The Innovative Medicines Initiative's New Drugs for Bad Bugs programme: European public-private partnerships for the development of new strategies to tackle antibiotic resistance.

Antibiotic resistance (ABR) is a global public health threat. Despite the emergence of highly resistant organisms and the huge medical need for new drugs, the development of antibacterials has slowed to an unacceptable level worldwide. Numerous government and non-government agencies have called for public-private partnerships and innovative funding mechanisms to address this problem. To respond to this public health crisis, the Innovative Medicines Initiative Joint Undertaking programme has invested more than €660 million, with a goal of matched contributions from the European Commission and the European Federation of Pharmaceutical Industries and Associations, in the development of new antibacterial strategies. The New Drugs for Bad Bugs (ND4BB) programme, an Innovative Medicines Initiative, has the ultimate goal to boost the fight against ABR at every level from basic science and drug discovery, through clinical development to new business models and responsible use of antibiotics. Seven projects have been launched within the ND4BB programme to achieve this goal. Four of them will include clinical trials of new anti-infective compounds, as well as epidemiological studies on an unprecedented scale, which will increase our knowledge of ABR and specific pathogens, and improve the designs of the clinical trials with new investigational drugs. The need for rapid concerted action has driven the funding of seven topics, each of which should add significantly to progress in the fight against ABR. ND4BB unites expertise and provides a platform where the commitment and resources required by all parties are streamlined into a joint public-private partnership initiative of unprecedented scale.

Synthesis and AT2 receptor-binding properties of angiotensin II analogues.

The present study investigates the importance of the amino acid side chains in the octapeptide angiotensin II (Ang II) for binding to the AT2 receptor. A Gly scan was performed where each amino acid in Ang II was substituted one-by-one with glycine. The resulting set of peptides was tested for affinity to the AT2 receptor (porcine myometrial membranes). For a comparison, the peptides were also tested for affinity to the AT1 receptor (rat liver membranes). Only the substitution of Arg2 reduced affinity to the AT2 receptor considerably (92-fold when compared with Ang II). For the other Gly-substituted analogues the affinity to the AT2 receptor was only moderately affected. To further investigate the role of the Arg2 side chain for receptor binding, we synthesized some N-terminally modified Ang II analogues. According to these studies a positive charge in the N-terminal end of angiotensin III [Ang II (2-8)] is not required for high AT2 receptor affinity but seems to be more important in Ang II. With respect to the AT1 receptor, [Gly2]Ang II and [Gly8]Ang II lacked binding affinity (Ki > 10 microM). Replacement of the Val3 or Ile5 residues with Gly produced only a slight decrease in affinity. Interestingly, substitution of Tyr4 or His6, which are known to be very important for AT1 receptor binding, resulted in only 48 and 14 times reduction in affinity, respectively.

Effects of cholesterol and model transmembrane proteins on drug partitioning into lipid bilayers as analysed by immobilized-liposome chromatography.

We have analysed how cholesterol and transmembrane proteins in phospholipid bilayers modulate drug partitioning into the bilayers. For this purpose we determined the chromatographic retention of drugs on liposomes or proteoliposomes entrapped in gel beads. The drug retention per phospholipid amount (the capacity factor Ks) reflects the drug partitioning. Cholesterol in the bilayers decreased the Ks value and hence the partitioning into the membrane in proportion to the cholesterol fraction. On average this cholesterol effect decreased with increasing temperature. Model transmembrane proteins, the glucose transporter GLUT1 and bacteriorhodopsin, interacted electrostatically with charged drugs to increase or decrease the drug partitioning into the bilayers. Bacteriorhodopsin proteoliposomes containing cholesterol combined the effects of the protein and the cholesterol and approached the partitioning properties of red blood cell membranes. For positively charged drugs the correlation between calculated intestinal permeability and log Ks was fair for both liposomes and bacteriorhodopsin-cholesterol proteoliposomes. Detailed modeling of solute partitioning into biological membranes may require an extensive knowledge of their structures.

Synthesis, receptor binding affinities and conformational properties of cyclic methylenedithioether analogues of angiotensin II.

Cyclic 12-, 13- and 14-membered ring angiotensin II analogues related to disulfides but encompassing methylene-dithioether bridges have been prepared. The affinity data from these derivatives were compared to those from the disulfides. The methylenedithioether analogues displayed good binding affinities to rat liver AT1 receptors although in most cases somewhat lower than their disulfide counterparts. One of the methylenedithioethers with a 13-membered ring system demonstrated the highest binding affinity among the thioethers. Theoretical conformational analysis of model compounds of the two series were performed suggesting a similarity between the disulfide and the corresponding methylenedithioether analogues and also between the ring size homologues. This analysis also suggested that some of the model compounds were prone to adopt inverse gamma-turn conformations, which was further supported by use of NMR spectroscopy of the 12-membered ring analogue in the series. The easily executed methylenedithioether cyclization should constitute a valuable complement to the common disulfide methodology for fine-tuning and for probing the bioactive conformation of peptides.

Competitive CYP2C9 inhibitors: enzyme inhibition studies, protein homology modeling, and three-dimensional quantitative structure-activity relationship analysis.

This study describes the generation of a three-dimensional quantitative structure activity relationship (3D-QSAR) model for 29 structurally diverse, competitive CYP2C9 inhibitors defined experimentally from an initial data set of 73 compounds. In parallel, a homology model for CYP2C9 using the rabbit CYP2C5 coordinates was built. For molecules with a known interaction mode with CYP2C9, this homology model, in combination with the docking program GOLD, was used to select conformers to use in the 3D-QSAR analysis. The remaining molecules were docked, and the GRID interaction energies for all conformers proposed by GOLD were calculated. This was followed by a principal component analysis (PCA) of the GRID energies for all conformers of all compounds. Based on the similarity in the PCA plot to the inhibitors with a known interaction mode, the conformer to be used in the 3D-QSAR analysis was selected. The compounds were randomly divided into two groups, the training data set (n = 21) to build the model and the external validation set (n = 8). The PLS (partial least-squares) analysis of the interaction energies against the K(i) values generated a model with r(2) = 0.947 and a cross-validation of q(2) = 0.730. The model was able to predict the entire external data set within 0.5 log units of the experimental K(i) values. The amino acids in the active site showed complementary features to the grid interaction energies in the 3D-QSAR model and were also in agreement with mutagenesis studies.

Synthesis and comparative molecular field analysis (CoMFA) of symmetric and nonsymmetric cyclic sulfamide HIV-1 protease inhibitors.

We have previously reported on the unexpected flipped conformation in the cyclic sulfamide class of inhibitors. An attempt to induce a symmetric binding conformation by introducing P2/P2' substituents foreseen to bind preferentially in the S2/S2' subsite was unsuccessful. On the basis of the flipped conformation we anticipated that nonsymmetric sulfamide inhibitors, with P2/P2' side chains modified individually for the S1' and S2 subsites, should be more potent than the corresponding symmetric analogues. To test this hypothesis, a set of 18 cyclic sulfamide inhibitors (11 nonsymmetric and 7 symmetric) with different P2/P2' substituents was prepared and evaluated in an enzyme assay. To rationalize the structure-activity relationship (SAR) and enable the alignment of the nonsymmetric inhibitors, i.e., which of the P2/P2' substituents of the nonsymmetric inhibitors interact with which subsite, a CoMFA study was performed. The CoMFA model, constructed from the 18 inhibitors in this study along with seven inhibitors from previous work by our group, has successfully been used to rationalize the SAR of the cyclic sulfamide inhibitors. Furthermore, from the information presented herein, the SAR of the cyclic sulfamide class of inhibitors seems to differ from the SAR of the related cyclic urea inhibitors reported by DuPont and DuPont-Merck.

Characterization of a set of HIV-1 protease inhibitors using binding kinetics data from a biosensor-based screen.

The interaction between 290 structurally diverse human immunodeficiency virus type 1 (HIV-1) protease inhibitors and the immobilized enzyme was analyzed with an optical biosensor. Although only a single concentration of inhibitor was used, information about the kinetics of the interaction could be obtained by extracting binding signals at discrete time points. The statistical correlation between the biosensor binding data, inhibition of enzyme activity (K(i)), and viral replication (EC(50)) revealed that the association and dissociation rates for the interaction could be resolved and that they were characteristic for the compounds. The most potent inhibitors, with respect to K(i) and EC(50) values, including the clinically used drugs, all exhibited fast association and slow dissociation rates. Selective or partially selective binders for HIV-1 protease could be distinguished from compounds that showed a general protein-binding tendency by using three reference target proteins. This biosensor-based direct binding assay revealed a capacity to efficiently provide high-resolution information on the interaction kinetics and specificity of the interaction of a set of compounds with several targets simultaneously.

Comparison of three gamma-turn mimetic scaffolds incorporated into angiotensin II.

Rigidification of peptides by cyclization and iterative incorporation of well-defined secondary structure mimetics constitutes one approach to the design of non-peptidergic structures with better defined conformations. We herein present the synthesis of a potential gamma-turn mimetic scaffold, and its incorporation in the 3-5 position of angiotensin II. Two analogues of angiotensin II (Ang II) incorporating this 1,3,5-trisubstituted benzene gamma-turn scaffold were synthesized. Evaluation of the compounds in a radioligand binding assay showed that they lacked affinity to the AT1 receptor. To rationalize these results a geometrical and electrostatical comparison with Ang II analogues encompassing a bicyclic scaffold that delivered inactive pseudo peptides and an azepine scaffold producing highly active ligands was made. This analysis did not provide a clear rationale for the inactivity of the benzene gamma-turn scaffolds.

Angiotensin II analogues encompassing 5,9- and 5,10-fused thiazabicycloalkane tripeptide mimetics.

A simple experimental procedure on solid phase for the construction of new tripeptidic 5,9- and 5,10-fused thiazabicycloalkane scaffolds that adopt beta-turns has been developed. This N-terminal-directed bicyclization, relying on masked aldehyde precursors derived from glutamic acid as key building blocks, provides a complement to the related bicyclization previously reported, where an aspartic acid-derived precursor was employed to induce cyclization toward the C-terminal end of the peptide. Thus, the regioselectivity of the bicyclization can be altered simply by varying the chain length of the incorporated aldehyde precursor. Four analogues of the hypertensive octapeptide angiotensin II, comprising the new scaffolds in the 3-5- and 5-7-positions, were synthesized. One of these conformationally constrained angiotensin II analogues exhibited AT(1) receptor affinity (K(i) = 750 nM). Results from theoretical conformational analysis of model compounds of the bicyclic tripeptide mimetics are presented, and they demonstrate that subtle differences in geometry have a strong impact on the affinity to the AT(1) receptor.

Inhibitors of the C(2)-symmetric HIV-1 protease: nonsymmetric binding of a symmetric cyclic sulfamide with ketoxime groups in the P2/P2' side chains.

Symmetric cyclic sulfamides, substituted in the P2/P2' position with functional groups foreseen to bind preferentially to the S2/S2' subsites of HIV-1 protease, have been prepared. Despite efforts to promote a symmetric binding, the sulfamides seemed prone to bind nonsymmetrically, as deduced from X-ray crystal structure analysis of one of the most potent inhibitors, possessing ketoxime groups in the P2/P2' side chains. Ab initio calculations suggested that the nonsymmetric conformation of the cyclic sulfamide scaffold had lower energy than the corresponding symmetric, cyclic urea-like conformation.

Bicyclic tripeptide mimetics with reverse turn inducing properties.

Analogues of the hypertensive octapeptide angiotensin II, comprising novel constrained 5,8-bicyclic and 5,9-bicyclic tripeptide units adopting nonclassical beta-turn geometries, as deduced from theoretical conformational analysis, have been synthesized. Spontanous bicyclization upon acid-catalyzed deprotection of a model peptide, encompassing a protected omega-formyl alpha-amino acid in position 5 and cysteine residues in positions 3 and 7, revealed a strong preference for bicyclization toward the C-terminus. The bicyclic thiazolidine related angiotensin II analogues synthesized exhibited no affinity for the angiotensin II AT1 receptor.

Correlation of human jejunal permeability (in vivo) of drugs with experimentally and theoretically derived parameters. A multivariate data analysis approach.

The effective permeability (Peff) in the human jejunum (in vivo) of 22 structurally diverse compounds was correlated with both experimentally determined lipophilicity values and calculated molecular descriptors. The permeability data were previously obtained by using a regional in vivo perfusion system in the proximal jejunum in humans as part of constructing a biopharmaceutical classification system for oral immediate-release products. pKa, log P, and, where relevant, log Pion values were determined using the pH-metric technique. On the basis of these experiments, log D values were calculated at pH 5.5, 6.5, and 7.4. Multivariate data analysis was used to derive models that correlate passive intestinal permeability to physicochemical descriptors. The best model obtained, based on 13 passively transcellularly absorbed compounds, used the variables HBD (number of hydrogen bond donors), PSA (polar surface area), and either log D5.5 or log D6.5 (octanol/water distribution coefficient at pH 5.5 and 6.5, respectively). Statistically good models for prediciting human in vivo Peff values were also obtained by using only HBD and PSA or HBD, PSA, and CLOGP. These models can be used to predict passive intestinal membrane diffusion in humans for compounds that fit within the defined property space. We used one of the models obtained above to predict the log Peff values for an external validation set consisting of 34 compounds. A good correlation with the absorption data of these compounds was found.

Cyclic HIV-1 protease inhibitors derived from mannitol: synthesis, inhibitory potencies, and computational predictions of binding affinities.

Ten C2-symmetric cyclic urea and sulfamide derivatives have been synthesized from L-mannonic gamma-lactone and D-mannitol. The results of experimental measurement of their inhibitory potencies against HIV-1 protease were compared to calculated free energies of binding derived from molecular dynamics (MD) simulations. The compounds were selected, firstly, to enable elucidation of the role of stereochemistry for binding affinity (1a-d) and, secondly, to allow evaluation of the effects of variation in the link to the P1 and P1' phenyl groups on affinity (1a and 2-5). Thirdly, compounds with hydrogen bond-accepting or-donating groups attached to the phenyl groups in the P2 and P2' side chains (6 and 7) were selected. Binding free energies were estimated by a linear response method, whose predictive power for estimating binding affinities from MD simulations was demonstrated.

Unexpected binding mode of a cyclic sulfamide HIV-1 protease inhibitor.

Two cyclic, C2-symmetric HIV-1 protease inhibitors, one sulfamide and one urea derivative, both comprising phenyl ether groups in the P1/P1' positions, were cocrystallized with HIV-1 protease, and the crystal structures were determined to 2.0 A resolution. The structure of the urea 2 showed a conformation similar to that reported for the related urea 3 by Lam et al., while the sulfamide 1 adopted an unanticipated conformation in which the P1' and P2' side chains were transposed.

Design, synthesis, and biological activities of four angiotensin II receptor ligands with gamma-turn mimetics replacing amino acid residues 3-5.

Disulfide cyclization is a powerful method for reducing the conformational space of a peptide. This in turn may enable the study of its bioactive conformation. Several analogues of angiotensin II (Ang II) containing a disulfide bridge between amino acids 3 and 5 have been reported. Among these the cyclic octapeptides c[Hcy3,5]-Ang II, c[Cys3,5]-Ang II, and c[Pen 3,5]-Ang II showed significant activity at Ang II receptors. We have performed conformational analysis studies using theoretical calculations and 1H-NMR spectroscopy on tripeptide model compounds of these cyclic octapeptides which show that the cyclic moieties of c[Cys3,5]-Ang II and c[Pen3,5]-Ang II preferentially assume an inverse gamma-turn conformation. On the basis of these results, we substituted amino acid residues 3-5 in Ang II with two different gamma-turn mimetics giving four diastereomeric Ang II analogues. Interestingly, two of these are equipotent to Ang II in binding to AT1 receptors. In the contractile test using rabbit aorta rings, one of the analogues is an agonist with full contractile activity approximately equipotent to c[Pen3,5]-Ang II but 300-fold less potent than Ang II. This low potency may suggest that Ang II does not adopt a gamma-turn in the 3-5 region when interacting with the receptor.

Dopamine D2 receptor agonists: an analysis of indirect models.

To evaluate a previously suggested "dopamine (DA) D2 receptor agonist pharmacophore model" the N-propyl derivatives (1S,2S)-5-hydroxy-1-methyl-2-propylaminotetralin [(1S,2S)-3)] and trans-(+/-)-5-hydroxy-3-methyl-2-propylaminotetralin [(+/-)-4] were synthesized and their conformational preferences were studied by molecular mechanics calculations. The new compounds were evaluated for affinity to striatal DA D2 receptors labelled by [3H]spiroperidol and [3H]N-propylnorapomorphine. In comparison to the corresponding N,N-dipropyl derivatives, compounds (1S,2S)-3 and (+/-)-4 more readily adopt pharmacophore conformations and have higher affinity for DA D2 receptors. Thus, the pharmacophore model for C5-oxygenated 2-aminotetralins appears to be accurate. The DA D2 receptor binding affinities of a series of C1- or C3-methyl substituted 5- or 7-hydroxy-2-dipropylaminotetralin derivatives were also determined to enable a comparison of three different modes of superposition, of 7- and 5-hydroxylated 2-aminotetralin derivatives. The results show that the relative spatial positions of the C1- and C3-methyl groups resulting from the various superpositions of 5- and 7-hydroxylated regioisomers differ and the fact that the bulk of a methyl group may be readily accepted by the receptor does not necessarily indicate that additional bulk may be tolerated. Consequently, a definitive choice of mode of superposition of 5- and 7-hydroxylated regioisomers can not be made at present and has to wait until the binding site itself has been better defined.

Conformational properties of melatonin and two conformationally restricted agonists: a molecular mechanics and NMR spectroscopic study.

The hormone melatonin (N-acetyl-5-methoxytryptamine) has been implicated in the regulation of several neural and endocrine processes that are cued by the daily change in photoperiod. The conformational properties of melatonin and two conformationally restricted agonists, 8-methoxy-2-acetamidotetralin and cis-1-methyl-8-methoxy-2-acetamidotetralin, were examined as a starting point for SAR studies. The conformational analysis was carried out by means of molecular mechanics calculations (MM2-85 force field) using a step-wise build-up procedure and the molecular modelling program MacroModel (which has a modified version of the MM2 force field) for Monte Carlo and systematic tree searches. NMR spectroscopy was used to evaluate the conformational behaviour in solution. The three search methods were complementary and produced a good description of the conformational characteristics. The two force fields produced different geometries of some low energy conformations and also different relative steric energies. When using the MM2-85 force field, an intra-molecular interaction between the amide-H and the aromatic system lowers the relative energies of several conformations of both melatonin and the 2-amidotetralins. However, NMR experiments indicated that these interactions, which may be quite important in vacuo, are not important in solution.

Topography and conformational preferences of 6,7,8,9-tetrahydro-1-hydroxy-N,N-dipropyl-5H-benzocyclohepten-6- ylamin e. A rationale for the dopaminergic inactivity.

In an attempt to rationalize the inability of phenolic benzocycloheptenylamines to activate dopamine (DA) D2 receptors, we have studied the conformational preferences and topography of 6,7,8,9-tetrahydro-1-hydroxy-N,N-dipropyl-5H-benzocyclohepten-6-++ +ylamine (1). Preferred conformations of 1 have been defined by use of experimental (NMR spectroscopic studies) and theoretical (MMP2, AM1, and MNDO calculations) methods. Topographical characteristics were studied by use of molecular graphics. Results from the MMP2 calculations agree with those from the AM1 calculations and the NMR spectroscopic study--1 seems to preferentially adopt chair conformations with a pseudoequatorial C6 substituent. The MNDO calculations, however, produced results that deviate considerably from those of the other theoretical methods. Most likely, pharmacophore conformations of (S)-1 do not present volumes that are part of the DA D2 receptor essential volume. Therefore, it appears that the energy penalty paid by the pharmacophore conformations is responsible for the dopaminergic inactivity of (S)-1. The inactivity of (R)-1 may be due to high energies of pharmacophore conformations and/or to steric factors since these conformations produce large excess volumes that may be part of the DA D2 receptor essential volume. The model used in the present report--a flexible pharmacophore combined with a partial receptor-excluded volume--might be used in the design of new DA D2 receptor agonists.