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1.  [No title available] 
PMCID: PMC4082686  PMID: 24648045
2.  INTEGRATING COMPUTATIONAL PROTEIN FUNCTION PREDICTION INTO DRUG DISCOVERY INITIATIVES 
Drug development research  2011;72(1):4-16.
Pharmaceutical researchers must evaluate vast numbers of protein sequences and formulate innovative strategies for identifying valid targets and discovering leads against them as a way of accelerating drug discovery. The ever increasing number and diversity of novel protein sequences identified by genomic sequencing projects and the success of worldwide structural genomics initiatives have spurred great interest and impetus in the development of methods for accurate, computationally empowered protein function prediction and active site identification. Previously, in the absence of direct experimental evidence, homology-based protein function annotation remained the gold-standard for in silico analysis and prediction of protein function. However, with the continued exponential expansion of sequence databases, this approach is not always applicable, as fewer query protein sequences demonstrate significant homology to protein gene products of known function. As a result, several non-homology based methods for protein function prediction that are based on sequence features, structure, evolution, biochemical and genetic knowledge have emerged. Herein, we review current bioinformatic programs and approaches for protein function prediction/annotation and discuss their integration into drug discovery initiatives. The development of such methods to annotate protein functional sites and their application to large protein functional families is crucial to successfully utilizing the vast amounts of genomic sequence information available to drug discovery and development processes.
doi:10.1002/ddr.20397
PMCID: PMC4270266  PMID: 25530654
function prediction; protein annotation; structural comparison; drug discovery; structural genomics; bioinformatics
3.  Ensemble Classification of Cancer Types and Biomarker Identification 
Drug development research  2012;73(7):414-419.
Cancer classification is an important step in biomarker identification. Developing machine learning methods that correctly predict cancer subtypes/types can help in identifying potential cancer biomarkers. In this commentary, we presented ensemble classification approach and compared its performance with single classification approaches. Additionally, the application of cancer classification in identifying biomarkers for drug design was discussed.
doi:10.1002/ddr.21032
PMCID: PMC4162531  PMID: 25221378
cancer classification; gene expression; ensemble; biomarker; drug design
4.  Anti-Oligomannose Antibodies as Potential Serum Biomarkers of Aggressive Prostate Cancer 
Drug development research  2013;74(2):65-80.
Strategy, Management and Health PolicyEnabling Technology, Genomics, ProteomicsPreclinical ResearchPreclinical Development Toxicology, Formulation Drug Delivery, PharmacokineticsClinical Development Phases I-III Regulatory, Quality, ManufacturingPostmarketing Phase IV
This study bridges a carbohydrate microarray discovery and a large-scale serological validation of anti-oligomannose antibodies as novel serum biomarkers of aggressive prostate cancer (PCa). Experimentally, a Man9-cluster-specific enzyme-linked immunosorbent assay was established to enable sensitive detection of anti-Man9 antibodies in human sera. A large-cohort of men with PCa or benign prostatic hyperplasia (BPH) whose sera were banked at Stanford University was characterized using this assay. Subjects included patients with 100% Gleason grade 3 cancer (n = 84), with Gleason grades 4 and/or 5 cancer (n = 204), and BPH controls (n = 135). Radical prostatectomy Gleason grades and biochemical (PSA) recurrence served as key parameters for serum biomarker evaluation. It was found that IgGMan9 and IgMMan9 were widely present in the sera of men with BPH, as well as those with cancer. However, these antibody reactivities were significantly increased in the subjects with the largest volumes of high grade cancer. Detection of serum IgGMan9 and IgMMan9 significantly predicted the clinical outcome of PCa post-radical prostatectomy. Given these results, we suggest that IgGMan9 and IgMMan9 are novel serum biomarkers for monitoring aggressive progression of PCa. The potential of oligomannosyl antigens as targets for PCa subtyping and targeted immunotherapy is yet to be explored.
doi:10.1002/ddr.21063
PMCID: PMC4139012  PMID: 25152555
autoantibodies; carbohydrate microarray; oligomannose; prostate cancer
5.  Uncovering Cryptic Glycan Markers in Multiple Sclerosis (MS) and Experimental Autoimmune Encephalomyelitis (EAE) 
Drug development research  2014;75(3):172-188.
Strategy, Management and Health PolicyEnabling Technology, Genomics, ProteomicsPreclinical ResearchPreclinical Development Toxicology, Formulation Drug Delivery, PharmacokineticsClinical Development Phases I-III Regulatory, Quality, ManufacturingPostmarketing Phase IV
Using an integrated antigen microarray approach, we observed epitope-spreading of autoantibody responses to a variety of antigenic structures in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) and in the serum of mice with experimental autoimmune encephalomyelitis (EAE). These included previously described protein- and lipid-based antigenic targets and newly discovered autoimmunogenic sugar moieties, notably, autoantibodies specific for the oligomannoses in both MS patient CSF and the sera of mice with EAE. These glycans are often masked by other sugar moieties and belong to a class of cryptic autoantigens. We further determined that these targets are highly expressed on multiple cell types in MS and EAE lesions. Co-immunization of SJL/J mice with a Man9-KLH conjugate at the time of EAE induction elicited highly significant levels of anti-Man9-cluster autoantibodies. Nevertheless, this anti-glycan autoantibody response was associated with a significantly reduced clinical severity of EAE. The potential of these cryptic glycan markers and targeting antibodies for diagnostic and therapeutic interventions of neurological disorders has yet to be explored.
doi:10.1002/ddr.21169
PMCID: PMC4019697  PMID: 24648292
glycomics; biomarkers; multiple sclerosis; encephalomyelitis; cryptic glycans; cerebrospinal fluid; autoantibodies
6.  Immunoregulatory and Effector Activities in Human Cutaneous and Mucosal Leishmaniasis: Understanding Mechanisms of Pathology 
Drug development research  2011;72(6):430-436.
Individuals infected with Leishmania braziliensis may develop the relatively benign localized cutaneous (CL) form or the mucosal (ML) form of the disease, which represents a more severe and mutilating variation. Interaction between parasite and host cells, as well as the genetic background of the host, are important determinants of the immune response, which is critical in determining disease outcome. Our studies over the years have been designed to determine the immunoregulatory and effector functions that culminate in the formation of lesions in CL and ML disease and how these host response factors may be better understood for design of novel therapies and prophylaxis. By studying the immune response from CL and ML patients in both the peripheral blood and in situ, we have learned much concerning the dynamics of the host–pathogen interaction that leads to the development of CL and ML. We used multiparameter flow cytometry to study the immunoregulatory profiles of the peripheral blood leukocytes, as well as laser scanning confocal microscopy to examine in situ several aspects of the local response, including the intensity of the inflammatory infiltrate, the cellular composition, inflammatory and anti-inflammatory cytokine expression, and the expression of the effector cytotoxic molecule, granzyme A, in lesions from CL and ML patients. Moreover, the application of correlative analysis between these immunological parameters has helped shed light on disease progression in CL and ML. These findings are reviewed within the context of understanding cellular and molecular mechanisms associated with the development of pathology in these diseases through a comparative analysis of the clinical forms, CL and ML, as well as of studies derived from peripheral blood and lesions.
doi:10.1002/ddr.20449
PMCID: PMC3595558  PMID: 23504276
leishmaniasis; immunoregulation; pathology; human
7.  Activation and Desensitization of Rat A3-Adenosine Receptors by Selective Adenosine Derivatives and Xanthine-7-Ribosides 
Drug development research  1998;44(2-3):97-105.
Strategy, Management and Health PolicyVenture Capital Enabling TechnologyPreclinical ResearchPreclinical Development Toxicology, Formulation Drug Delivery, PharmacokineticsClinical Development Phases I-III Regulatory, Quality, ManufacturingPostmarketing Phase IV
Xanthine and adenosine derivatives, known to bind to recombinant rat A3 adenosine receptors stably expressed in Chinese hamster ovary cells, were characterized in a functional assay consisting of activation of A3 receptor-stimulated binding of [35S]GTPγS in rat RBL-2H3 cell membranes. 1,3-Dibutylxanthine-7-riboside-5′-N-methylcarboxamide (DBXRM, 7b), previously shown to inhibit adenylyl cyclase via rat A3 receptors with full efficacy, appeared to be a partial agonist at the rat A3 receptor of RBL-2H3 cells. Full agonists, such as Cl-IB-MECA or I-AB-MECA, were more potent and effective than the partial agonist DBXRM in causing desensitization of rat A3 receptors, as indicated by loss of [35S]GTPγS binding. At A1 receptors, antagonism of agonist-elicited inhibition of rat adipocyte adenylyl cyclase was observed for several xanthine-7-riboside derivatives that had been shown to be full agonists at rat A3 receptors. A new xanthine riboside (3′-deoxyDBXRM, 7c) was synthesized and found to be a partial agonist at rat A3 receptors and an antagonist at rat A1 receptors. Thus, it is possible for the same compound to stimulate one adenosine receptor subtype (A3) and block another subtype (A1) within the same species.
doi:10.1002/(SICI)1098-2299(199806/07)44:2/3<97::AID-DDR7>3.0.CO;2-5
PMCID: PMC3589805  PMID: 23487508
xanthines; adenosine derivatives; nucleosides; adenylyl cyclase; guanine nucleotides
8.  Uricases as therapeutic agents to treat refractory gout: Current states and future directions 
Drug development research  2011;73(2):66-72.
Treatment of refractory gout remains a challenge on drug development. While pegloticase, a recombinant mammalian uricase modified with monomethoxyl-poly(ethylene glycol) (mPEG) is effective in treating refractory gout, after continued treatment for three months biweekly at a therapeutic dose of 0.14 mg/kg body weight, it elicits an immune response against mPEG in nearly 20% of patients. For continued treatment of refractory gout PEGylated uricases at monthly therapeutic doses below 4 μg/kg body weight have promise. To formulate uricases to achieve monthly therapeutic regimens requires pharmacodynamics simulation and experimentation including: (a) molecular engineering of uricases based on rational design and evolution biotechnology in combination to improve their inherent catalytic efficiency, thermostability and selectivity for urate over xanthine and; (b) optimization of the number and distribution of accessible reactive amino acid residues in native uricases for site-specific PEGylation with PEG derivatives with lower of immunogenicity than mPEG to retain activity, minimize immunogenicity and enhance the pharmacokinetics of the PEGylated uricase. These issues are briefly reviewed as a means to stimulate the development of safer uricase formulations for continued treatment of refractory gout.
doi:10.1002/ddr.20493
PMCID: PMC3365602  PMID: 22665944
Hyperuricemia; uricase; refractory gout; molecular engineering
9.  Predictors of Medication Adherence in Patients with Rheumatoid Arthritis 
Drug development research  2011;72(8):756-763.
Medication adherence is a significant problem in patients with rheumatoid arthritis (RA), a prevalent autoimmune disease. Due to the equivocal results reported in the research, consistent predictors of medication adherence in patients with RA are undetermined. A cross-sectional descriptive, predictive study of 108 patients with RA was used to: 1) describe self-reported medication adherence to disease modifying anti-rheumatic drugs (DMARDs); 2) compare demographic (age, residence, marital status, employment status, years of education, and ethnicity) and clinical (duration of disease and number of medications) factors of adherent and non-adherent individuals; and 3) determine the predictive power of demographic and clinical factors for DMARD adherence using various cut-points (research-based, mean, and median) on a validated, self-report scale measuring medication adherence. Independent samples t-tests, Chi square analyses, and logistic regression modeling were used to analyze these data. Approximately 90% of the individuals with RA reported adherence with their prescribed DMARD prescriptions. The only demographic and clinical difference between the adherent and non-adherent group was ethnicity (p=0.04); nonwhite individuals reported significantly less adherence with their prescribed DMARDs when compared to white individuals. Logistic regression models identified ethnicity (OR= 3.34-10.1; p< 0.05) and the number of medications taken (OR=1.7; p< 0.05) as predictors of medication non-adherence. These data provide evidence that ethnicity and taking an increased number of prescribed medications are independent predictors of medication adherence in patients with RA. These findings confirm the presence of a health disparity and an area where further research is needed to optimize patient outcomes.
doi:10.1002/ddr.20484
PMCID: PMC3261653  PMID: 22267889
medication adherence; predictors; rheumatoid arthritis
10.  Beyond transitional selection: New roles for BLyS in peripheral tolerance 
Drug development research  2011;72(8):779-787.
B cell targeted therapies have enjoyed recent success in the treatment of systemic autoimmune diseases. Among these, Belimumab, which blocks the B cell survival cytokine BLyS, was recently approved for the treatment of Systemic Lupus Erythematosus. It is therefore important to consider the roles BLyS plays in B cell tolerance. Herein, we review how BLyS contributes to the negative selection of autoreactive B cell clones from the preimmune repertoire as well as its role in regulating both germinal center and extrafollicular peripheral B cell responses. We further examine the complex role of Toll-like receptors (TLRs) in humoral autoimmunity, pointing out potential crosstalk between BLyS and TLR pathways.
doi:10.1002/ddr.20487
PMCID: PMC3272642  PMID: 22323842
BLyS; BAFF; B lymphocyte; Autoimmunity; Tolerance; Toll like receptors; Belimumab
11.  The Lymphocyte Potassium Channels Kv1.3 and KCa3.1 as Targets for Immunosuppression 
Drug development research  2011;72(7):573-584.
The voltage-gated Kv1.3 and the calcium-activated KCa3.1 potassium channel modulate many calcium-dependent cellular processes in immune cells, including T-cell activation and proliferation, and have therefore been proposed as novel therapeutic targets for immunomodulation. Kv1.3 is highly expressed in CCR7− effector memory T cells and is emerging as a target for T-cell mediated diseases like multiple sclerosis, rheumatoid arthritis, type-1 diabetes mellitus, allergic contact dermatitis, and psoriasis. KCa3.1 in contrast is expressed in CCR7+ naïve and central memory T cells, as well as in mast cells, macrophages, dedifferentiated vascular smooth muscle cells, fibroblasts, vascular endothelium, and airway epithelium. Given this expression pattern, KCa3.1 is a potential therapeutic target for conditions ranging from inflammatory bowel disease, multiple sclerosis, arthritis, and asthma to cardiovascular diseases like atherosclerosis and post-angioplasty restenosis. Results from animal studies have been supportive of the therapeutic potential of both Kv1.3 and KCa3.1 blockers and have also not shown any toxicities associated with pharmacological Kv1.3 and KCa3.1 blockade. To date, two compounds targeting Kv1.3 are in preclinical development but, so far, no Kv1.3 blocker has advanced into clinical trials. KCa3.1 blockers, on the other hand, have been evaluated in clinical trials for sickle cell anemia and exercise-induced asthma, but have so far not shown efficacy. However, the trial results support KCa3.1 as a safe therapeutic target, and will hopefully help enable clinical trials for other medical conditions that might benefit from KCa3.1 blockade.
doi:10.1002/ddr.20467
PMCID: PMC3253536  PMID: 22241939
immunosuppression; potassium channel; Kv1.3; KCa3.1; TRAM-34; PAP-1
12.  Evidence for the Recognition of Non-Nucleotide Antagonists Within the Transmembrane Domains of the Human P2Y1 Receptor 
Drug development research  2003;57(4):173-181.
Site-directed mutagenesis was used to search for amino acid residues of the human P2Y1 receptor involved in the binding of the P2 receptor antagonists pyridoxal-5′-phosphate-6-azophenyl-2,4-disulfonate (PPADS), its analogue 6-(2′-chloro-phenylazo)-pyridoxal-α5-phosphate (MRS 2210), the suramin analogue 8-8′-[carbonylbis(imino-3,1-phenylene)]bis(1,3,5-naphthalene-trisulfonate) (NF023), and Reactive blue 2. Receptors containing single amino acid replacements at positions in transmembrane helical domains (TMs) 3, 5, 6, and 7 critical for the activation of the receptor by nucleotide agonists were expressed in COS-7 (African green monkey kidney) cells. Inositol phosphate accumulation was induced by 2-methylthioadenosine 5′-diphosphate (2-MeSADP). In wild type human P2Y1 receptors, PPADS (10 to 60 µM), MRS 2210 (10 µM), NF023 (100 µM), and Reactive blue 2 (10 µM) shifted the concentration-response curve of 2-MeSADP in a parallel manner to the right. For PPADS, a pA2 value of 5.2 was estimated. The shifts caused by MRS 2210, NF023, and Reactive blue 2 corresponded to apparent pKB values of 5.6, 5.0, and 5.8, respectively. In K280A mutant receptors, the affinities of PPADS, MRS 2210, NF023, and Reactive blue 2 were about 6- to 60-fold lower than those observed at wild type receptors. The K280A mutation also caused an approximately 1,000-fold increase in the EC50 value of the agonist 2-MeSADP, similar to previous observations. In contrast, no major change in antagonistic potency was observed at receptors with other mutations in TMs 3, 5, 6, and 7. Thus, the residue Lys280 (6.55), which is located within the upper third of TM 6 of the human P2Y1 receptor, is not only critical for the activation of the receptor but also plays an important role in the binding of pyridoxal derivatives and a number of other chemically unrelated P2 receptor antagonists. Lys280 seems to belong to an overlapping region of the respective binding sites.
doi:10.1002/ddr.10145
PMCID: PMC3479677  PMID: 23105165
antagonists; G protein-coupled receptors; mutagenesis; phospholipase C; nucleotides
13.  Activation of Phosphoinositide Breakdown and Elevation of Intracellular Calcium in a Rat RBL-2H3 Mast Cell Line by Adenosine Analogs: Involvement of A3-Adenosine Receptors? 
Drug development research  1996;39(1):36-46.
A variety of adenosine analogs activate phosphoinositide breakdown in a rat RBL-2H3 mast cell line. It is presumed that an A3-adenosine receptor is involved, since the phosphoinositide response is insensitive to xanthines. However, the very potent A3- receptor agonist 2-chloro-N6-iodobenzyl-N-methylcarboxamidoadenosine (2-CI-IBMECA) with an EC50 of 4.1 µM is about twofold less potent (and less efficacious) than N-ethylcarboxamidoadenosine (NECA) with an EC50 of 2.1 µM. The other agents consisting of N6-p-aminophenylethyladenosine (APNEA), N6-iodobenzylMECA (IB-MECA), N6-R- phenylisopropyladenosine (R-PIA), 2-chloroadenosine, N6-benzyladenosine, N6- cyclohexyladenosine (CHA), N6-cyclohexylNECA (CHNECA), 2-(p- carboxyethylphenyl-ethylaminoNECA (CGS 21680), 1,3-dibutylxanthine 7-riboside-5′-N-methylcarboxamide (DBXRM), adenosine, and 8-bromoadenosine are all nearly equipotent with EC50 values of 5.5-13.9 µM. The rank order of potencies of the analogs in causing an elevation of intracellular calcium is quite different. The potent A3 receptor agonists 2-CI-IBMECA and IB-MECA with EC50 values of 0.07 and 0.11 µM, respectively, are about fourfold more potent than N6-cyclohexylNECA and about 15-fold more potent than NECA. The other analogs are comparable or somewhat less potent than NECA, some are less efficacious, and 8-bromoadenosine is inactive. The results suggest that stimulation of phosphoinositide breakdown by adenosine analogs in RBL-2H3 cells as measured by IP1 accumulation is not predictive of IP3-mediated elevations of intracellular calcium. Rank order of potency for the calcium response is consonant with intermediacy of A3-adenosine receptors, while the former, as measured by [3H]IP1-formation, probably reflects contributions from both an A3-mediated response and some other mechanism. Combinations of subthreshold concentrations of 2-CI-IBMECA with either the A1-selective agonist CHA or the A2A-selective agonist CGS 21680 caused a marked stimulation of phosphoinositide breakdown, providing further evidence for dual mechanisms. The selective A3-adenosine receptor antagonist 3,6-dichloro-2′-(isopropyloxy)-4′-methylflavone (MRS 1067) inhibits 2-CI-IBMECA- and NECA-elicited elevation of calcium levels, and had differential effects on phosphoinositide breakdown, blocking [3H]IP3 accumulation and either blocking (NECA) or having no effect (2-CI-IBMECA) on [3H]IP1 accumulation.
PMCID: PMC3475513  PMID: 23087534
adenosine receptors; phosphoinositides; calcium; xanthines
14.  Molecular Characterization of A1 and A2a Adenosine Receptors 
Drug development research  2004;28(3):226-231.
Detailed amino acid sequence analyses of A1 and A2a adenosine receptors were assembled by analogy to other G-protein-coupled receptors and correlated with pharmacological observations. Sites for phosphorylation, palmitoylation, and sodium binding have been proposed. Striatal A2a receptors from human and other species were photoaffinity-labeled using the selective, radioiodinated agonist PAPA-APEC. Selective chemical affinity labels for A1 and A2a receptors have been introduced. For example, an isothiocyanate, p-DITC-APEC (100 nM), irreversibly diminished the Bmax for [3H]CGS 21680 (2-[4-[(2-carboxyethyl) phenyl] ethylamino]-5’-N-ethylcarboxamidoadenosine) binding in rabbit striatal membranes by 71% (Kd unaffected), suggesting a direct modification of the ligand binding site. Novel trifunctional affinity labels have been designed. Rabbit and human A2a receptors were characterized using [3H]XAC binding in the presence of 50 or 25 nM CPX (8-cyclopentyl-l,3-dipropylxanthine), respectively. The inhibition of A2 radioligand binding by the histidyl-modifying reagent diethylpyrocarbonate suggested the involvement of His residues in interactions with adenosine agonists and antagonists. Properties of transiently expressed mutants of bovine A1 receptors in which either His251 or His278 residues have been substituted with Leu suggest that both histidines are important in binding.
doi:10.1002/ddr.430280307
PMCID: PMC3446818  PMID: 23002320
affinity labeling; sequence analysis; xanthines; chemical modification; mutagenesis
15.  Structure Activity Relationships for Derivatives of Adenosine-5′-Triphosphate as Agonists at P2 Purinoceptors: Heterogeneity Within P2X and P2Y Subtypes 
Drug development research  2004;31(3):206-219.
The structure-activity relationships for a variety of adenine nucleotide analogues at P2x- and P2Y-purinoceptors were investigated. Compounds formed by structural modifications of the ATP molecule including substitutions of the purine ring (C2, C8, N1, and N6-substituents, and a uridine base instead of adenine), the ribose moiety (2′ and 3′-positions), and the triphosphate group (lower phosphates, bridging oxygen substitution, and cyclization) were prepared. Pharmacological activity at P2Y-purinoceptors was assayed in the guinea pig taenia coli, endothelial cells of the rabbit aorta, smooth muscle of the rabbit mesenteric artery, and turkey erythrocyte membranes. Activity at P2X-purinoceptors was assayed in the rabbit saphenous artery and the guinea-pig vas deferens and urinary bladder. Some of the analogues displayed selectivity, or even specificity, for either the P2X- or the P2Y-purinoceptors. Certain analogues displayed selectivity or specificity within the P2X- or P2Y-purinoceptor superfamilies, giving hints about possible subclasses. For example, 8-(6-aminohexylamino)ATP and 2′,3′-isopropylidene-AMP were selective for endothelial Pzypurinoceptors over P2Y-purinoceptors in the guinea pig taenia coli, rabbit aorta, and turkey erythrocytes. These compounds were both inactive at P2X-purinoceptors. The potent agonist N6-methyl ATP and the somewhat less potent agonist 2′-deoxy-ATP were selective for P2Y-purinoceptors in the guinea pig taenia coli, but were inactive at P2X-purinoceptors and the vascular P2Y-purinoceptors. 3′-Benzylamino-3′-deoxyATP was very potent at the P2X-purinoceptors in the guinea pig vas deferens and bladder, but not in the rabbit saphenous artery and was inactive at P2Y receptors. These data suggest that specific compounds can be developed that can be utilized to activate putative subtypes of the P2X- and P2Y-purinoceptor classes.
doi:10.1002/ddr.430310308
PMCID: PMC3434461  PMID: 22962511
ATP; purinoceptors; smooth muscle; nucleotides; phospholipase C
16.  Clinical aspects of Chagas disease and implications for novel therapies 
Drug development research  2011;72(6):471-479.
The interaction between the protozoan parasite Trypanosoma cruzi and the human host dates back 9000 years, as demonstrated by molecular analysis of material obtained from Andean mummies indicating the presence of the parasite’s kinetoplast DNA in populations from Chile and Peru. This long-established interaction, which persists today, demonstrates that T. cruzi has established a very well adapted relationship with the human host. From a host-parasite relationship point-of-view this is desirable, however, such a high degree of adaptation is perhaps the foundation for many of the unknowns that surround this disease. Unveiling of the immunological mechanisms that underlie the establishment of pathology, identification of parasite-associated factors that determine strain-differential tissue tropism, discovery of host genetic elements that influence the development of different clinical forms of the disease, and understanding environmental factors that may influence the host-parasite interactions, are some of the key questions remaining to be answered. The response to these questions will aid in addressing some of the current challenges in Chagas disease: fulfilling the need for efficient diagnosis, developing effective prophylactic measures, discovering effective therapeutics, and finding methods to control disease progression.
doi:10.1002/ddr.20454
PMCID: PMC3259567  PMID: 22267887
Chagas disease; pathology; cardiomyopathy; treatment; Trypanosoma cruzi
17.  New regimens for reducing the duration of the treatment of drug-susceptible pulmonary tuberculosis 
Drug development research  2011;72(6):501-508.
Tuberculosis (TB) remains an important health problem worlwide. The structure necessary for delivering TB treatment and implementing the directly observed treatment accounts for more than two-thirds of its final cost. Furthemore, although with efficacy greater than 90%, the effectiveness of present treatment regimens ranges from 55–85%, depending on the setting, mainly due to poor adherence. Duration of treatment with the current first-line anti-TB drugs is a minimum of 6 months. Reducing the duration of the treatment from six to two months or less could result in significant increase of adherence to treatment and cost reduction. The aim of this review is to highlight potential new agents or new drug combinations that could reduce the time of treatment of drug-susceptible TB, currently under study or recently evaluated through clinical trials. We conducted a literature search in the English language for clinical studies as well as an electronic computer-assisted and manual search. The literature search was conducted on November 2010, using MEDLINE (2000–2010), EMBASE (2000–2010) and the National Institute of Health (NIH) Clinical Trials Register database (2000–2010). Most of the new agents identified as anti-TB drug candidates are still in the preclinical phases. Nitroimidazole-PA-824 and fluoroquinolones are evaluated while two first line drugs - rifampicin and rifapentine -are re-evaluated to optimize their efficacy in new ultra-short anti-TB regimens through phases II/III clinical studies. A summary of the studies are presented, with their potential to change recommendations for TB treatment in the near future.
doi:10.1002/ddr.20456
PMCID: PMC3259568  PMID: 22267888
tuberculosis; drugs; treatment; rifapentine; fluoroquinolones; clinical trial
18.  Synthesis and Structure-Activity Relationships of Pyridoxal-6-arylazo-5′-phosphate and Phosphonate Derivatives as P2 Receptor Antagonists 
Drug development research  1998;45(2):52-66.
Novel analogs of the P2 receptor antagonist pyridoxal-5′-phosphate-6-phenylazo-2′,4′-disulfonate (PPADS) were synthesized. Modifications were made through functional group substitution on the sulfophenyl ring and at the phosphate moiety through the inclusion of phosphonates, demonstrating that a phosphate linkage is not required for P2 receptor antagonism. Substituted 6-phenylazo and 6-naphthylazo derivatives were also evaluated. Among the 6-phenylazo derivatives, 5′-methyl, ethyl, propyl, vinyl, and allyl phosphonates were included. The compounds were tested as antagonists at turkey erythrocyte and guinea-pig taenia coli P2Y1 receptors, in guinea-pig vas deferens and bladder P2X1 receptors, and in ion flux experiments by using recombinant rat P2X2 receptors expressed in Xenopus oocytes. Competitive binding assay at human P2X1 receptors in differentiated HL-60 cell membranes was carried out by using [35S]ATP-γ-S. A 2′-chloro-5′-sulfo analog of PPADS (C14H12O9N3ClPSNa), a vinyl phosphonate derivative (C15H12O11N3PS2Na3), and a naphthylazo derivative (C18H14O12N3PS2Na2), were particularly potent in binding to human P2X1 receptors. The potencies of phosphate derivatives at P2Y1 receptors were generally similar to PPADS itself, except for the p-carboxyphenylazo phosphate derivative C15H13O8N3PNa and its m-chloro analog C15H12O8N3ClPNa, which were selective for P2X vs. P2Y1 receptors. C15H12O8N3ClPNa was very potent at rat P2X2 receptors with an IC50 value of 0.82 μM. Among the phosphonate derivatives, [4-formyl-3-hydroxy-2-methyl-6-(2-chloro-5-sulfonylphenylazo)-pyrid-5-yl]methylphosphonic acid (C14H12-O8N3ClPSNa) showed high potency at P2Y1 receptors with an IC50 of 7.23 μM. The corresponding 2,5-disulfonylphenyl derivative was nearly inactive at turkey erythrocyte P2Y1 receptors, whereas at recombinant P2X2 receptors had an IC50 value of 1.1 μM. An ethyl phosphonate derivative (C15H15O11N3PS2Na3), whereas inactive at turkey erythrocyte P2Y1 receptors, was particularly potent at recombinant P2X2 receptors.
doi:10.1002/(SICI)1098-2299(199810)45:2<52::AID-DDR2>3.0.CO;2-V
PMCID: PMC3424000  PMID: 22922976
ATP; nucleotides; ion channels; phospholipase C; smooth muscle; guinea pig; turkey erythrocytes
19.  Northern Ring Conformation of Methanocarba-Adenosine 5′-Triphosphate Required for Activation of P2X Receptors 
Drug development research  2004;61(4):227-232.
Strategy, Management and Health PolicyEnabling Technology, Genomics, ProteomicsPreclinical ResearchPreclinical Development Toxicology, Formulation Drug Delivery, PharmacokineticsClinical Development Phases I-III Regulatory, Quality, ManufacturingPostmarketing Phase IV
Replacement of the ribose moiety of adenosine 5′-triphosphate (ATP) with a carbocyclic ring constrained in either the Northern (N) or Southern (S) conformation produces agonists with widely differing activities at P2Y receptors (Kim et al. [2002] J Med Chem 45:208–218). We have used whole cell patch clamp recording to investigate the agonist activity of these two methanocarba analogs of ATP at four different P2X receptors (P2X1, P2X2, P2X3, and P2X2/3). On dorsal root ganglion neurons, (N) methanocarba-ATP ((1′S,2′R,3′S,4′R,5′S)-4-(6-amino-9H-purin-9-yl)-1-[triphosphoryloxymethyl] bicyclo[ 3.1.0]hexane-2,3-diol; MRS 2340) activated rapidly-desensitizing (P2X3) and slowly-desensitizing (P2X2/3) receptors with a similar potency to ATP. In contrast, (S) methanocarba-ATP ((±)-5-(6-amino-9H-purin-9-yl)-1-[triphosphoryloxymethyl] bicycle [3.1.0]hexane-2,3-diol MRS 2312) was devoid of agonist activity. On nodose ganglion neurones, that express mainly P2X2/3 receptors, ATP evoked a slowly desensitizing inward current with an EC50 value of 26 μM. MRS 2340 was an effective agonist, but less potent than ATP, while MRS 2312 at concentrations up to 100 μM produced a barely detectable response. On mammalian cell lines expressing recombinant hP2X1 and hP2X2 receptors, MRS 2340 evoked inward currents similar in amplitude to those produced by the same concentration of ATP or α,β-mATP. In contrast, MRS 2312 failed to give a detectable response. Although the conformation of the ribose affects agonist activity at P2Y receptors, there is a strong requirement for the (N) conformation for the activation of these P2X receptors. Furthermore, the region of the agonist binding site that accommodates the ribose moiety appears to be highly conserved among different P2X receptors. Drug Dev Res 61:227–232, 2004.
doi:10.1002/ddr.10381
PMCID: PMC3402081  PMID: 22833693
ATP; P2X receptors; structure activity relationship
20.  Activity of Novel Adenine Nucleotide Derivatives as Agonists and Antagonists at Recombinant Rat P2X Receptors 
Drug development research  2000;49(4):253-259.
Strategy, Management and Health PolicyVenture Capital Enabling TechnologyPreclinical ResearchPreclinical Development Toxicology, Formulation Drug Delivery, PharmacokineticsClinical Development Phases I-III Regulatory, Quality, ManufacturingPostmarketing Phase IV
The effects of structural modifications of adenine nucleotides previously shown to enhance either agonist (2-thioether groups) or antagonist (additional phosphate moieties at the 3′- or 2′-position) properties at P2Y1 receptors were examined at recombinant rat P2X1, P2X2, P2X3, and P2X4 receptors expressed in Xenopus oocytes. The potency of P2Y1 agonists HT-AMP (2-(hexylthio)adenosine-5′-monophosphate) and PAPET (2-[2-(4-aminophenyl)ethylthio]adenosine-5′-triphosphate) was examined at P2X receptors. Both nucleotides showed a preference for the Group I (α,β-meATP-sensitive, fast-inactivating) P2X sub-units. HT-AMP was 5-fold more potent than ATP at P2X3 receptors and a partial agonist at all except P2X2 receptors, at which it was a full agonist. The efficacy of HT-AMP was as low as 23% at P2X4 receptors. PAPET was a weak partial agonist at rat P2X4 receptors and a nearly full agonist at the other subtypes. At rat P2X3 receptors, PAPET was more potent than any other known agonist (EC50 = 17 ± 3 nM). MRS 2179 (N6-methyl-2′-deoxyadenosine 3′, 5-bisphosphate, a potent P2Y1 receptor antagonist) inhibited ATP-evoked responses at rat P2X1 receptors with an IC50 value of 1.15 ± 0.21 μM. MRS 2179 was a weak antagonist at rat P2X3 receptors, with an IC50 value of 12.9 ± 0.1 μM, and was inactive at rat P2X2 and P2X4 receptors. Thus, MRS 2179 was 11-fold and 130-fold selective for P2Y1 receptors vs. P2X1 and P2X3 receptors, respectively. MRS 2209, the corresponding 3′-deoxy-2′-phosphate isomer, was inactive at rat P2X1 receptors, thus demonstrating its greater selectivity as a P2Y1 receptor antagonist. Various adenine bisphosphates in the family of MRS 2179 containing modifications of either the adenine (P2Y1 antagonists with 2- and 6-substitutions), the phosphate (a 3′,5′-cyclic diphosphate, inactive at P2Y1 receptors), or the ribose moieties (antagonist carbocyclic analogue), were inactive at both rat P2X1 and P2X3 receptors. An anhydrohexitol derivative (MRS 2269) and an acyclic derivative (MRS 2286), proved to be selective antagonists at P2Y1 receptors, since they were inactive as agonist or antagonist at P2X1 and P2X3 receptors.
doi:10.1002/1098-2299(200004)49:4<253::AID-DDR4>3.0.CO;2-1
PMCID: PMC3393598  PMID: 22791931
ion channel; oocytes; purines; ATP derivatives; bisphosphates; deoxyadenosine derivatives
21.  8-(3-Isothiocyanatostyryl)caffeine Is a Selective, Irreversible Inhibitor of Striatal A2-Adenosine Receptors 
Drug development research  2004;29(4):292-298.
8-(3-Isothiocyanatostyryl)caffeine (ISC) was synthesized and shown to inhibit selectively the binding of [3H]CGS 21680 (an A2a-selective agonist) at adenosine receptors in striatal membranes. The Ki value at A2a-receptors was found to be 110 nM (rat), with selectivity ratios for A2a versus A1-receptors in rat, guinea pig, bovine, and rabbit striatum of >100-fold. Preincubation of membranes with ISC caused a dose-dependent, irreversible antagonism of the binding of [3H]CGS 21680, with an IC50 value of 3 μM. The irreversibility is likely due to the presence of the chemically reactive isothiocyanate group, since the binding of the corresponding analogue in which the isothiocyanate was replaced with a chloro group was completely reversible. The potency of ISC to irreversibly inhibit the binding of [3H]CGS 21680 in several species varied in the order rat ≈ guinea pig > bovine ≈ rabbit. In all four species, binding of the A1-selective agonist [3H]R-N6-phenylisopropyladenosine was not diminished by pre-treatment with 2 μM ISC. The kinetics of irreversible inhibition of rat A2a-receptors by 2 μM ISC gave a t1/2 of approximately 3 min. Following partial inactivation, the remaining rat A2a-binding sites retained the same Kd value as in control membranes for saturation by [3H]CGS 21680. Thus, ISC appears to be a selective affinity label for A2a- versus A1-receptors in the brain.
doi:10.1002/ddr.430290407
PMCID: PMC3392128  PMID: 22787287
xanthines; affinity label; adenosine receptors; radioligand binding
22.  Riboflavin: Inhibitory Effects on Receptors, G-Proteins, and Adenylate Cyclase 
Drug development research  1998;42(2):98-108.
Riboflavin inhibited binding of both agonist and antagonist radioligands to rat brain A1-adenosine receptors with Ki values of approximately 10 µM. In an adenylate cyclase assay with membrane preparations from either rat adipocytes or DDT MF-2 cells, both of which contain A1-adenosine receptors, riboflavin inhibited isoproterenol-stimulated cyclase activity with an IC50 of approximately 20 µM. However, the inhibition of cyclase by riboflavin was not reversed by an A1-selective antagonist, nor by pretreatment with pertussis toxin. Thus, neither A1-receptors nor Gi-proteins appear critically involved in the inhibition of cyclase by riboflavin. Riboflavin did block the stimulation by an adenosine analog of [35S]GTPγS binding in rat cerebral cortical membranes. However, riboflavin also inhibited the stimulation by fMLP of [35S]GTPγS binding in HL-60 cell membranes. Riboflavin inhibited forskolin-stimulated cyclase in membranes from DDT MF-2 cells > rat adipocytes > PC12 cells, hamster CHO M2 cells, and wild-type S49 cells. There was virtually no inhibition of forskolin-stimulated cyclase in membranes of human platelets, rat cerebral cortex, or cyc−S49 cells lacking Gs-proteins. The calcium-stimulated cyclase in rat cerebral cortical membranes was inhibited by riboflavin. A preincubation of membranes with riboflavin markedly enhanced the inhibition for DDT MF-2 and wild-type and cyc−S49 membranes. The extent of inhibition in the different cell lines was dependent on the agent used to stimulate cyclase. Riboflavin, like the P-site inhibitor 2´,5´-dideoxyadenosine, was more potent and efficacious when manganese instead of forskolin was used as the stimulant. However, unlike the P-site inhibitor, riboflavin did not markedly inhibit GppNHp- or fluoride-stimulated cyclase. Riboflavin at low micromolar concentrations appears to have three possibly interrelated effects on second messenger systems subserved by G-proteins. These are antagonism at A1-adenosine receptors, inhibition of turnover of guanyl nucleotides at G-proteins, and inhibition of adenylate cyclase.
doi:10.1002/(SICI)1098-2299(199710)42:2<98::AID-DDR6>3.0.CO;2-K
PMCID: PMC3389815  PMID: 22772360
adenylate cyclase; P-site; adenosine analogs; xanthines; forskolin; G-proteins; A1-adenosine receptors
23.  Purification and Recognition of Recombinant Mouse P2X1 Receptors Expressed in a Baculovirus System 
Drug development research  2000;51(1):7-19.
Strategy, Management and Health Policy Venture Capital Enabling TechnologyPreclinical ResearchPreclinical Development Toxicology, Formulation Drug Delivery, PharmacokineticsClinical Development Phases I–III Regulatory, Quality, ManufacturingPostmarketing Phase IV
The hexahistidine-tagged mouse P2X1 receptor (H-mP2X1R), an ATP-gated ion channel receptor, was expressed in a baculovirus system using the pAcHLT-B transfer vector containing a hexahistidine tag. Both widely used denaturing (8M urea) and nondenaturing (such as 1% Triton X-100) solubilization conditions were compared, resulting in about 30% of the P2X1 receptors being solubilized (S1). However, at pH 13 most of the H-mP2X1R from the initially insoluble pellet fraction was solubilized (S2) and remained in the soluble fraction (S3) after dialyzing against a nondenaturing buffer. H-mP2X1Rs were purified sequentially through cobalt and ATP affinity columns. Receptors purified from S3 had higher purity than those from S1 (i.e., ~90% vs. ~75%). Circular dichroism spectra indicated identical protein secondary structures of the receptors from both sources. Autoradiographic data showed that the purified receptors from S3 had higher affinity for 8-azido-ATP-γ-32P than the receptors from S1. The binding of 8-azido-ATP-γ-32P to H-mP2X1R was inhibited by ATP-γ-S, α,β-me-ATP, and PPADS, but not by a nucleoside analog (N6-methyl-2′-deoxy-adenosine). In the presence of 2 mM Ca2+ or Mg2+ the binding was increased, but not when using a partially purified receptor fraction, in which unidentified proteins bound 8-azido-ATP-γ-32P or were phosphorylated at 4°C in the presence of 2 mM Mg2+. These data suggest that the decrease in potency of ATP in the presence of Ca2+ and Mg2+, as observed in functional studies, is not due to a direct effect of the cations on the binding of ATP to the receptor. Both cyanogen bromide and hydroxylamine cleavage further confirmed the peptide structure of the purified H-mP2X1R. Autoradiographic analysis of the cleavage products showed that 8-azido-ATP-γ-32P was crosslinked to the carboxyl side of the extracellular domain of the receptor.
doi:10.1002/1098-2299(20000901)51:1<7::AID-DDR2>3.0.CO;2-W
PMCID: PMC3383047  PMID: 22745520
ion channels; nucleotides; affinity chromatography; agonist; binding site
24.  In Silico Augmentation of the Drug Development Pipeline: Examples from the study of Acute Inflammation 
Drug development research  2011;72(2):187-200.
The clinical translation of promising basic biomedical findings, whether derived from reductionist studies in academic laboratories or as the product of extensive high-throughput and –content screens in the biotechnology and pharmaceutical industries, has reached a period of stagnation in which ever higher research and development costs are yielding ever fewer new drugs. Systems biology and computational modeling have been touted as potential avenues by which to break through this logjam. However, few mechanistic computational approaches are utilized in a manner that is fully cognizant of the inherent clinical realities in which the drugs developed through this ostensibly rational process will be ultimately used. In this article, we present a Translational Systems Biology approach to inflammation. This approach is based on the use of mechanistic computational modeling centered on inherent clinical applicability, namely that a unified suite of models can be applied to generate in silico clinical trials, individualized computational models as tools for personalized medicine, and rational drug and device design based on disease mechanism.
doi:10.1002/ddr.20415
PMCID: PMC3086282  PMID: 21552346
inflammation; mathematical model; sepsis; trauma; rational drug design; in silico clinical trial
25.  A Discovery Funnel for Nucleic Acid Binding Drug Candidates 
Drug development research  2011;72(2):178-186.
Computational approaches are becoming increasingly popular for the discovery of drug candidates against a target of interest. Proteins have historically been the primary targets of many virtual screening efforts. While in silico screens targeting proteins has proven successful, other classes of targets, in particular DNA, remain largely unexplored using virtual screening methods. With the realization of the functional importance of many non-cannonical DNA structures such as G-quadruplexes, increased efforts are underway to discover new small molecules that can bind selectively to DNA structures. Here, we describe efforts to build an integrated in silico and in vitro platform for discovering compounds that may bind to a chosen DNA target. Millions of compounds are initially screened in silico for selective binding to a particular structure and ranked to identify several hundred best hits. An important element of our strategy is the inclusion of an array of possible competing structures in the in silico screen. The best hundred or so hits are validated experimentally for binding to the actual target structure by a high-throughput 96-well thermal denaturation assay to yield the top ten candidates. Finally, these most promising candidates are thoroughly characterized for binding to their DNA target by rigorous biophysical methods, including isothermal titration calorimetry, differential scanning calorimetry, spectroscopy and competition dialysis.This platform was validated using quadruplex DNA as a target and a newly discovered quadruplex binding compound with possible anti-cancer activity was discovered. Some considerations when embarking on virtual screening and in silico experiments are also discussed.
doi:10.1002/ddr.20414
PMCID: PMC3090163  PMID: 21566705
drug discovery; in silico screening; SURFLEX-DOCK; DNA; G-quadruplex; high-throughput screening

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