Career development in fragment-based drug discovery.

The pharmaceutical industry is highly reliant on researchers who not only possess the technical knowledge but also the professional skills to collaborate in drug development. To prepare future practitioners to thrive in this interdisciplinary environment, Innovative Training Networks (ITNs) have become increasingly important in doctoral training. In this piece, we explore the benefits of these ITNs in training future practitioners in drug discovery. Through a bibliometric review, we find that the top researchers in fragment-based drug discovery have a high degree of collaboration and mobility across institutes. We then investigate which aspects of the ITN training program enable PhD students to gain these skills. We find that secondments, the short-term stays that students have in partner research institutes, are useful in preparing students to have both broad knowledge of drug discovery and specialization in their field of interest. Aside from imparting technical skills, we find that the collaborative environment in ITNs enables students to communicate better and to work effectively in teams. Doctoral students benefit by being exposed to relevant experiences that they can later apply as they navigate through the complex web of relationships and competencies in the industry. We conclude by recommending best practices to further improve ITNs in the training of future practitioners.

Micro-foundations of dynamic capabilities to facilitate university technology transfer.

Within the university-industry ecosystem, improvement and innovation of technology transfer involve implementing appropriate dynamic capabilities. To answer the question-What are the micro-foundations of dynamic capabilities in university technology transfer?-this study investigates in-depth organizational-level dynamic capabilities in transferring university-based knowledge to business and society. Two qualitative case studies were deployed at organizational entities at Vrije Universiteit Amsterdam: the Industry Alliance Office, and the Demonstrator Lab. These two organizations stimulate science- and business-oriented university technology transfer. In this context, the micro-foundations of the dynamic capabilities "sensing", "seizing" and "reconfiguring" are identified and discussed. For "sensing", which is the university's ability to explore the opportunities in the ecosystem, the micro-foundations are "selecting internal competency" and "sensing external partners". For "seizing", which supports universities in managing complementarity with industry and society, micro-foundations include "resource co-allocation" and "collaborative business model". The micro-foundations of "reconfiguring", through which universities maintain evolutionary fitness in the innovation ecosystem, are "strategic renewal", "establishing a university technology transfer-friendly environment", and "asset orchestration". This study provides researchers with a better understanding of how dynamic capabilities facilitate university technology transfer. Industrial practitioners and policymakers can consider the suggestions of the present study when pursuing collaboration with universities.

VUF10166, a novel compound with differing activities at 5-HT3A and 5-HT3AB receptors.

The actions of a novel, potent 5-HT3 receptor ligand, [2-chloro-(4-methylpiperazine-1-yl)quinoxaline (VUF10166)], were examined at heterologously expressed human 5-HT3A and 5-HT3AB receptors. VUF10166 displaced [³H]granisetron binding to 5-HT3A receptors expressed in human embryonic kidney cells with high affinity (K(i) = 0.04 nM) but was less potent at 5-HT3AB receptors (K(i) = 22 nM). Dissociation of [³H]granisetron in the presence of VUF10166 was best fit with a single time constant (t(1/2) = 53 min) at 5-HT3A receptors, but with two time constants (t(1/2) = 55 and 2.4 min) at 5-HT3AB receptors. Electrophysiological studies in oocytes revealed that VUF10166 inhibited 5-HT-induced responses at 5-HT3A receptors at nanomolar concentrations, but inhibition and recovery were too slow to determine an IC50. At 5-HT3AB receptors, inhibition and recovery were faster, yielding an IC50 of 40 nM. Cysteine substitutions in the complementary (-), but not the principal (+), face of the 5-HT3B subunit produced heteromeric receptors in which the actions of VUF10166 resembled those at homomeric receptors. At 5-HT3A receptors, VUF10166 at higher concentrations also behaved as a partial agonist (EC50 = 5.2 µM; R(max) = 0.24) but did not elicit significant responses at 5-HT3AB receptors at ≤100 µM. Thus, we propose that VUF10166 binds to the common A+A- site of both receptor types and to a second A+B- modulatory site in the heteromeric receptor. The ability of VUF10166 to distinguish between 5-HT3A and 5-HT3AB receptors could help evaluate differences between these receptor types and has potential therapeutic value.

C(X)CR in silico: Computer-aided prediction of chemokine receptor-ligand interactions.

This review will focus on the construction, refinement, and validation of chemokine receptor models for the purpose of structure-based virtual screening and ligand design. The review will present a comparative analysis of ligand binding pockets in chemokine receptors, including a review of the recently released CXCR4 X-ray structures, and their implication on chemokine receptor (homology) modeling. The recommended protein-ligand modeling procedure as well as the use of experimental anchors to steer the modeling procedure is discussed and an overview of several successful structure-based ligand discovery and design studies is provided. This review shows that receptor models, despite structural inaccuracies, can be efficiently used to find novel ligands for chemokine receptors.:

Structure-based exploration and pharmacological evaluation of N-substituted piperidin-4-yl-methanamine CXCR4 chemokine receptor antagonists.

Using the available structural information of the chemokine receptor CXCR4, we present hit finding and hit exploration studies that make use of virtual fragment screening, design, synthesis and structure-activity relationship (SAR) studies. Fragment 2 was identified as virtual screening hit and used as a starting point for the exploration of 31 N-substituted piperidin-4-yl-methanamine derivatives to investigate and improve the interactions with the CXCR4 binding site. Additionally, subtle structural ligand changes lead to distinct interactions with CXCR4 resulting in a full to partial displacement of CXCL12 binding and competitive and/or non-competitive antagonism. Three-dimensional quantitative structure-activity relationship (3D-QSAR) and binding model studies were used to identify important hydrophobic interactions that determine binding affinity and indicate key ligand-receptor interactions.

[From gene to drug: recent developments in medicinal chemistry]. / Van gen tot medicijn: moderne ontwikkelingen in de farmacochemie.

The development ofa new drug often takes more than ten years. Until recently, researchers were obliged to carry out experimental research on the efficacy of variants of those molecules of which the effect was known. It took a long time before a structure-activity relationship could be established. Currently, the full effect of the stormy developments in the Life Sciences, including the sequencing of the human genome, has not yet been fully felt in clinical practice. However, in laboratories researching medicinal products, the technology and the strategies being applied are changing drastically. Understanding disease processes and pharmaconprotein interactions at molecular level make it possible to develop biologically active pharmaceuticals using efficient developmental techniques involving crystallography and computer models. At the same time it is becoming clear that more research is necessary in order to understand the pharmacokinetic characteristics and toxicological activity of medicines under development. This second phase in the development of medicinal products too, is being strongly stimulated by the recent changes.

Development of a pharmacophore model for histamine H3 receptor antagonists, using the newly developed molecular modeling program SLATE.

New molecular modeling tools were developed to construct a qualitative pharmacophore model for histamine H3 receptor antagonists. The program SLATE superposes ligands assuming optimum hydrogen bond geometry. One or two ligands are allowed to flex in the procedure, thereby enabling the determination of the bioactive conformation of flexible H3 antagonists. In the derived model, four hydrogen-bonding site points and two hydrophobic pockets available for binding antagonists are revealed. The model results in a better understanding of the structure-activity relationships of H3 antagonists. To validate the model, a series of new antagonists was synthesized. The compounds were designed to interact with all four hydrogen-bonding site points and the two hydrophobic pockets simultaneously. These ligands have high H3 receptor affinity, thereby illustrating how the model can be used in the design of new classes of H3 antagonists.

Characterization of the binding site of the histamine H3 receptor. 1. Various approaches to the synthesis of 2-(1H-imidazol-4-yl)cyclopropylamine and histaminergic activity of (1R,2R)- and (1S,2S)-2-(1H-imidazol-4-yl)-cyclopropylamine.

Various approaches to the synthesis of all four stereoisomers of 2-(1H-imidazol-4-yl)cyclopropylamine (cyclopropylhistamine) are described. The rapid and convenient synthesis and resolution of trans-cyclopropylhistamine is reported. The absolute configuration of its enantiomers was determined by single-crystal X-ray crystallographic analysis. The distinct trans-cyclopropylhistamine enantiomers were tested for their activity and affinity on the histamine H3 receptor. (1S,2S)-Cyclopropylhistamine (VUF 5297) acts as an agonist both on the rat cortex (pD2 = 7.1; alpha = 0.75) and on guinea pig jejunum (pD2 = 6.6; alpha = 0.75). Its enantiomer, (1R, 2R)-cyclopropylhistamine (VUF 5296), is about 1 order of magnitude less active. Both enantiomers show weak activity on H1 and H2 receptors. All synthetic attempts to cis-cyclopropylhistamine were unsuccessful. Nevertheless, the results of this study provide an ideal template for molecular modeling studies of histamine H3 receptor ligands.

Histamine H3 receptor agonists.

The SAR of H3 ligands has been difficult to evaluate because of species differences, multiple isoforms and constitutive activity, among other complicating factors. A review is given of the sometimes-conflicting affinity, activity and efficacy data of H3 agonists that has been described in literature to date.

A structural chemogenomics analysis of aminergic GPCRs: lessons for histamine receptor ligand design.

BACKGROUND AND PURPOSE: Chemogenomics focuses on the discovery of new connections between chemical and biological space leading to the discovery of new protein targets and biologically active molecules. G-protein coupled receptors (GPCRs) are a particularly interesting protein family for chemogenomics studies because there is an overwhelming amount of ligand binding affinity data available. The increasing number of aminergic GPCR crystal structures now for the first time allows the integration of chemogenomics studies with high-resolution structural analyses of GPCR-ligand complexes. EXPERIMENTAL APPROACH: In this study, we have combined ligand affinity data, receptor mutagenesis studies, and amino acid sequence analyses to high-resolution structural analyses of (hist)aminergic GPCR-ligand interactions. This integrated structural chemogenomics analysis is used to more accurately describe the molecular and structural determinants of ligand affinity and selectivity in different key binding regions of the crystallized aminergic GPCRs, and histamine receptors in particular. KEY RESULTS: Our investigations highlight interesting correlations and differences between ligand similarity and ligand binding site similarity of different aminergic receptors. Apparent discrepancies can be explained by combining detailed analysis of crystallized or predicted protein-ligand binding modes, receptor mutation studies, and ligand structure-selectivity relationships that identify local differences in essential pharmacophore features in the ligand binding sites of different receptors. CONCLUSIONS AND IMPLICATIONS: We have performed structural chemogenomics studies that identify links between (hist)aminergic receptor ligands and their binding sites and binding modes. This knowledge can be used to identify structure-selectivity relationships that increase our understanding of ligand binding to (hist)aminergic receptors and hence can be used in future GPCR ligand discovery and design.

Design and pharmacological characterization of VUF14480, a covalent partial agonist that interacts with cysteine 98(3.36) of the human histamine H4 receptor.

BACKGROUND AND PURPOSE: The recently proposed binding mode of 2-aminopyrimidines to the human (h) histamine H4 receptor suggests that the 2-amino group of these ligands interacts with glutamic acid residue E182(5.46) in the transmembrane (TM) helix 5 of this receptor. Interestingly, substituents at the 2-position of this pyrimidine are also in close proximity to the cysteine residue C98(3.36) in TM3. We hypothesized that an ethenyl group at this position will form a covalent bond with C98(3.36) by functioning as a Michael acceptor. A covalent pyrimidine analogue will not only prove this proposed binding mode, but will also provide a valuable tool for H4 receptor research. EXPERIMENTAL APPROACH: We designed and synthesized VUF14480, and pharmacologically characterized this compound in hH4 receptor radioligand binding, G protein activation and ß-arrestin2 recruitment experiments. The ability of VUF14480 to act as a covalent binder was assessed both chemically and pharmacologically. KEY RESULTS: VUF14480 was shown to be a partial agonist of hH4 receptor-mediated G protein signalling and ß-arrestin2 recruitment. VUF14480 bound covalently to the hH4 receptor with submicromolar affinity. Serine substitution of C98(3.36) prevented this covalent interaction. CONCLUSION AND IMPLICATIONS: VUF14480 is thought to bind covalently to the hH4 receptor-C98(3.36) residue and partially induce hH4 receptor-mediated G protein activation and ß-arrestin2 recruitment. Moreover, these observations confirm our previously proposed binding mode of 2-aminopyrimidines. VUF14480 will be a useful tool to stabilize the receptor into an active confirmation and further investigate the structure of the active hH4 receptor.

Pharmacological characterization of a small-molecule agonist for the chemokine receptor CXCR3.

BACKGROUND AND PURPOSE: The chemokine receptor CXCR3 is a GPCR found predominantly on activated T cells. CXCR3 is activated by three endogenous peptides; CXCL9, CXCL10 and CXCL11. Recently, a small-molecule agonist, VUF10661, has been reported in the literature and synthesized in our laboratory. The aim of the present study was to provide a detailed pharmacological characterization of VUF10661 by comparing its effects with those of CXCL11. EXPERIMENTAL APPROACH: Agonistic properties of VUF10661 were assessed in a chemotaxis assay with murine L1.2 cells transiently transfected with cDNA encoding the human CXCR3 receptor and in binding studies, with [(125)I]-CXCL10 and [(125)I]-CXCL11, on membrane preparations from HEK293 cells stably expressing CXCR3. [(35)S]-GTPγS binding was used to determine its potency to induce CXCR3-mediated G protein activation and BRET-based assays to investigate its effects on intracellular cAMP levels and ß-arrestin recruitment. KEY RESULTS: VUF10661 acted as a partial agonist in CXCR3-mediated chemotaxis, bound to CXCR3 in an allosteric fashion in ligand binding assays and activated G(i) proteins with the same efficacy as CXCL11 in the [(35)S]-GTPγS binding and cAMP assay, while it recruited more ß-arrestin1 and ß-arrestin2 to CXCR3 receptors than the chemokine. CONCLUSIONS AND IMPLICATIONS: VUF10661, like CXCL11, activates both G protein-dependent and -independent signalling via the CXCR3 receptor, but probably exerts its effects from an allosteric binding site that is different from that for CXCL11. It could stabilize different receptor and/or ß-arrestin conformations leading to differences in functional output. Such ligand-biased signalling might offer interesting options for the therapeutic use of CXCR3 agonists.

Antinociceptive activity of furan-containing congeners of improgan and ranitidine.

Furan-containing congeners of the histamine H(2) receptor antagonist ranitidine were synthesized and tested for improgan-like antinociceptive activity. The most potent ligand of the series, VUF5498, is the most potent improgan-like agent described to date (ED(50)=25 nmol, icv). This compound is approximately equal in potency with morphine. These non-imidazole, improgan-like pain relievers further define the structural requirements for analgesics of this class and are important tools for ongoing mechanism-based studies.

Virus-encoded G-protein-coupled receptors: constitutively active (dys)regulators of cell function and their potential as drug target.

G-protein-coupled receptors encoded by herpesviruses such as EBV, HCMV and KSHV are very interesting illustrations of the (patho)physiological importance of constitutive GPCR activity. These viral proteins are expressed on the cell surface of infected cells and often constitutively activate a variety of G-proteins. For some virus-encoded GPCRs, the constitutive activity has been shown to occur in vivo, i.e., in infected cells. In this paper, we will review the occurrence of virus-encoded GPCRs and describe their known signaling properties. Moreover, we will also review the efforts, directed towards the discovery of small molecule antagonist, that so far have been mainly focused on the HCMV-encoded GPCR US28. This virus-encoded receptor might be involved in cardiovascular diseases and cancer and seems an interesting target for drug intervention.

A qualitative model for the histamine H3 receptor explaining agonistic and antagonistic activity simultaneously.

A pharmacophore model for histamine H3 ligands is derived that reveals the putative interaction of both H3 agonists and antagonists with an aspartate residue of the receptor. This interaction is determined by applying the density functional theory implemented in a program package adapted for parallel computers. The model reveals a molecular determinant explaining efficacy as the conformation of the aspartic acid residue differs according to whether it is binding to agonists or antagonists. The differences in structure-activity relationships (SAR) observed for the lipophilic tails of different classes of H3 antagonists are now explained, since the model reveals two distinct lipophilic pockets available for antagonist binding.

SLATE: a method for the superposition of flexible ligands.

A novel program for the superposition of flexible molecules, SLATE, is presented. It uses simulated annealing to minimise the difference between the distance matrices calculated from the hydrogen-bonding and aromaticring properties of two ligands. A method for generating a molecular stack using multiple pairwise matches is illustrated. These stacks are used by the program DOH to predict the relative positions of receptor atoms that could form hydrogen bonds to two or more ligands in the dataset. The methodology has been applied to ligands binding to dihydrofolate reductase, thermolysin. H3 histamine receptors, alpha2 adrenoceptors and 5-HT1D receptors. When there are sufficient numbers and diversity of molecules in the dataset, the prediction of receptor-atom positions is applicable to compound design.

Synthesis and histamine H3 receptor activity of 4-(n-alkyl)-1H-imidazoles and 4-(omega-phenylalkyl)-1H-imidazoles.

The influence of lipophilic moieties attached to a 4-1H-imidazole ring on the histamine H3 receptor activity was systematically investigated. Series of 4-(n-alkyl)-1H-imidazoles and 4-(omega-phenylalkyl)-1H-imidazoles were prepared, with an alkyl chain varying from 2-9 methylene groups and from 1-9 methylene groups, respectively. The compounds were tested for their activity on the H3 receptor under in vitro conditions. For the 4-(n-alkyl)-1H-imidazoles the activity is proportional to chain length, ranging from a pA2 value of 6.3 +/- 0.2 for 4-(n-propyl)-1H-imidazole to a pA2 value of 7.2 +/- 0.1 for 4-(n-decyl)-1H-imidazole. For the series 4-(omega-phenylalkyl)-4H-imidazoles an optimum in H3 activity was found for the pentylene spacer: 4-(omega-phenylpentyl)-1H-imidazole has a pA2 value of 7.8 +/- 0.1.