RepurposeDrugs: an interactive web-portal and predictive platform for repurposing mono- and combination therapies.

RepurposeDrugs (https://repurposedrugs.org/) is a comprehensive web-portal that combines a unique drug indication database with a machine learning (ML) predictor to discover new drug-indication associations for approved as well as investigational mono and combination therapies. The platform provides detailed information on treatment status, disease indications and clinical trials across 25 indication categories, including neoplasms and cardiovascular conditions. The current version comprises 4314 compounds (approved, terminated or investigational) and 161 drug combinations linked to 1756 indications/conditions, totaling 28 148 drug-disease pairs. By leveraging data on both approved and failed indications, RepurposeDrugs provides ML-based predictions for the approval potential of new drug-disease indications, both for mono- and combinatorial therapies, demonstrating high predictive accuracy in cross-validation. The validity of the ML predictor is validated through a number of real-world case studies, demonstrating its predictive power to accurately identify repurposing candidates with a high likelihood of future approval. To our knowledge, RepurposeDrugs web-portal is the first integrative database and ML-based predictor for interactive exploration and prediction of both single-drug and combination approval likelihood across indications. Given its broad coverage of indication areas and therapeutic options, we expect it accelerates many future drug repurposing projects.

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.

Mitoxantrone, pixantrone and mitoxantrone (2-hydroxyethyl)piperazine are toll-like receptor 4 antagonists, inhibit NF-κB activation, and decrease TNF-alpha secretion in primary microglia.

Toll-like receptor 4 (TLR4) recognizes various endogenous and microbial ligands and is an essential part in the innate immune system. TLR4 signaling initiates transcription factor NF-κB and production of proinflammatory cytokines. TLR4 contributes to the development or progression of various diseases including stroke, neuropathic pain, multiple sclerosis, rheumatoid arthritis and cancer, and better therapeutics are currently sought for these conditions. In this study, a library of 140 000 compounds was virtually screened and a resulting hit-list of 1000 compounds was tested using a cellular reporter system. The topoisomerase II inhibitor mitoxantrone and its analogues pixantrone and mitoxantrone (2-hydroxyethyl)piperazine were identified as inhibitors of TLR4 and NF-κB activation. Mitoxantrone was shown to bind directly to the TLR4, and pixantrone and mitoxantrone (2-hydroxyethyl)piperazine were shown to inhibit the production of proinflammatory cytokines such as tumor necrosis factor alpha (TNFα) in primary microglia. The inhibitory effect on NF-κB activation or on TNFα production was not mediated through cytotoxity at ≤ 1 µM concentration for pixantrone and mitoxantrone (2-hydroxyethyl)piperazine treated cells, as assessed by ATP counts. This study thus identifies a new mechanism of action for mitoxantrone, pixantrone, and mitoxantrone (2-hydroxyethyl)piperazine through the TLR4.

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.

Deciphering Imidazoline Off-targets by Fishing in the Class A of GPCR field.

Based on the finding that a central antihypertensive agent with high affinity for I1-type imidazoline receptors - rilmenidine, shows cytotoxic effects on cultured cancer cell lines, it has been suggested that imidazoline receptors agonists might have a therapeutic potential in the cancer therapy. Nevertheless, potential rilmenidine side effects caused by activation of α-adrenoceptors, or other associated receptors and enzymes, might hinder its therapeutic benefits. Considering that human α-adrenoceptors belong to the rhodopsin-like class A of G-protein-coupled receptors (GPCRs) it is reasonable to assume that imidazolines might have the affinity for other receptors from the same class. Therefore, to investigate possible off-target effects of imidazoline ligands we have prepared a reverse docking protocol on class A GPCRs, using imidazoline ligands and their decoys. To verify our in silico results, three ligands with high scores and three ligands with low scores were tested for antagonistic activity on α2 - adrenoceptors.

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.

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.

Stapled truncated orexin peptides as orexin receptor agonists.

The peptides orexin-A and -B, the endogenous agonists of the orexin receptors, have similar 19-amino-acid C-termini which retain full maximum response as truncated peptides with only marginally reduced potency, while further N-terminal truncations successively reduce the activity. The peptides have been suggested to bind in an α-helical conformation, and truncation beyond a certain critical length is likely to disrupt the overall helical structure. In this study, we set out to stabilize the α-helical conformation of orexin-A15-33 via peptide stapling at four different sites. At a suggested hinge region, we varied the length of the cross-linker as well as replaced the staple with two α-aminoisobutyric acid residues. Modifications close to the peptide C-terminus, which is crucial for activity, were not allowed. However, central and N-terminal modifications yielded bioactive peptides, albeit with decreased potencies. This provides evidence that the orexin receptors can accommodate and be activated by α-helical peptides. The decrease in potency is likely linked to a stabilization of suboptimal peptide conformation or blocking of peptide backbone-receptor interactions at the hinge region by the helical stabilization or the modified amino acids.

Orexin receptor agonist Yan 7874 is a weak agonist of orexin/hypocretin receptors and shows orexin receptor-independent cytotoxicity.

Two promising lead structures of small molecular orexin receptor agonist have been reported, but without detailed analyses of the pharmacological properties. One of them, 1-(3,4-dichlorophenyl)-2-[2-imino-3-(4-methylbenzyl)-2,3-dihydro-1H-benzo[d]imidazol-1-yl]ethan-1-ol (Yan 7874), is commercially available, and we set out to analyze its properties. As test system we utilized human OX1 and OX2 orexin receptor-expressing Chinese hamster ovary (CHO) K1 cells as well as control CHO-K1 and neuro-2a neuroblastoma cells. Gq-coupling was assessed by measurement of intracellular Ca2+ and phospholipase C activity, and the coupling to Gi and Gs by adenylyl cyclase inhibition and stimulation, respectively. At concentrations above 1 µM, strong Ca2+ and low phospholipase C responses to Yan 7874 were observed in both OX1- and OX2-expressing cells. However, a major fraction of the response was not mediated by orexin receptors, as determined utilizing the non-selective orexin receptor antagonist N-biphenyl-2-yl-1-{[(1-methyl-1H-benzimidazol-2-yl)sulfanyl]acetyl}-L-prolinamide (TCS 1102) as well as control CHO-K1 cells. Yan 7874 did not produce any specific adenylyl cyclase response. Some experiments suggested an effect on cell viability by Yan 7874, and we thus analyzed this. Within a few hours of exposure, Yan 7874 markedly changed cell morphology (shrunken, rich in vacuoles), reduced growth, promoted cell detachment, and induced necrotic cell death. The effect was equal in cells expressing orexin receptors or not. Thus, Yan 7874 is a weak partial agonist of orexin receptors. It also displays strong off-target effects in the same concentration range, culminating in necrotic cell demise. This makes Yan 7874 unsuitable as orexin receptor agonist.

[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.

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.

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.

Impact of probe compound in MRP2 vesicular transport assays.

MRP2 is an efflux transporter that is expressed mainly in the canalicular membrane of hepatocytes, where it expels polar and ionic compounds into the bile. MRP2 is also present in the apical membrane of enterocytes and epithelial cells of proximal tubules of the kidney. Inhibition of MRP2 transport can lead to the accumulation of metabolites and other MRP2 substrates up to toxic levels in these cells. The transport properties of MRP2 are frequently studied with the vesicular transport assay. The assay identifies compounds that interact with MRP2 by measuring the effect of a compound on the transport of a radioactively labeled or fluorescent probe. We have compared the effect of eight selected test compounds (quercetin, disopyramide, paracetamol, indomethacin, diclofenac, estrone-3-sulfate, budesonide, and thioridazine) on the MRP2-mediated transport of three commonly used probes: 5(6)-carboxy-2,7-dichlorofluorescein, leukotriene C4 and estradiol-17-ß-d-glucuronide (E217ßG). Five of the test compounds had different probe-dependent effects on the MRP2-mediated transport, suggesting differences in the transport mechanism of the probes. Our results underline the complexity of substrate recognition by these efflux transporters and the difficulties in directly comparing results obtained with different assays, especially when different probes are used.

Molecular mechanisms of ligand-receptor interactions in transmembrane domain V of the alpha2A-adrenoceptor.

1. The structural determinants of catechol hydroxyl interactions with adrenergic receptors were examined using 12 alpha2-adrenergic agonists and a panel of mutated human alpha2A-adrenoceptors. The alpha2ASer201 mutant had a Cys --> Ser201 (position 5.43) amino-acid substitution, and alpha2ASer201Cys200 and alpha2ASer201Cys204 had Ser --> Cys200 (5.42) and Ser --> Cys204 (5.46) substitutions, respectively, in addition to the Cys --> Ser201 substitution. 2. Automated docking methods were used to predict the receptor interactions of the ligands. Radioligand-binding assays and functional [35S]GTPgammaS-binding assays were performed using transfected Chinese hamster ovary cells to experimentally corroborate the predicted binding modes. 3. The hydroxyl groups of phenethylamines were found to have different effects on ligand affinity towards the activated and resting forms of the wild-type alpha2A-adrenoceptor. Substitution of Ser200 or Ser204 with cysteine caused a deterioration in the capability of catecholamines to activate the alpha2A-adrenoceptor. The findings indicate that (i) Cys201 plays a significant role in the binding of catecholamine ligands and UK14,304 (for the latter, by a hydrophobic interaction), but Cys201 is not essential for receptor activation; (ii) Ser200 interacts with the meta-hydroxyl group of phenethylamine ligands, affecting both catecholamine binding and receptor activation; while (iii) substituting Ser204 with a cysteine interferes both with the binding of catecholamine ligands and with receptor activation, due to an interaction between Ser204 and the para-hydroxyl group of the catecholic ring.