on the same biological system. AVAILABILITY AND IMPLEMENTATION: Relevant data and the KNeMAP function is available at: https://github.com/fhaive/KNeMAP and 10.5281/zenodo.7334711.

Azulene as a biphenyl mimetic in orexin/hypocretin receptor agonists.

Azulene is a rare ring structure in drugs, and we investigated whether it could be used as a biphenyl mimetic in known orexin receptor agonist Nag 26, which is binding to both orexin receptors OX1 and OX2 with preference towards OX2. The most potent azulene-based compound was identified as an OX1 orexin receptor agonist (pEC50 = 5.79 ± 0.07, maximum response = 81 ± 8% (s.e.m. of five independent experiments) of the maximum response to orexin-A in Ca2+ elevation assay). However, the azulene ring and the biphenyl scaffold are not identical in their spatial shape and electron distribution, and their derivatives may adopt different binding modes in the binding site.

Macrodomain Binding Compound MRS 2578 Inhibits Alphavirus Replication.

Alphaviruses are positive-strand RNA viruses causing febrile disease. Macrodomain-containing proteins, involved in ADP-ribose-mediated signaling, are encoded by both host cells and several virus groups, including alphaviruses. In this study, compound MRS 2578 that targets the human ADP-ribose glycohydrolase MacroD1 inhibited Semliki Forest virus production as well as viral RNA replication and replicase protein expression. The inhibitor was similarly active in alphavirus trans-replication systems, indicating that it targets the viral RNA replication stage.

Modeling membrane proteins: The importance of cysteine amino-acids.

Computational modeling of membrane proteins is critical to understand biochemical systems and to support chemical biology. In this work, we use a dataset of 448 non-redundant membrane protein chains to expose a "rule" that governs membrane protein structure: free cysteine thiols are not found accessible to oxidative compartments such as the extracellular space, but are rather involved in disulphide bridges. Taking as examples the 1018 three-dimensional models produced during the GPCR Dock 2008, 2010 and 2013 competitions and 390 models for a GPCR target in CASP13, we show that this rule was not accounted for by the modeling community. We thus highlight a new direction for model development that should lead to more accurate membrane protein models, especially in the loop domains.

Binding Site Interactions of Modulators of Breast Cancer Resistance Protein, Multidrug Resistance-Associated Protein 2, and P-Glycoprotein Activity.

ATP-binding cassette (ABC)-transporters protect tissues by pumping their substrates out of the cells in many physiological barriers, such as the blood-brain barrier, intestine, liver, and kidney. These substrates include various endogenous metabolites, but, in addition, ABC transporters recognize a wide range of compounds, therefore affecting the disposition and elimination of clinically used drugs and their metabolites. Although numerous ABC-transporter inhibitors are known, the underlying mechanism of inhibition is not well characterized. The aim of this study is to deepen our understanding of transporter inhibition by studying the molecular basis of ligand recognition. In the current work, we compared the effect of 44 compounds on the active transport mediated by three ABC transporters: breast cancer resistance protein (BCRP and ABCG2), multidrug-resistance associated protein (MRP2 and ABCC2), and P-glycoprotein (P-gp and ABCB1). Eight compounds were strong inhibitors of all three transporters, while the activity of 36 compounds was transporter-specific. Of the tested compounds, 39, 25, and 11 were considered as strong inhibitors, while 1, 4, and 11 compounds were inactive against BCRP, MRP2, and P-gp, respectively. In addition, six transport-enhancing stimulators were observed for P-gp. In order to understand the observed selectivity, we compared the surface properties of binding cavities in the transporters and performed structure-activity analysis and computational docking of the compounds to known binding sites in the transmembrane domains and nucleotide-binding domains. Based on the results, the studied compounds are more likely to interact with the transmembrane domain than the nucleotide-binding domain. Additionally, the surface properties of the substrate binding site in the transmembrane domains of the three transporters were in line with the observed selectivity. Because of the high activity toward BCRP, we lacked the dynamic range needed to draw conclusions on favorable interactions; however, we identified amino acids in both P-gp and MRP2 that appear to be important for ligand recognition.

Adenosine analogs bearing phosphate isosteres as human MDO1 ligands.

The human O-acetyl-ADP-ribose deacetylase MDO1 is a mono-ADP-ribosylhydrolase involved in the reversal of post-translational modifications. Until now MDO1 has been poorly characterized, partly since no ligand is known besides adenosine nucleotides. Here, we synthesized thirteen compounds retaining the adenosine moiety and bearing bioisosteric replacements of the phosphate at the ribose 5'-oxygen. These compounds are composed of either a squaryldiamide or an amide group as the bioisosteric replacement and/or as a linker. To these groups a variety of substituents were attached such as phenyl, benzyl, pyridyl, carboxyl, hydroxy and tetrazolyl. Biochemical evaluation showed that two compounds, one from both series, inhibited ADP-ribosyl hydrolysis mediated by MDO1 in high concentrations.

Structural Isosteres of Phosphate Groups in the Protein Data Bank.

We developed a computational workflow to mine the Protein Data Bank for isosteric replacements that exist in different binding site environments but have not necessarily been identified and exploited in compound design. Taking phosphate groups as examples, the workflow was used to construct 157 data sets, each composed of a reference protein complexed with AMP, ADP, ATP, or pyrophosphate as well other ligands. Phosphate binding sites appear to have a high hydration content and large size, resulting in U-shaped bioactive conformations recurrently found across unrelated protein families. A total of 16 413 replacements were extracted, filtered for a significant structural overlap on phosphate groups, and sorted according to their SMILES codes. In addition to the classical isosteres of phosphate, such as carboxylate, sulfone, or sulfonamide, unexpected replacements that do not conserve charge or polarity, such as aryl, aliphatic, or positively charged groups, were found.

[Pyrophosphate in medicine]. / Pyrofosfaatti lääketieteessä.

In all organisms from bacteria to humans, specific hydrolases--pyrophosphatases--hydrolyse inorganic pyrophosphate to phosphate. Without this, DNA, RNA and protein synthesis stops. Pyrophosphatases are thus essential for all life. In humans, disorders in pyrophosphate metabolism cause chondrocalcinosis and hypophosphatasia. Currently, pyrophosphate analogues, e.g. alendronate, are in clinical use in osteoporosis and Paget's disease but also for e.g. complications of prostate cancer. In bacteria and protozoan parasites, membrane-bound pyrophosphatases (mPPases), which do not occur in humans, convert pyrophosphate to a proton or sodium gradient. mPPases, which are crucial for protozoan parasites, are thus promising drug targets e.g. for malaria and leishmaniasis.

Modeling of the OX1R-orexin-A complex suggests two alternative binding modes.

BACKGROUND: Interactions between the orexin peptides and their cognate OX1 and OX2 receptors remain poorly characterized. Site-directed mutagenesis studies on orexin peptides and receptors have indicated amino acids important for ligand binding and receptor activation. However, a better understanding of specific pairwise interactions would benefit small molecule discovery. RESULTS: We constructed a set of three-dimensional models of the orexin 1 receptor based on the 3D-structures of the orexin 2 receptor (released while this manuscript was under review), neurotensin receptor 1 and chemokine receptor CXCR4, conducted an exhaustive docking of orexin-A16-33 peptide fragment with ZDOCK and RDOCK, and analyzed a total of 4301 complexes through multidimensional scaling and clustering. The best docking poses reveal two alternative binding modes, where the C-terminus of the peptide lies deep in the binding pocket, on average about 5-6 Å above Tyr(6.48) and close to Gln(3.32). The binding modes differ in the about 100° rotation of the peptide; the peptide His26 faces either the receptor's fifth transmembrane helix or the seventh helix. Both binding modes are well in line with previous mutation studies and partake in hydrogen bonding similar to suvorexant. CONCLUSIONS: We present two binding modes for orexin-A into orexin 1 receptor, which help rationalize previous results from site-directed mutagenesis studies. The binding modes should serve small molecule discovery, and offer insights into the mechanism of receptor activation.

PockDrug: A Model for Predicting Pocket Druggability That Overcomes Pocket Estimation Uncertainties.

Predicting protein druggability is a key interest in the target identification phase of drug discovery. Here, we assess the pocket estimation methods' influence on druggability predictions by comparing statistical models constructed from pockets estimated using different pocket estimation methods: a proximity of either 4 or 5.5 Å to a cocrystallized ligand or DoGSite and fpocket estimation methods. We developed PockDrug, a robust pocket druggability model that copes with uncertainties in pocket boundaries. It is based on a linear discriminant analysis from a pool of 52 descriptors combined with a selection of the most stable and efficient models using different pocket estimation methods. PockDrug retains the best combinations of three pocket properties which impact druggability: geometry, hydrophobicity, and aromaticity. It results in an average accuracy of 87.9% ± 4.7% using a test set and exhibits higher accuracy (∼5-10%) than previous studies that used an identical apo set. In conclusion, this study confirms the influence of pocket estimation on pocket druggability prediction and proposes PockDrug as a new model that overcomes pocket estimation variability.

Exploring the structure-activity relationships of ABCC2 modulators using a screening approach.

ABCC2 is a transporter with key influence on liver and kidney pharmacokinetics. In order to explore the structure-activity relationships of compounds that modulate ABCC2, and by doing so gain insights into drug-drug interactions, we screened a library of 432 compounds for modulators of radiolabeled ß-estradiol 17-(ß-d-glucuronide) (EG) and fluorescent 5(6)-carboxy-2',7'-dichlorofluorescein transport (CDCF) in membrane vesicles. Following the primary screen at 80µM, dose-response curves were used to investigate in detail 86 compounds, identifying 16 low µM inhibitors and providing data about the structure-activity relationships in four series containing 19, 24, 10, and eight analogues. Measurements with the CDCF probe were consistently more robust than for the EG probe. Only one compound was clearly probe-selective with a 50-fold difference in the IC50s obtained by the two assays. We built 24 classification models using the SVM and fused-XY Kohonen methods, revealing molecular descriptors related to number of rings, solubility and lipophilicity as important to distinguish inhibitors from inactive compounds. This study is to the best of our knowledge the first to provide details about structure-activity relationships in ABCC2 modulation.

SVM classification and CoMSIA modeling of UGT1A6 interacting molecules.

The human UDP-glucuronosyltransferase 1A6 (UGT1A6) plays important roles in elimination of many xenobiotics, including drugs. We have experimentally assessed inhibitory properties of 46 compounds toward UGT1A6 catalyzing the glucuronidation of 1-naphthol and built models for predicting compounds interactions with the enzyme. The tested compounds were divided into a training set (n = 31; evaluated by 10-fold cross-validation) and an external test set (n = 15), both of which yielded similar accuracies (80-81%) and Matthews correlation coefficients (0.61-0.63) when classified using support vector machines. Comparative molecular similarity index analysis (CoMSIA) modeling was conducted for nine methods of compound alignment. The most predictive CoMSIA model was analyzed in the light of a homology modeled UGT1A6 structure, with leave-one-out cross-validation, yielding a q² of 0.62 and r² of 0.91 on the training set and a r²(pred) of 0.82 on the test set. The CoMSIA contour plots highlighted the importance of H-bond donors and electrostatic field interactions, accounting for 28% and 25% contribution of the model, respectively.

Nostosins, Trypsin Inhibitors Isolated from the Terrestrial Cyanobacterium Nostoc sp. Strain FSN.

Two new trypsin inhibitors, nostosin A (1) and B (2), were isolated from a hydrophilic extract of Nostoc sp. strain FSN, which was collected from a paddy field in the Golestan Province of Iran. Nostosins A (1) and B (2) are composed of three subunits, 2-hydroxy-4-(4-hydroxyphenyl)butanoic acid (Hhpba), L-Ile, and L-argininal (1) or argininol (2). Nostosins A (1) and B (2) exhibited IC50 values of 0.35 and 55 µM against porcine trypsin, respectively, suggesting that the argininal aldehyde group plays a crucial role in the efficient inhibition of trypsin. Molecular docking of nostosin A (1) (449 Da), leupeptin (426 Da, IC50 0.5 µM), and spumigin E (610 Da, IC50 < 0.1 µM) with trypsin suggested prominent binding similarity between nostosin A (1) and leupeptin but only partial binding similarity with spumigin E. The number of hydrogen bonds between ligands and trypsin increased according to the length and size of the ligand molecule, and the docking affinity values followed the measured IC50 values. Nostosin A (1) is the first highly potent three-subunit trypsin inhibitor with potency comparable to the known commercial trypsin inhibitor leupeptin. These findings expand the known diversity of short-chain linear peptide protease inhibitors produced by cyanobacteria.

Characterization of a putative orexin receptor in Ciona intestinalis sheds light on the evolution of the orexin/hypocretin system in chordates.

Tunicates are evolutionary model organisms bridging the gap between vertebrates and invertebrates. A genomic sequence in Ciona intestinalis (CiOX) shows high similarity to vertebrate orexin receptors and protostome allatotropin receptors (ATR). Here, molecular phylogeny suggested that CiOX is divergent from ATRs and human orexin receptors (hOX1/2). However, CiOX appears closer to hOX1/2 than to ATR both in terms of sequence percent identity and in its modelled binding cavity, as suggested by molecular modelling. CiOX was heterologously expressed in a recombinant HEK293 cell system. Human orexins weakly but concentration-dependently activated its Gq signalling (Ca2+ elevation), and the responses were inhibited by the non-selective orexin receptor antagonists TCS 1102 and almorexant, but only weakly by the OX1-selective antagonist SB-334867. Furthermore, the 5-/6-carboxytetramethylrhodamine (TAMRA)-labelled human orexin-A was able to bind to CiOX. Database mining was used to predict a potential endogenous C. intestinalis orexin peptide (Ci-orexin-A). Ci-orexin-A was able to displace TAMRA-orexin-A, but not to induce any calcium response at the CiOX. Consequently, we suggested that the orexin signalling system is conserved in Ciona intestinalis, although the relevant peptide-receptor interaction was not fully elucidated.

Visually interpretable models of kinase selectivity related features derived from field-based proteochemometrics.

Achieving selectivity for small organic molecules toward biological targets is a main focus of drug discovery but has been proven difficult, for example, for kinases because of the high similarity of their ATP binding pockets. To support the design of more selective inhibitors with fewer side effects or with altered target profiles for improved efficacy, we developed a method combining ligand- and receptor-based information. Conventional QSAR models enable one to study the interactions of multiple ligands toward a single protein target, but in order to understand the interactions between multiple ligands and multiple proteins, we have used proteochemometrics, a multivariate statistics method that aims to combine and correlate both ligand and protein descriptions with affinity to receptors. The superimposed binding sites of 50 unique kinases were described by molecular interaction fields derived from knowledge-based potentials and Schrödinger's WaterMap software. Eighty ligands were described by Mold(2), Open Babel, and Volsurf descriptors. Partial least-squares regression including cross-terms, which describe the selectivity, was used for model building. This combination of methods allows interpretation and easy visualization of the models within the context of ligand binding pockets, which can be translated readily into the design of novel inhibitors.

Exploring cooperative molecular contacts using a PostgreSQL database system.

Cooperative molecular contacts play an important role in protein structure and ligand binding. Here, we constructed a PostgreSQL database that stores structural information in the form of atomic environments and allows flexible mining of molecular contacts. Taking the Ser-His-Asp/Glu catalytic triad as a first test case, we demonstrate that the presence of a carboxylate oxygen atom in the vicinity of a His is associated with shorter Ser-OH..N-His bond in the PDB30 subset. We prospectively mine catalytic triads in unannotated proteins, suggesting catalytic functions for unannotated proteins. As a second test case, we demonstrate that this database system can include ligand atoms, represented by Sybyl atom types, by evaluating the proportion of counter-ions for ligand carboxylate oxygens.

Expanding the Paradigm of Structure-Based Drug Design: Molecular Dynamics Simulations Support the Development of New Pyridine-Based Protein Kinase C-Targeted Agonists.

Protein kinase C (PKC) modulators hold therapeutic potential for various diseases, including cancer, heart failure, and Alzheimer's disease. Targeting the C1 domain of PKC represents a promising strategy; the available protein structures warrant the design of PKC-targeted ligands via a structure-based approach. However, the PKC C1 domain penetrates the lipid membrane during binding, complicating the design of drug candidates. The standard docking-scoring approach for PKC lacks information regarding the dynamics and the membrane environment. Molecular dynamics (MD) simulations with PKC, ligands, and membranes have been used to address these shortcomings. Previously, we observed that less computationally intensive simulations of just ligand-membrane interactions may help elucidate C1 domain-binding prospects. Here, we present the design, synthesis, and biological evaluation of new pyridine-based PKC agonists implementing an enhanced workflow with ligand-membrane MD simulations. This workflow holds promise to expand the approach in drug design for ligands targeted to weakly membrane-associated proteins.

Analysis of Biologics Molecular Descriptors towards Predictive Modelling for Protein Drug Development Using Time-Gated Raman Spectroscopy.

Pharmaceutical proteins, compared to small molecular weight drugs, are relatively fragile molecules, thus necessitating monitoring protein unfolding and aggregation during production and post-marketing. Currently, many analytical techniques take offline measurements, which cannot directly assess protein folding during production and unfolding during processing and storage. In addition, several orthogonal techniques are needed during production and market surveillance. In this study, we introduce the use of time-gated Raman spectroscopy to identify molecular descriptors of protein unfolding. Raman spectroscopy can measure the unfolding of proteins in-line and in real-time without labels. Using K-means clustering and PCA analysis, we could correlate local unfolding events with traditional analytical methods. This is the first step toward predictive modeling of unfolding events of proteins during production and storage.

Multisubstituted pyrimidines effectively inhibit bacterial growth and biofilm formation of Staphylococcus aureus.

Biofilms are multicellular communities of microorganisms that generally attach to surfaces in a self-produced matrix. Unlike planktonic cells, biofilms can withstand conventional antibiotics, causing significant challenges in the healthcare system. Currently, new chemical entities are urgently needed to develop novel anti-biofilm agents. In this study, we designed and synthesized a set of 2,4,5,6-tetrasubstituted pyrimidines and assessed their antibacterial activity against planktonic cells and biofilms formed by Staphylococcus aureus. Compounds 9e, 10d, and 10e displayed potent activity for inhibiting the onset of biofilm formation as well as for killing pre-formed biofilms of S. aureus ATCC 25923 and Newman strains, with half-maximal inhibitory concentration (IC50) values ranging from 11.6 to 62.0 µM. These pyrimidines, at 100 µM, not only decreased the number of viable bacteria within the pre-formed biofilm by 2-3 log10 but also reduced the amount of total biomass by 30-50%. Furthermore, these compounds were effective against planktonic cells with minimum inhibitory concentration (MIC) values lower than 60 µM for both staphylococcal strains. Compound 10d inhibited the growth of S. aureus ATCC 25923 in a concentration-dependent manner and displayed a bactericidal anti-staphylococcal activity. Taken together, our study highlights the value of multisubstituted pyrimidines to develop novel anti-biofilm agents.

Exploration of Pyrazolo[1,5-a]pyrimidines as Membrane-Bound Pyrophosphatase Inhibitors.

Inhibition of membrane-bound pyrophosphatase (mPPase) with small molecules offer a new approach in the fight against pathogenic protozoan parasites. mPPases are absent in humans, but essential for many protists as they couple pyrophosphate hydrolysis to the active transport of protons or sodium ions across acidocalcisomal membranes. So far, only few nonphosphorus inhibitors have been reported. Here, we explore the chemical space around previous hits using a combination of screening and synthetic medicinal chemistry, identifying compounds with low micromolar inhibitory activities in the Thermotoga maritima mPPase test system. We furthermore provide early structure-activity relationships around a new scaffold having a pyrazolo[1,5-a]pyrimidine core. The most promising pyrazolo[1,5-a]pyrimidine congener was further investigated and found to inhibit Plasmodium falciparum mPPase in membranes as well as the growth of P. falciparum in an ex vivo survival assay.