History and Prospects of Drug Discovery and Development Collaboration between Industry and Academia.

Research collaborations and licensing deals are critical for the discovery and development of life-saving drugs. This practice has been ongoing since the inception of the pharmaceutical industry. The current process of drug discovery and development is complex, regulated, and highly regimented, having evolved over time. Academia excels in the discovery of fundamental scientific concepts and biological processes, while industry excels in translational science and product development. Potential for collaboration exists at every step of the drug discovery and development continuum. This perspective walks through such collaborative activities, provides examples, and offers tips for potential collaborations.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) affects gene expression pathways beyond cholesterol metabolism in liver cells.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) induces degradation of low-density lipoprotein receptor (LDLR) in the liver. It is being pursued as a therapeutic target for LDL-cholesterol reduction. Earlier genome-wide gene expression studies showed that PCSK9 over-expression in HepG2 cells resulted in up-regulation of genes in cholesterol biosynthesis and down-regulation of genes in stress response pathways; however, it was not known whether these changes were directly regulated by PCSK9 or were secondary to PCSK9-induced changes to the intracellular environment. In order to further understand the biological function of PCSK9 we treated HepG2 cells with purified recombinant wild type (WT) and D374Y gain-of-function PCSK9 proteins for 8, 24, and 48 h, and used microarray analysis to identify genome-wide expression changes and pathways. These results were compared to the changes induced by culturing HepG2 cells in cholesterol-free medium, mimicking the intracellular environment of cholesterol starvation. We determined that PCSK9-induced up-regulation of cholesterol biosynthesis genes resulted from intracellular cholesterol starvation. In addition, we identified novel pathways that are presumably regulated by PCSK9 and are independent of its effects on cholesterol uptake. These pathways included "protein ubiquitination," "xenobiotic metabolism," "cell cycle," and "inflammation and stress response." Our results indicate that PCSK9 affects metabolic pathways beyond cholesterol metabolism in HepG2 cells.

Short-chain fatty acids act as antiinflammatory mediators by regulating prostaglandin E(2) and cytokines.

AIM: To investigate the effect of short-chain fatty acids (SCFAs) on production of prostaglandin E(2) (PGE(2)), cytokines and chemokines in human monocytes. METHODS: Human neutrophils and monocytes were isolated from human whole blood by using 1-Step Polymorph and RosetteSep Human Monocyte Enrichment Cocktail, respectively. Human GPR41 and GPR43 mRNA expression was examined by quantitative real-time polymerase chain reaction. The calcium flux assay was used to examine the biological activities of SCFAs in human neutrophils and monocytes. The effect of SCFAs on human monocytes and peripheral blood mononuclear cells (PBMC) was studied by measuring PGE(2), cytokines and chemokines in the supernatant. The effect of SCFAs in vivo was examined by intraplantar injection into rat paws. RESULTS: Human GPR43 is highly expressed in human neutrophils and monocytes. SCFAs induce robust calcium flux in human neutrophils, but not in human monocytes. In this study, we show that SCFAs can induce human monocyte release of PGE(2) and that this effect can be enhanced in the presence of lipopolysaccharide (LPS). In addition, we demonstrate that PGE(2) production induced by SCFA was inhibited by pertussis toxin, suggesting the involvement of a receptor-mediated mechanism. Furthermore, SCFAs can specifically inhibit constitutive monocyte chemotactic protein-1 (MCP-1) production and LPS-induced interleukin-10 (IL-10) production in human monocytes without affecting the secretion of other cytokines and chemokines examined. Similar activities were observed in human PBMC for the release of PGE(2), MCP-1 and IL-10 after SCFA treatment. In addition, SCFAs inhibit LPS-induced production of tumor necrosis factor-alpha and interferon-gamma in human PBMC. Finally, we show that SCFAs and LPS can induce PGE(2) production in vivo by intraplantar injection into rat paws (P < 0.01). CONCLUSION: SCFAs can have distinct antiinflammatory activities due to their regulation of PGE(2), cytokine and chemokine release from human immune cells.

Characterizing cannabinoid CB2 receptor ligands using DiscoveRx PathHunter beta-arrestin assay.

The authors have characterized a set of cannabinoid CB(2) receptor ligands, including triaryl bis sulfone inverse agonists, in a cell-based receptor/beta-arrestin interaction assay (DiscoveRx PathHunter). The results were compared with results using a competitive ligand binding assay, and with effects on forskolin-stimulated cAMP levels (PerkinElmer LANCE). The authors show good correlation between the 3 assay systems tested, with the beta-arrestin protein complementation assay exhibiting a more robust signal than the cAMP assay for cannabinoid CB(2) agonists. Further assay validation shows that DiscoveRx PathHunter HEK293 CB(2) beta-arrestin assay can be carried out from cryopreserved cell suspensions, eliminating variations caused by the need for multiple cell pools during live cell screening campaigns. These results, and the authors' results evaluating a test set of random library compounds, validate the use of ligand-induced interaction between the human cannabinoid CB(2) receptor and beta-arrestin as an appropriate and valuable screening platform for compounds specific for the cannabinoid CB(2) receptor.

Discovery of novel chemotypes to a G-protein-coupled receptor through ligand-steered homology modeling and structure-based virtual screening.

Melanin-concentrating hormone receptor 1 (MCH-R1) is a G-protein-coupled receptor (GPCR) and a target for the development of therapeutics for obesity. The structure-based development of MCH-R1 and other GPCR antagonists is hampered by the lack of an available experimentally determined atomic structure. A ligand-steered homology modeling approach has been developed (where information about existing ligands is used explicitly to shape and optimize the binding site) followed by docking-based virtual screening. Top scoring compounds identified virtually were tested experimentally in an MCH-R1 competitive binding assay, and six novel chemotypes as low micromolar affinity antagonist "hits" were identified. This success rate is more than a 10-fold improvement over random high-throughput screening, which supports our ligand-steered method. Clearly, the ligand-steered homology modeling method reduces the uncertainty of structure modeling for difficult targets like GPCRs.

P518/Qrfp sequence polymorphisms in SAMP6 osteopenic mouse.

Mice lacking GPR103A expression display osteopenia. Analysis of mouse quantitative trait loci literature associated with bone mineral density suggested GPR103A ligand P518/Qrfp (chromosome 2qB) as a candidate osteoporosis gene. Promoter and coding regions of mouse P518/Qrfp were sequenced from genomic DNA obtained from the osteoporosis-prone strain SAMP6 and control strains SAMR1, A/J, AKR/J, BALB/c, C3H/HeJ, C57BL/6J, and DBA/2J. Four single-nucleotide polymorphisms (SNPs) were identified in only SAMP6 genomic DNA, g.-1773 T-->C, g.110 A-->G (N37S), g.188 G-->A (R63K), and g.135 T-->C (H45H). The promoter SNP generated a novel neuron-restrictive silencing factor binding site, a repressor that decreases gene expression in nonneuronal tissues. TaqMan analysis demonstrated fivefold lower P518/Qrfp liver expression in SAMP6 versus SAMR1 or C57BL/6J control strains. Tissue distribution of human, mouse, and rat P518/Qrfp and its receptors showed expression in bone and spinal cord. A direct role for P518/Qrfp function in maintaining bone mineral density is suggested.

Sequence variation in NPC1L1 and association with improved LDL-cholesterol lowering in response to ezetimibe treatment.

Niemann-Pick C1-like 1 (NPC1L1) is an intestinal cholesterol transporter and the molecular target of ezetimibe, a cholesterol absorption inhibitor demonstrated to reduce LDL-cholesterol (LDL-C) both as monotherapy and when co-administered with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins). Interestingly, significant interindividual variability has been observed for rates of intestinal cholesterol absorption and LDL-C reductions at both baseline and post ezetimibe treatment. To test the hypothesis that genetic variation in NPC1L1 could influence the LDL-C response to ezetimibe, we performed extensive resequencing of the gene in 375 apparently healthy individuals and genotyped hypercholesterolemic patients from clinical trial cohorts. No association was observed between NPC1L1 single-nucleotide polymorphism and baseline cholesterol. However, significant associations to LDL-C response to treatment with ezetimibe were observed in patients treated with ezetimibe in two large clinical trials. Our data demonstrate that DNA sequence variants in NPC1L1 are associated with an improvement in response to ezetimibe pharmacotherapy and suggest that detailed analysis of genetic variability in clinical trial cohorts can lead to improved understanding of factors contributing to variable drug response.

PepPat, a pattern-based oligopeptide homology search method and the identification of a novel tachykinin-like peptide.

UNLABELLED: PepPat, a hybrid method that combines pattern matching with similarity scoring, is described. We also report PepPat's application in the identification of a novel tachykinin-like peptide. PepPat takes as input a query peptide and a user-specified regular expression pattern within the peptide. It first performs a database pattern match and then ranks candidates on the basis of their similarity to the query peptide. PepPat calculates similarity over the pattern spanning region, enhancing PepPat's sensitivity for short query peptides. PepPat can also search for a user-specified number of occurrences of a repeated pattern within the target sequence. We illustrate PepPat's application in short peptide ligand mining. As a validation example, we report the identification of a novel tachykinin-like peptide, C14TKL-1, and show it is an NK1 (neuokinin receptor 1) agonist whose message is widely expressed in human periphery. AVAILABILITY: PepPat is offered online at: http://peppat.cbi.pku.edu.cn.

Identification and characterization of a novel RF-amide peptide ligand for orphan G-protein-coupled receptor SP9155.

Orphan G-protein-coupled receptors are a large class of receptors whose cognate ligands are unknown. SP9155 (also referred to as AQ27 and GPR103) is an orphan G-protein-coupled receptor originally cloned from a human brain cDNA library. SP9155 was found to be predominantly expressed in brain, heart, kidney, retina, and testis. Phylogenetic analysis shows that SP9155 shares high homology with Orexin, NPFF, and cholecystokinin (CCK) receptors, but identification of the endogenous ligand for SP9155 has not been reported. In this study, we have used a novel method to predict peptides from genome data bases. From these predicted peptides, a novel RF-amide peptide, P52 was shown to selectively activate SP9155-transfected cells. We subsequently cloned the precursor gene of the P52 ligand and characterized the activity of other possible peptides encoded by the precursor. This revealed an extended peptide, P518, which exhibited high affinity for SP9155 (EC50 = 7 nm). mRNA expression analysis revealed that the peptide P518 precursor gene is predominantly expressed in various brain regions, coronary arteries, thyroid and parathyroid glands, large intestine, colon, bladder, testes, and prostate. These results indicate the existence of a novel RF-amide neuroendocrine peptide system, and suggest that SP9155 is likely the relevant G-protein-coupled receptor for this peptide.

Cloning and characterization of murine neuromedin U receptors.

Neuromedin U (NmU) is a neuropeptide involved in various physiological functions such as feeding behavior, muscle contractile activity, and regulation of intestinal ion transport. Recently, two human G protein-coupled receptors have been identified as NmU-specific receptors, NmU-R1 and NmU-R2, which share 55% amino acid identity. It is unclear however, which of the two receptors mediates responses to NmU observed in rodent models. Attempts to define the pharmacological profile of the two receptors are confounded by overlapping expression of the two receptors and a lack of subtype-selective compounds. In order to establish a basis to further our understanding of the function of these receptors, we cloned and characterized the mouse homologues of the two human NmU receptors. Mouse NmU-R1 and mouse NmU-R2 are 79 and 81% identical to their respective human homologues. Expression of NmU-R1 was mainly observed in testis, gastrointestinal (GI) tract, and immune system, while NmU-R2 was primarily expressed in brain tissues. Each mouse receptor was independently expressed in HEK293 cells and demonstrated a dose-dependent calcium flux in response to NmU-8, NmU-23 and NmU-25. In an attempt to identify a synthetic NmU peptide that would exhibit selectivity at one of the two receptors, we examined the functional activity of eight alanine-substituted NmU-8 peptides. These experiments demonstrated that alanine substitution at positions 5 and 7 affects the functional activity of the peptide at both receptors. The arginine residue at position 7 is required for NmU-8 activity at either receptor while alanine substitution at position 5 selectively affects the potency and the efficacy at mNmU-R1. These experiments validate the use of rodent models to characterize NmU function relative to humans and suggest that substitution at Arginine-5 of NmU-8 may provide a receptor selective peptide.

Molecular modeling and site-specific mutagenesis of the histamine-binding site of the histamine H4 receptor.

The histamine H4 receptor is a novel G-protein-coupled receptor with a unique pharmacological profile. The distribution of H4 mRNA suggests that it may play a role in the regulation of immune function, particularly with respect to allergy and asthma. To define the histamine-binding site of this receptor, molecular modeling and site-directed mutagenesis were used to predict and alter amino acids residing in the histamine-binding pocket. The effects of these alterations on histamine binding and receptor activation were then assessed. Our results indicate that Asp94 (3.32) in transmembrane region (TM) 3 and Glu182 (5.46) in TM5 are critically involved in histamine binding. Asp94 probably serves as a counter-anion to the cationic amino group of histamine, whereas Glu182 (5.46) interacts with the N(tau) nitrogen atom of the histamine imidazole ring via an ion pair. In contrast, Thr178 (5.42) and Ser179 (5.43) in TM5 are not significantly involved in either histamine binding or receptor activation. These results resemble those for the analogous residues in the H1 histamine receptor but contrast with findings regarding the H2 histamine receptor. Our results also demonstrate that Asn147 (4.57) in TM4 and Ser320 (6.52) in TM6 play a role in receptor activation but are not involved in histamine binding. Taken together, these data indicate that although histamine seems to bind to the H4 receptor in a fashion similar to that predicted for the other histamine receptor subtypes, there are also important differences that can probably be exploited for the discovery of novel H4-selective compounds.

P2Y(13): identification and characterization of a novel Galphai-coupled ADP receptor from human and mouse.

We have identified an orphan G protein-coupled receptor, SP174, that shares a high degree of homology with the recently described ADP receptor P2Y(12). mRNA for SP174 is abundant in the brain and in cells of the immune system. In the present study, we demonstrate that SP174 is also a receptor for ADP, which is coupled to Galphai. ADP potently stimulates SP174 with an EC(50) of 60 nM, and other related nucleotides are active as well, with a rank order of potency 2-methylthio-ADP tetrasodium = adenosine 5'-O-2-(thio)diphosphate = 2-methylthio-ATP tetrasodium > ADP > AP3A >ATP > IDP. This pharmacological profile is similar to that for P2Y(12). We have also identified the murine homolog of SP174, which exhibits 75% homology to the human receptor. ADP is also a potent agonist at the murine receptor, and its pharmacological profile is similar to its human counterpart, but ADP and related nucleotides are more potent at the murine receptor than the human receptor. In keeping with the general nomenclature for the purinergic receptors, we propose designating this novel receptor P2Y(13).