Supplementary Materials and Methods, Tables S1 through S3 and Figures S1 and S2 - Supplementary Materials and Methods: The sources, application and dilution of the antibodies used for Western blotting (WB) and immunohistochemical (IHC) analyses in this study are listed. Supplementary Tables: The supplementary tables demonstrate the data and statistics from x-ray co-crystallization experiments with BI 885578 and the human INSR kinase domain (Tables S1 and S2) and the kinase selectivity of BI 885578 on a large panel of human kinases (Table S3). Supplementary Figures: The supplementary figures demonstrate the efficacy and tolerability of BI 885578 in the CL-14 colon cancer xenograft model (Figure S1) and the PD effects of BI 885578 on the phosphorylation of the IGF1R and INSR in GEO tumors (Figure S2).

Pharmacological probes are important tools for exploring disease biology and discovering new therapies. Often molecules of insufficient quality are used instead, leading to spurious and misleading results. The Boehringer Ingelheim open innovation portal opnMe.com addresses this deficiency by sharing extensively validated pharmacological probes with the scientific community.

In the current study we have evaluated the applicability of ligand-based virtual screening (LBVS) methods for the identification of small fragment-like biologically active molecules using different similarity descriptors and different consensus scoring approaches. For this purpose, we have evaluated the performance of 14 chemical similarity descriptors in retrospective virtual screening studies to discriminate fragment-like ligands of three membrane-bound receptors from fragments that are experimentally determined to have no affinity for these proteins (true inactives). We used a complete fragment affinity data set of experimentally determined ligands and inactives for two G protein-coupled receptors (GPCRs), the histamine H-1 receptor (H1R) and the histamine H-4 receptor (H4R), and one ligand-gated ion channel (LGIC), the serotonin receptor (5-HT(3)AR), to validate our retrospective virtual screening studies. We have exhaustively tested consensus scoring strategies that combine the results of multiple actives (group fusion) or combine different similarity descriptors (similarity fusion), and for the first time systematically evaluated different combinations of group fusion and similarity fusion approaches. Our studies show that for these three case study protein targets both consensus scoring approaches can increase virtual screening enrichments compared to single chemical similarity search methods. Our cheminformatics analyses recommend to use a combination of both group fusion and similarity fusion for prospective ligand-based virtual fragment screening.

Abstract Inhibition of the IGF1R, INSRA, and INSRB receptor tyrosine kinases represents an attractive approach of pharmacologic intervention in cancer, owing to the roles of the IGF1R and INSRA in promoting cell proliferation and survival. However, the central role of the INSRB isoform in glucose homeostasis suggests that prolonged inhibition of this kinase could result in metabolic toxicity. We describe here the profile of the novel compound BI 885578, a potent and selective ATP-competitive IGF1R/INSR tyrosine kinase inhibitor distinguished by rapid intestinal absorption and a short in vivo half-life as a result of rapid metabolic clearance. BI 885578, administered daily per os, displayed an acceptable tolerability profile in mice at doses that significantly reduced the growth of xenografted human GEO and CL-14 colon carcinoma tumors. We found that treatment with BI 885578 is accompanied by increases in circulating glucose and insulin levels, which in turn leads to compensatory hyperphosphorylation of muscle INSRs and subsequent normalization of blood glucose within a few hours. In contrast, the normalization of IGF1R and INSR phosphorylation in GEO tumors occurs at a much slower rate. In accordance with this, BI 885578 led to a prolonged inhibition of cell proliferation and induction of apoptosis in GEO tumors. We propose that the remarkable therapeutic window observed for BI 885578 is achieved by virtue of the distinctive pharmacokinetic properties of the compound, capitalizing on the physiologic mechanisms of glucose homeostasis and differential levels of IGF1R and INSR expression in tumors and normal tissues. Mol Cancer Ther; 14(12); 2762–72. ©2015 AACR.

In the current study we have evaluated the applicability of ligand-based virtual screening (LBVS) methods for the identification of small fragment-like biologically active molecules using different similarity descriptors and different consensus scoring approaches. For this purpose, we have evaluated the performance of 14 chemical similarity descriptors in retrospective virtual screening studies to discriminate fragment-like ligands of three membrane-bound receptors from fragments that are experimentally determined to have no affinity for these proteins (true inactives). We used a complete fragment affinity data set of experimentally determined ligands and inactives for two G protein-coupled receptors (GPCRs), the histamine H1 receptor (H1R) and the histamine H4 receptor (H4R), and one ligand-gated ion channel (LGIC), the serotonin receptor (5-HT3AR), to validate our retrospective virtual screening studies. We have exhaustively tested consensus scoring strategies that combine the results of multiple actives (group fusion) or combine different similarity descriptors (similarity fusion), and for the first time systematically evaluated different combinations of group fusion and similarity fusion approaches. Our studies show that for these three case study protein targets both consensus scoring approaches can increase virtual screening enrichments compared to single chemical similarity search methods. Our cheminformatics analyses recommend to use a combination of both group fusion and similarity fusion for prospective ligand-based virtual fragment screening.

The increasing number of G protein-coupled receptor (GPCR) crystal structures offers new opportunities for histamine receptor homology modeling. However, computational prediction of ligand binding modes in GPCRs such as the histamine H4 receptor (H4R), a receptor that plays an important role in inflammation, remains a challenging task. In the current work we have combined complementary in silico receptor modeling approaches with in vitro ligand structure–activity relationship (SAR) and protein site-directed mutagenesis studies to elucidate the binding modes of different ligand classes in H4R. By systematically considering different H4R modelling templates, ligand binding poses, and ligand protonation states in combination with docking and MD simulations we are able to explain ligand-specific mutation effects and subtle differences in ligand SAR. Our studies confirm that a combined theoretical and experimental approach represents a powerful strategy to map ligand–protein interactions.

Background and Purpose The recently proposed binding mode of 2-aminopyrimidines to the human (h) histamine H-4 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 H-4 receptor research. Experimental Approach We designed and synthesized VUF14480, and pharmacologically characterized this compound in hH(4) receptor radioligand binding, G protein activation and -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 hH(4) receptor-mediated G protein signalling and -arrestin2 recruitment. VUF14480 bound covalently to the hH(4) 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 hH(4) receptor-C98(3.36) residue and partially induce hH(4) receptor-mediated G protein activation and -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 hH(4) receptor.

The basic methylpiperazine moiety is considered a necessary substructure for high histamine H4 receptor (H4R) affinity. This moiety is however also the metabolic hot spot for various classes of H4R ligands (e.g., indolcarboxamides and pyrimidines). We set out to investigate whether mildly basic 2-aminopyrimidines in combination with the appropriate linker can serve as a replacement for the methylpiperazine moiety. In the series of 2-aminopyrimidines, the introduction of an additional 2-aminopyrimidine moiety in combination with the appropriate linker lead to bispyrimidines displaying pKi values for binding the human H4R up to 8.2. Furthermore, the methylpiperazine replacement results in compounds with improved metabolic properties. The attempt to transfer the knowledge generated in the class of bispyrimidines to the indolecarboxamides failed. Combining the derived structure-activity relationships with homology modeling leads to new detailed insights in the molecular aspects of ligand-H4R binding in general and the binding mode of the described bispyrimidines in specific.

Virtual screening (VS) has become an integral part of fragment-based drug discovery (FBDD). In this study we have evaluated the applicability of ligand-based virtual screening (LBVS) methods for identifying small fragment-like biologically active molecules using different similarity descriptors and different consensus scoring approaches. For this purpose we have evaluated the performance of 14 chemical similarity descriptors in retrospective virtual screening studies to discriminate fragment-like ligands of three membrane bound receptors from fragments that are experimentally determined to have no affinity for these proteins (true inactives). We used a consistent fragment affinity data set for two G Protein-Coupled Receptors (GPCRs), the histamine H4 receptor (H4R) and the histamine H1 receptor (H1R), and one Ligand-Gated Ion Channel (LGIC), the serotonin receptor (5HT3AR), to validate our systematic retrospective virtual screening studies. We further tested different consensus scoring methods to combine the results for multiple actives and all possible combinations of different similarity descriptors. Our studies show that LBVS methods are able to discriminate active from inactive fragments and allow the identification of scaffolds that are different from the reference template structure. Best enrichments were observed with quite large reference actives. These actives however rank larger actives higher than smaller ones. This indicates that LBVS for especially small fragments is a challenging task. Consensus scoring of multiple actives (group fusion) or multiple similarity descriptors (similarity fusion) is very effective to increase enrichments. Whereas the enrichments of individual similarity descriptors are quite target dependent, most combinations gave better enrichments than the average enrichment. For prospective virtual screening our results highly recommend to use a combination of both, group fusion and data fusion.

SAR beyond protein-ligand interactions: By combining structure-affinity relationships, protein-ligand modeling studies, and quantum mechanical calculations, we show that ligand conformational energies and basicity play critical roles in ligand binding to the histamine H4 receptor, a GPCR that plays a key role in inflammation.

A series of 76 derivatives of the indolecarboxamide 1 were synthesized, which allows a detailed SAR investigation of this well known scaffold. The data enable the definition of a predictive QSAR model which identifies several compounds with an activity comparable to 1. A selection of these new H(4)R antagonists was synthesized and a comparison of predicted and measured values demonstrates the robustness of the model (47-55). In addition to the H(4)-receptor activity general CMC and DMPK properties were investigated. Some of the new analogs are not only excellently soluble, but display a significantly increased half-life in mouse liver microsomes as well. These properties qualify these compounds as a possible new standard for future in vivo studies (e.g 51, 52 and 55). Moreover, the current studies also provide valuable information on the potential receptor ligand interactions between the indolcarboxamides and the H(4)R protein.

The influence of simple molecular descriptors on the melting point temperature (T-m) has been investigated using a matched molecular pair (MMP) analysis. This method has been used to identify small structural differences between pairs of molecules which are related to changes in T-m. Our analysis shows that the number of hydrogen bond donors, hydrogen bond acceptors and rotatable bonds has a significant effect on the T-m of a molecule. Hydrogen bond donors have the most pronounced effect. Furthermore, the studies reveal that not ClogP but rather the number of bromine and iodine atoms has a marked effect on the T-m. The results of our MMP analysis are discussed within the context of drug solubility optimization.

Deregulation of the ErbB (proto-oncogene B of the avian erythroblastosis virus AEV-H strain) receptor network is well recognized as an oncogenic driver in epithelial cancers. Several targeted drugs have been developed, including antibodies and small-molecule kinase inhibitors, each of them characterized by distinct patterns of ErbB receptor interactions. Understanding the precise pharmacological properties of these compounds is important for optimal use in clinical practice. Afatinib [BIBW 2992; N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[[(3S)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4-(dimethylamino)-2-butenamide] is an ATP-competitive anilinoquinazoline derivative harboring a reactive acrylamide group. It was designed to covalently bind and irreversibly block enzymatically active ErbB receptor family members. Here, we show by X-ray crystallography the covalent binding of afatinib to wild-type epidermal growth factor receptor (EGFR) and by mass spectrometry the covalent interaction with EGFR, EGFRL858R/T790M, human epidermal growth factor receptor 2 (HER2), and ErbB-4. Afatinib potently inhibits the enymatic activity of ErbB-4 (EC50=1 nM) and the proliferation of cancer cell lines driven by multiple ErbB receptor aberrations at concentrations below 100 nM. N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[[(3S)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4-(dimethylamino)-2-butanamide (BI 37781), a close analog of afatinib lacking the acrylamide group and thus incapable of covalent bond formation, had similar potency on cells driven by EGFR or EGFRL858R, but less or no detectable activity on cells expressing EGFRL858R/T790M HER2 or ErbB-4. These results stress the importance of the acrylamide group and show that afatinib differs from approved ErbB targeting agents by irreversibly inhibiting the kinase activity of all ErbB family members. They provide a mechanistic rationale for the distinct pharmacological features of this compound and explain the clinical activity seen in some patients who are resistant to antibody or kinase inhibitor therapy because of secondary mutations or ErbB receptor "reprogramming."

Deregulation of the ErbB receptor network is well recognized as an oncogenic driver in epithelial cancers. Several targeted drugs have been developed, including antibodies and small molecule kinase inhibitors, each of them characterized by distinct patterns of ErbB receptor interactions. Understanding the precise pharmacological properties of these compounds is important for optimal use in clinical practice. Afatinib (BIBW 2992) is an ATP-competitive anilinoquinazoline derivative harboring a reactive acrylamide group and was designed to covalently bind and irreversibly block enzymatically active ErbB receptor family members. Here we show by X-ray crystallography the covalent binding of afatinib to wild-type EGFR and by mass spectrometry the covalent interaction with EGFR, EGFR, HER2 and ErbB-4. Afatinib potently inhibits the enymatic activity of ErbB-4 (EC50 = 1 nM) and the proliferation of cancer cell lines driven by multiple ErbB receptor aberrations at concentrations below 100 nM. BI 37781, a close analog of afatinib lacking the acrylamide group and thus incapable of covalent bond formation, had similar potency on cells driven by EGFR or EGFR, but less or no detectable activity on cells expressing EGFR T790M HER2 or ErbB-4. These results stress the importance of the acrylamide group and show that afatinib differs from approved ErbB targeting agents by irreversibly inhibiting the kinase activity of all ErbB family members. This article has not been copyedited and formatted. The final version may differ from this version. JPET Fast Forward. Published on August 10, 2012 as DOI: 10.1124/jpet.112.197756 at A PE T Jornals on A ril 0, 2017 jpet.asjournals.org D ow nladed from

Within the last decades, the detailed knowledge on the impact of membrane bound drug efflux transporters of the ATP binding cassette (ABC) protein family on the pharmacological profile of drugs has enormously increased. Especially, ABCB1 (P-glycoprotein, P-gp, MDR1) has attracted particular interest in medicinal chemistry, since it determines the clinical efficacy, side effects and toxicity risks of drug candidates. Based on this, the development of in silico models that provide rapid and cost-effective screening tools for the classification of substrates and nonsubstrates of ABCB1 is an urgent need in contemporary ADMET profiling. A characteristic hallmark feature of this transporter is its polyspecific ligand recognition pattern. In this study we describe a method for classifying ABCB1 ligands in terms of simple, conjunctive rules (RuleFit) based on interpretable ADMET features. The retrieved results showed that models based on large, very diverse data sets gave better classification performance than models based on smaller, more homogenous training sets. The best model achieved gave a correct classification rate of 0.90 for an external validation set. Furthermore, from the interpretation of the best performing model it could be concluded that in comparison to nonsubstrates ABCB1 substrates generally show a higher number of hydrogen-bond acceptors, are more flexible and exhibit higher logP values.

Fragment-based screening (FBS) has become an established approach for hit identification. Starting points identified by FBS, are small fragments that require substantial modification to become leads. As fragments are different from classical hits a process tailored for fragment evolution is required. Scores for ligand efficiency have been proposed as guides for this process. Here we review how these have been applied to guide the selection and optimization of fragment hits.

Previous studies have demonstrated that clobenpropit (N-(4-chlorobenzyl)-S-[3-(4(5)-imidazolyl)propyl]isothiourea) binds to both the human histamine H(3) receptor (H(3)R) and H(4) receptor (H(4)R). In this paper, we describe the synthesis and pharmacological characterization of a series of clobenpropit analogs, which vary in the functional group adjacent to the isothiourea moiety in order to study structural requirements for H(3)R and H(4)R ligands. The compounds show moderate to high affinity for both the human H(3)R and H(4)R. Furthermore, the changes in the functional group attached to the isothiourea moiety modulate the intrinsic activity of the ligands at the H(4)R, ranging from neutral antagonism to full agonism. QSAR models have been generated in order to explain the H(3)R and H(4)R affinities.

At the turn of the millennium, the DNA sequence encoding the histamine H4 receptor (H4R) was identified in data from human genome databases. Considering the clinical importance of H1R and H2R ligands, and the clinical trials that are ongoing for H3R ligands, the latest addition to the histamine receptor family was noted with interest by the pharmaceutical industry. Initial studies describing the expression of the H4R, and the activity of this receptor in (patho)physiology, suggested that the H4R played a role in the immune system. The introduction of the reference H4R antagonist JNJ-7777120 (Johnson & Johnson Pharmaceutical Research & Development LLC/Abbott Laboratories), and proof of the efficacy of this agent in models of asthma, allergic rhinitis and pruritus, highlighted the H4R as a novel drug target. The first clinical candidates targeting the H4R have been identified, and new H4R antagonists are expected to enter the clinic in the near future.

ABC-type drug efflux pumps, e.g., ABCB1 (=P-glycoprotein, =MDR1), ABCC1 (=MRP1), and ABCG2 (=MXR, =BCRP), confer a multi-drug resistance (MDR) phenotype to cancer cells. Furthermore, the important contribution of ABC transporters for bioavailability, distribution, elimination, and blood-brain barrier permeation of drug candidates is increasingly recognized. This review presents an overview on the different computational methods and models pursued to predict ABC transporter substrate properties of drug-like compounds. They encompass ligand-based approaches ranging from 'simple rule'-based efforts to sophisticated machine learning methods. Many of these models show excellent performance for the data sets used. However, due to the complex nature of the applied methods, useful interpretation of the models that can be directly translated into chemical structures by the medicinal chemist is rather difficult. Additionally, very recent and promising attempts in the field of structure-based modeling of ABC transporters, which embody homology modeling as well as recently published X-ray structures of murine ABCB1, will be discussed.