Structural basis for the bi-specificity of USP25 and USP28 inhibitors.

The development of cancer therapeutics is often hindered by the fact that specific oncogenes cannot be directly pharmaceutically addressed. Targeting deubiquitylases that stabilize these oncogenes provides a promising alternative. USP28 and USP25 have been identified as such target deubiquitylases, and several small-molecule inhibitors indiscriminately inhibiting both enzymes have been developed. To obtain insights into their mode of inhibition, we structurally and functionally characterized USP28 in the presence of the three different inhibitors AZ1, Vismodegib and FT206. The compounds bind into a common pocket acting as a molecular sink. Our analysis provides an explanation why the two enzymes are inhibited with similar potency while other deubiquitylases are not affected. Furthermore, a key glutamate residue at position 366/373 in USP28/USP25 plays a central structural role for pocket stability and thereby for inhibition and activity. Obstructing the inhibitor-binding pocket by mutation of this glutamate may provide a tool to accelerate future drug development efforts for selective inhibitors of either USP28 or USP25 targeting distinct binding pockets.

Assessment of fragment docking and scoring with the endothiapepsin model system.

Fragment-based screening has become indispensable in drug discovery. Yet, the weak binding affinities of these small molecules still represent a challenge for the reliable detection of fragment hits. The extent of this issue was illustrated in the literature for the aspartic protease endothiapepsin: When seven biochemical and biophysical in vitro screening methods were applied to screen a library of 361 fragments, very poor overlap was observed between the hit fragments identified by the individual approaches, resulting in high levels of false positive and/or false negative results depending on the mutually compared methods. Here, the reported in vitro findings are juxtaposed with the results from in silico docking and scoring approaches. The docking programs GOLD and Glide were considered with the scoring functions ASP, ChemScore, ChemPLP, GoldScore, DSXCSD, and GlideScore. First, the ranking power and scoring power were assessed for the named scoring functions. Second, the capability of reproducing the crystallized fragment binding modes was tested in a structure-based redocking approach. The redocking success notably depended on the ligand efficiency of the considered fragments. Third, a blinded virtual screening approach was employed to evaluate whether in silico screening can compete with in vitro methods in the enrichment of fragment databases.

MD-Based Assessment of Covalent Inhibitors in Noncovalent Association Complexes: Learning from Cathepsin K as a Test Case.

A sufficiently stable noncovalent association complex between a covalent inhibitor and its protein target is regarded as a prerequisite for the formation of a covalent complex. As this transient form can hardly be assessed experimentally, computational modeling is required to probe the suitability of a given ligand at this particular stage. To investigate which criteria should be fulfilled by suitable candidates in a molecular dynamics (MD) assessment, a systematic study was conducted with 20 complexes of cathepsin K, a papain-like cysteine protease of pharmaceutical relevance. The covalent inhibitors in these complexes were converted to their pre-reaction states, and the resulting noncovalent complexes were subjected to MD simulations. The critical distance between the electrophilic and nucleophilic reaction partners was monitored as a potential parameter to assess the suitability for covalent bond formation. Across various warhead types, a distance between 3.6 and 4.0 Å, comparable to the sum of the generalized Born radii of carbon and sulfur, was found to be stably maintained under appropriate conditions. The protonation state of the catalytic dyad and the resulting solvation pattern dramatically affected the noncovalent binding mode and the distance of the warhead to the active site. For several complexes, fluctuations in the orientation of the warhead were observed due to torsional rotations in adjacent bonds. This observation helped to explain the gradual transitions from noncovalent to covalent complexes observed in the crystal structures of three closely related nitrile-based inhibitors. According to comparative simulations conducted for a set of 13 cathepsin S complexes, the overall findings of the study appear to be transferable to related cysteine proteases as targets of covalent inhibitors.

Photoswitchable Pseudoirreversible Butyrylcholinesterase Inhibitors Allow Optical Control of Inhibition in Vitro and Enable Restoration of Cognition in an Alzheimer's Disease Mouse Model upon Irradiation.

To develop tools to investigate the biological functions of butyrylcholinesterase (BChE) and the mechanisms by which BChE affects Alzheimer's disease (AD), we synthesized several selective, nanomolar active, pseudoirreversible photoswitchable BChE inhibitors. The compounds were able to specifically influence different kinetic parameters of the inhibition process by light. For one compound, a 10-fold difference in the IC50-values (44.6 nM cis, 424 nM trans) in vitro was translated to an "all or nothing" response with complete recovery in a murine cognition-deficit AD model at dosages as low as 0.3 mg/kg.

PROTAC-mediated degradation reveals a non-catalytic function of AURORA-A kinase.

The mitotic kinase AURORA-A is essential for cell cycle progression and is considered a priority cancer target. Although the catalytic activity of AURORA-A is essential for its mitotic function, recent reports indicate an additional non-catalytic function, which is difficult to target by conventional small molecules. We therefore developed a series of chemical degraders (PROTACs) by connecting a clinical kinase inhibitor of AURORA-A to E3 ligase-binding molecules (for example, thalidomide). One degrader induced rapid, durable and highly specific degradation of AURORA-A. In addition, we found that the degrader complex was stabilized by cooperative binding between AURORA-A and CEREBLON. Degrader-mediated AURORA-A depletion caused an S-phase defect, which is not the cell cycle effect observed upon kinase inhibition, supporting an important non-catalytic function of AURORA-A during DNA replication. AURORA-A degradation induced rampant apoptosis in cancer cell lines and thus represents a versatile starting point for developing new therapeutics to counter AURORA-A function in cancer.

Predicting Bile and Lipid Interaction for Drug Substances.

Predicting biopharmaceutical characteristics and food effects for drug substances may substantially leverage rational formulation outcomes. We established a bile and lipid interaction prediction model for new drug substances and further explored the model for the prediction of bile-related food effects. One hundred and forty-one drugs were categorized as bile and/or lipid interacting and noninteracting drugs using 1H nuclear magnetic resonance (NMR) spectroscopy. Quantitative structure-property relationship modeling with molecular descriptors was applied to predict a drug's interaction with bile and/or lipids. Bile interaction, for example, was indicated by two descriptors characterizing polarity and lipophilicity with a high balanced accuracy of 0.8. Furthermore, the predicted bile interaction correlated with a positive food effect. Reliable prediction of drug substance interaction with lipids required four molecular descriptors with a balanced accuracy of 0.7. These described a drug's shape, lipophilicity, aromaticity, and hydrogen bond acceptor capability. In conclusion, reliable models might be found through drug libraries characterized for bile interaction by NMR. Furthermore, there is potential for predicting bile-related positive food effects.

Sesquiterpenes and sesquiterpenoids harbor modulatory allosteric potential and affect inhibitory GABAA receptor function in vitro.

Naturally occurring compounds such as sesquiterpenes and sesquiterpenoids (SQTs) have been shown to modulate GABAA receptors (GABAA Rs). In this study, the modulatory potential of 11 SQTs at GABAA Rs was analyzed to characterize their potential neurotropic activity. Transfected HEK293 cells and primary hippocampal neurons were functionally investigated using electrophysiological whole-cell recordings. Significantly different effects of ß-caryophyllene and α-humulene, as well as their respective derivatives ß-caryolanol and humulol, were observed in the HEK293 cell system. In neurons, the concomitant presence of phasic and tonic GABAA R configurations accounts for differences in receptor modulation by SQTs. The in vivo presence of the γ2 and δ subunits is important for SQT modulation. While phasic GABAA receptors in hippocampal neurons exhibited significantly altered GABA-evoked current amplitudes in the presence of humulol and guaiol, negative allosteric potential at recombinantly expressed α1 ß2 γ2 receptors was only verified for humolol. Modeling and docking studies provided support for the binding of SQTs to the neurosteroid-binding site of the GABAA R localized between transmembrane segments 1 and 3 at the (+ α)-(- α) interface. In sum, differences in the modulation of GABAA R isoforms between SQTs were identified. Another finding is that our results provide an indication that nutritional digestion affects the neurotropic potential of natural compounds.

Molecular Insights into Site-Specific Interferon-α2a Bioconjugates Originated from PEG, LPG, and PEtOx.

Conjugation of biologics with polymers modulates their pharmacokinetics, with polyethylene glycol (PEG) as the gold standard. We compared alternative polymers and two types of cyclooctyne linkers (BCN/DBCO) for bioconjugation of interferon-α2a (IFN-α2a) using 10 kDa polymers including linear mPEG, poly(2-ethyl-2-oxazoline) (PEtOx), and linear polyglycerol (LPG). IFN-α2a was azide functionalized via amber codon expansion and bioorthogonally conjugated to all cyclooctyne linked polymers. Polymer conjugation did not impact IFN-α2a's secondary structure and only marginally reduced IFN-α2a's bioactivity. In comparison to PEtOx, the LPG polymer attached via the less rigid cyclooctyne linker BCN was found to stabilize IFN-α2a against thermal stress. These findings were further detailed by molecular modeling studies which showed a modulation of protein flexibility upon PEtOx conjugation and a reduced amount of protein native contacts as compared to PEG and LPG originated bioconjugates. Polymer interactions with IFN-α2a were further assessed via a limited proteolysis (LIP) assay, which resulted in comparable proteolytic cleavage patterns suggesting weak interactions with the protein's surface. In conclusion, both PEtOx and LPG bioconjugates resulted in a similar biological outcome and may become promising PEG alternatives for bioconjugation.

C-2-Linked Dimeric Strychnine Analogues as Bivalent Ligands Targeting Glycine Receptors.

Strychnine is the prototypic antagonist of glycine receptors, a family of pentameric ligand-gated ion channels. Recent high-resolution structures of homomeric glycine receptors have confirmed the presence of five orthosteric binding sites located in the extracellular subunit interfaces of the receptor complex that are targeted by strychnine. Here, we report the synthesis and extensive pharmacological evaluation of bivalent ligands composed of two strychnine pharmacophores connected by appropriate spacers optimized toward simultaneous binding to two adjacent orthosteric sites of homomeric α1 glycine receptors. In all bivalent ligands, the two strychnine units were linked through C-2 by amide spacers of various lengths ranging from 6 to 69 atoms. Characterization of the compounds in two functional assays and in a radioligand binding assay indicated that compound 11a, with a spacer consisting of 57 atoms, may be capable of bridging the homomeric α1 GlyRs by simultaneous occupation of two adjacent strychnine-binding sites. The findings are supported by docking experiments to the crystal structure of the homomeric glycine receptor. Based on its unique binding mode, its relatively high binding affinity and antagonist potency, and its slow binding kinetics, the bivalent strychnine analogue 11a could be a valuable tool to study the functional properties of glycine receptors.

Controlling Supramolecular Structures of Drugs by Light.

Controlling physicochemical properties of light-unresponsive drugs, by light, prima facie, a paradox approach. We expanded light control by ion pairing light-unresponsive salicylate or ibuprofen to photoswitchable azobenzene counterions, thereby reversibly controlling supramolecular structures, hence the drugs' physicochemical and kinetic properties. The resulting ion pairs photoliquefied into room-temperature ionic liquids under ultraviolet light. Aqueous solutions showed trans-cis-dependent supramolecular structures under a light with wormlike aggregates decomposing into small micelles and vice versa. Light control allowed for permeation through membranes of cis-ibuprofen ion pairs within 12 h in contrast to the trans ion pairs requiring 72 h. In conclusion, azobenzene ion-pairing expands light control of physicochemical and kinetic properties to otherwise light-unresponsive drugs.

Melatonin receptor ligands: A pharmaco-chemical perspective.

Melatonin MT1 and MT2 receptor ligands have been vigorously explored for the last 4 decades. Inspection of approximately 80 publications in the field revealed that most melatonergic ligands were structural analogues of melatonin combining three essential features of the parent compound: an aromatic ring bearing a methoxy group and an amide side chain in a relative arrangement similar to that present in melatonin. While several series of MT2 -selective agents-agonists, antagonists, or partial agonists-were reported, the field was lacking MT1 -selective agents. Herein, we describe various approaches toward the development of melatonergic ligands, keeping in mind that most of the molecules/pharmacophores obtained were essentially melatonin copies, even though diverse tri- or tetra-cyclic compounds were explored. In addition to lack of structural diversity, only few studies examined the activity of the reported melatonergic ligands in vivo. Moreover, an extensive pharmacological characterization including biopharmaceutical stability, pharmacokinetic properties, specificity toward other major receptors to name a few remained scarce. For example, many of the antagonists described were not stable in vivo, were not selective for the melatonin receptor subtype of interest, and were not fully characterized from a pharmacological standpoint. Indeed, virtual screening of large compound libraries has led to the recent discovery of potent and selective melatonin receptor agonists and partial agonists of new chemotypes. Having said this, the melatonergic field is still lacking subtype-selective melatonin receptor antagonists "active" in vivo, which are critical to our understanding of melatonin and melatonin receptors' role in basic physiology and disease.

Tacrine-xanomeline and tacrine-iperoxo hybrid ligands: Synthesis and biological evaluation at acetylcholinesterase and M1 muscarinic acetylcholine receptors.

We synthesized a set of new hybrid derivatives (7-C8, 7-C10, 7-C12 and 8-C8, 8-C10, 8-C12), in which a polymethylene spacer chain of variable length connected the pharmacophoric moiety of xanomeline, an M1/M4-preferring orthosteric muscarinic agonist, with that of tacrine, a well-known acetylcholinesterase (AChE) inhibitor able to allosterically modulate muscarinic acetylcholine receptors (mAChRs). When tested in vitro in a colorimetric assay for their ability to inhibit AChE, the new compounds showed higher or similar potency compared to that of tacrine. Docking analyses were performed on the most potent inhibitors in the series (8-C8, 8-C10, 8-C12) to rationalize their experimental inhibitory power against AChE. Next, we evaluated the signaling cascade at M1 mAChRs by exploring the interaction of Gαq-PLC-ß3 proteins through split luciferase assays and the myo-Inositol 1 phosphate (IP1) accumulation in cells. The results were compared with those obtained on the known derivatives 6-C7 and 6-C10, two quite potent AChE inhibitors in which tacrine is linked to iperoxo, an exceptionally potent muscarinic orthosteric activator. Interestingly, we found that 6-C7 and 6-C10 behaved as partial agonists of the M1 mAChR, at variance with hybrids 7-Cn and 8-Cn containing xanomeline as the orthosteric molecular fragment, which were all unable to activate the receptor subtype response.

11-Aminostrychnine and N-(Strychnine-11-yl)propionamide: Synthesis, Configuration, and Pharmacological Evaluation at Glycine Receptors.

(11S)-11-Aminostrychnine (1) and N-[(11S)-strychnine-11-yl]propionamide (2) were synthesized and characterized as antagonists of homomeric α1 and heteromeric α1ß glycine receptors in a functional fluorescence-based assay and a patch-clamp assay and in radioligand binding studies. The absolute configuration at C-11 of 1 was determined based on vicinal coupling constants and NOESY data. Docking experiments to the orthosteric binding site of the α3 glycine receptor showed a binding mode of compound 2 analogous to that of strychnine, explaining its high antagonistic potency. The findings identify the C-11 amide function of strychnine as a suitable linker group for the future development of dimeric strychnine analogues targeting glycine receptors. The findings extend the SAR of strychnine at glycine receptors.

Extending the Scope of GTFR Glucosylation Reactions with Tosylated Substrates for Rare Sugars Synthesis.

Functionalized rare sugars were synthesized with 2-, 3-, and 6-tosylated glucose derivatives as acceptor substrates by transglucosylation with sucrose and the glucansucrase GTFR from Streptococcus oralis. The 2- and 3-tosylated glucose derivatives yielded the corresponding 1,6-linked disaccharides (isomaltose analogues), whereas the 6-tosylated glucose derivatives resulted in 1,3-linked disaccharides (nigerose analogue) with high regioselectivity in up to 95 % yield. Docking studies provided insight into the binding mode of the acceptors and suggested two different orientations that were responsible for the change in regioselectivity.

Conformational flexibility of DENV NS2B/NS3pro: from the inhibitor effect to the serotype influence.

The dengue virus (DENV) has four well-known serotypes, namely DENV1 to DENV4, which together cause 50-100 million infections worldwide each year. DENV NS2B/NS3pro is a protease recognized as a valid target for DENV antiviral drug discovery. However, NS2B/NS3pro conformational flexibility, involving in particular the NS2B region, is not yet completely understood and, hence, a big challenge for any virtual screening (VS) campaign. Molecular dynamics (MD) simulations were performed in this study to explore the DENV3 NS2B/NS3pro binding-site flexibility and obtain guidelines for further VS studies. MD simulations were done with and without the Bz-nKRR-H inhibitor, showing that the NS2B region stays close to the NS3pro core even in the ligand-free structure. Binding-site conformational states obtained from the simulations were clustered and further analysed using GRID/PCA, identifying four conformations of potential importance for VS studies. A virtual screening applied to a set of 31 peptide-based DENV NS2B/NS3pro inhibitors, taken from literature, illustrated that selective alternative pharmacophore models can be constructed based on conformations derived from MD simulations. For the first time, the NS2B/NS3pro binding-site flexibility was evaluated for all DENV serotypes using homology models followed by MD simulations. Interestingly, the number of NS2B/NS3pro conformational states differed depending on the serotype. Binding-site differences could be identified that may be crucial to subsequent VS studies.

Development, synthesis and structure-activity-relationships of inhibitors of the macrophage infectivity potentiator (Mip) proteins of Legionella pneumophila and Burkholderia pseudomallei.

The bacteria Burkholderia pseudomallei and Legionella pneumophila cause severe diseases like melioidosis and Legionnaire's disease with high mortality rates despite antibiotic treatment. Due to increasing antibiotic resistances against these and other Gram-negative bacteria, alternative therapeutical strategies are in urgent demand. As a virulence factor, the macrophage infectivity potentiator (Mip) protein constitutes an attractive target. The Mip proteins of B. pseudomallei and L. pneumophila exhibit peptidyl-prolyl cis/trans isomerase (PPIase) activity and belong to the PPIase superfamily. In previous studies, the pipecolic acid moiety proved to be a valuable scaffold for inhibiting this PPIase activity. Thus, a library of pipecolic acid derivatives was established guided by structural information and computational analyses of the binding site and possible binding modes. Stability and toxicity considerations were taken into account in iterative extensions of the library. Synthesis and evaluation of the compounds in PPIase assays resulted in highly active inhibitors. The activities can be interpreted in terms of a common binding mode obtained by docking calculations.

Oxime Ethers of (E)-11-Isonitrosostrychnine as Highly Potent Glycine Receptor Antagonists.

A series of (E)-11-isonitrosostrychnine oxime ethers, 2-aminostrychnine, (strychnine-2-yl)propionamide, 18-oxostrychnine, and N-propylstrychnine bromide were synthesized and evaluated pharmacologically at human α1 and α1ß glycine receptors in a functional fluorescence-based and a whole-cell patch-clamp assay and in [3H]strychnine binding studies. 2-Aminostrychnine and the methyl, allyl, and propargyl oxime ethers were the most potent α1 and α1ß antagonists in the series, displaying IC50 values similar to those of strychnine at the two receptors. Docking experiments to the strychnine binding site of the crystal structure of the α3 glycine receptor indicated the same orientation of the strychnine core for all analogues. For the most potent oxime ethers, the ether substituent was accommodated in a lipophilic receptor binding pocket. The findings identify the oxime hydroxy group as a suitable attachment point for linking two strychnine pharmacophores by a polymethylene spacer and are, therefore, important for the design of bivalent ligands targeting glycine receptors.

Experimental and theoretical investigations into the stability of cyclic aminals.

Background: Cyclic aminals are core features of natural products, drug molecules and important synthetic intermediates. Despite their relevance, systematic investigations into their stability towards hydrolysis depending on the pH value are lacking. Results: A set of cyclic aminals was synthesized and their stability quantified by kinetic measurements. Steric and electronic effects were investigated by choosing appropriate groups. Both molecular mechanics (MM) and density functional theory (DFT) based studies were applied to support and explain the results obtained. Rapid decomposition is observed in acidic aqueous media for all cyclic aminals which occurs as a reversible reaction. Electronic effects do not seem relevant with regard to stability, but the magnitude of the conformational energy of the ring system and pKa values of the N-3 nitrogen atom. Conclusion: Cyclic aminals are stable compounds when not exposed to acidic media and their stability is mainly dependent on the conformational energy of the ring system. Therefore, for the preparation and work-up of these valuable synthetic intermediates and natural products, appropriate conditions have to be chosen and for application as drug molecules their sensitivity towards hydrolysis has to be taken into account.

An ordered water channel in Staphylococcus aureus FabI: unraveling the mechanism of substrate recognition and reduction.

One third of all drugs in clinical use owe their pharmacological activity to the functional inhibition of enzymes, highlighting the importance of enzymatic targets for drug development. Because of the close relationship between inhibition and catalysis, understanding the recognition and turnover of enzymatic substrates is essential for rational drug design. Although the Staphylococcus aureus enoyl-acyl carrier protein reductase (saFabI) involved in bacterial fatty acid biosynthesis constitutes a very promising target for the development of novel, urgently needed anti-staphylococcal agents, the substrate binding mode and catalytic mechanism remained unclear for this enzyme. Using a combined crystallographic, kinetic, and computational approach, we have explored the chemical properties of the saFabI binding cavity, obtaining a consistent mechanistic model for substrate binding and turnover. We identified a water-molecule network linking the active site with a water basin inside the homo-tetrameric protein, which seems to be crucial for the closure of the flexible substrate binding loop as well as for an effective hydride and proton transfer during catalysis. On the basis of our results, we also derive a new model for the FabI-ACP complex that reveals how the ACP-bound acyl-substrate is injected into the FabI binding crevice. These findings support the future development of novel FabI inhibitors that target the FabI-ACP interface leading to the disruption of the interaction between these two proteins.

Rational design of broad spectrum antibacterial activity based on a clinically relevant enoyl-acyl carrier protein (ACP) reductase inhibitor.

Determining the molecular basis for target selectivity is of particular importance in drug discovery. The ideal antibiotic should be active against a broad spectrum of pathogenic organisms with a minimal effect on human targets. CG400549, a Staphylococcus-specific 2-pyridone compound that inhibits the enoyl-acyl carrier protein reductase (FabI), has recently been shown to possess human efficacy for the treatment of methicillin-resistant Staphylococcus aureus infections, which constitute a serious threat to human health. In this study, we solved the structures of three different FabI homologues in complex with several pyridone inhibitors, including CG400549. Based on these structures, we rationalize the 65-fold reduced affinity of CG400549 toward Escherichia coli versus S. aureus FabI and implement concepts to improve the spectrum of antibacterial activity. The identification of different conformational states along the reaction coordinate of the enzymatic hydride transfer provides an elegant visual depiction of the relationship between catalysis and inhibition, which facilitates rational inhibitor design. Ultimately, we developed the novel 4-pyridone-based FabI inhibitor PT166 that retained favorable pharmacokinetics and efficacy in a mouse model of S. aureus infection with extended activity against Gram-negative and mycobacterial organisms.