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Background: Mouse models that recapitulate key features of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection are important tools for understanding complex interactions between host genetics, immune responses, and SARS-CoV-2 pathogenesis. Little is known about how predominantly cellular (Th1 type) versus humoral (Th2 type) immune responses influence SARS-CoV-2 dynamics, including infectivity and disease course., Methods: We generated knock-in (KI) mice expressing human ACE2 (hACE2) and/or human TMPRSS2 (hTMPRSS2) on Th1-biased (C57BL/6; B6) and Th2-biased (BALB/c) genetic backgrounds. Mice were infected intranasally with SARS-CoV-2 Delta (B.1.617.2) or Omicron BA.1 (B.1.1.529) variants, followed by assessment of disease course, respiratory tract infection, lung histopathology, and humoral and cellular immune responses., Findings: In both B6 and BALB/c mice, hACE2 expression was required for infection of the lungs with Delta, but not Omicron BA.1. Disease severity was greater in Omicron BA.1-infected hTMPRSS2-KI and double-KI BALB/c mice compared with B6 mice, and in Delta-infected double-KI B6 and BALB/c mice compared with hACE2-KI mice. hACE2-KI B6 mice developed more severe lung pathology and more robust SARS-CoV-2-specific splenic CD8 T cell responses compared with hACE2-KI BALB/c mice. There were no notable differences between the two genetic backgrounds in plasma cell, germinal center B cell, or antibody responses to SARS-CoV-2., Interpretation: SARS-CoV-2 Delta and Omicron BA.1 infection, disease course, and CD8 T cell response are influenced by the host genetic background. These humanized mice hold promise as important tools for investigating the mechanisms underlying the heterogeneity of SARS-CoV-2-induced pathogenesis and immune response., Funding: This work was funded by NIH U19 AI142790-02S1, the GHR Foundation, the Arvin Gottleib Foundation, and the Overton family (to SS and EOS); Prebys Foundation (to SS); NIH R44 AI157900 (to KJ); and by an American Association of Immunologists Career Reentry Fellowship (FASB)., Competing Interests: Declaration of interests CC, NS, and KJ were employees of Synbal, Inc. DOC is employed by and holds stock in TransViragen Inc., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Monoclonal antibodies (mAbs) are one of the fastest-growing classes of drugs and have been approved to treat several diseases, including cancers and autoimmune disorders. Preclinical pharmacokinetics studies are performed to determine the therapeutically meaningful dosages and efficacy of candidate drugs. These studies are typically performed in non-human primates; however, using primates is costly and raises ethical considerations. As a result, rodent models that better mimic human-like pharmacokinetics have been generated and remain an area of active investigation. Pharmacokinetic characteristics of a candidate drug, such as half-life, are partly controlled by antibody binding to the human neonatal receptor hFCRN. Due to the abnormally high binding of human antibodies to mouse FCRN, traditional laboratory rodents do not accurately model the pharmacokinetics of human mAbs. In response, humanized rodents expressing hFCRN have been generated. However, these models generally use large inserts randomly integrated into the mouse genome. Here, we report the production and characterization of a CRISPR/Cas9-mediated hFCRN transgenic mouse termed SYNB-hFCRN. Using CRISPR/Cas9-assisted gene targeting, we prepared a strain with a simultaneous knockout of mFcrn and insertion of a hFCRN mini-gene under the control of the endogenous mouse promoter. These mice are healthy and express hFCRN in the appropriate tissues and immune cell subtypes. Pharmacokinetic evaluation of human IgG and adalimumab (Humira®) demonstrate hFCRN-mediated protection. These newly generated SYNB-hFCRN mice provide another valuable animal model for use in preclinical pharmacokinetics studies during early drug development., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kurt Jarnagin reports financial support was provided by Bristol Myers Squibb Co. David R. Webb reports a relationship with Synbal, Inc. that includes: employment and equity or stocks. Kurt Jarnagin reports a relationship with Synbal, Inc. that includes: employment and equity or stocks. Shannon Xu reports a relationship with Synbal, Inc. that includes: employment and equity or stocks. Don van Fossan reports a relationship with Synbal Inc. that includes: employment and equity or stocks. Kristen Read reports a relationship with Synbal Inc. that includes: employment and equity or stocks. Dale O. Cowley reports a relationship with Transviragen, Inc. that includes: employment and equity or stocks. Oscar Alvarez reports a relationship with Synbal, Inc. that includes: employment and equity or stocks. Christopher M. Conner reports a relationship with Synbal, Inc. that includes: employment and equity or stocks., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Objective: The aim of the study is to determine relationships between lockdowns and excess mortality, unemployment, and employment growth., Methods: Each US states' mortality data for 2020 were compared with the prior 3 years to determine excess mortality. Data were compared using measures of lockdowns, or state openness scores and adjusted for age, sex, race/ethnicity, and cardiovascular disease. Comparisons were made with unemployment rates and employment growth rates., Results: The 2020 excess mortality ranged from -9% to 46%. The average openness score was not significant ( P = 0.20). However, openness was strongly associated with both unemployment ( P = 0.01) and employment growth ( P = 0.0008)., Conclusions: There was no statistical relationship between excess mortality and openness scores, while there were strong relationships with employment measures. These results suggest that lockdowns are not sufficiently beneficial for future use in this pandemic and raise concerns for use in future pandemics., Competing Interests: Conflicts of Interest: Dr Hegmann is an editor of the American College of Occupational and Environmental Medicine practice guidelines., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American College of Occupational and Environmental Medicine.)

Introduction: There is an urgent need for new animal models of SARS CoV-2 infection to improve research and drug development. This brief commentary examines the deficits of current models and proposes several improved alternates. The existing single transgene mouse models poorly mimic the clinical features of COVID-19; those strains get a milder disease than human COVID-19 disease. Many of the current transgenic models utilize random integration of several copies of single ACE2 transgenes, resulting in unnatural gene expression and exhibit rapid lethality. We suggest preparing precision knock-in of selected human mini genes at the mouse initiation codon and knock-out of the mouse homolog as a better option. Three genes critical for infection are suggested targets, ACE2 (the viral cellular receptor), its co-infection protease TMRPSS2, and the primary antibody clearance receptor FcγRT. To offer the best platform for COVID 19 research, preparation of single, double, and triple humanized combinations offers the researcher the opportunity to better understand the contributions of these receptors, coreceptors to therapeutic efficacy. In addition, we propose to create the humanized strains in the C57BL/6J and BALB/c backgrounds. These two backgrounds are Th1 responders and Th2 responders, respectively, and allow modeling of the variability seen in human pathology including lung pathology and late sequelae of COVID-19 disease (BALB/c). We suggest the need to do a thorough characterization of both the short-term and long-term effects of SAR-CoV-2 infection at the clinical, virologic, histopathologic, hematologic, and immunologic levels. We expect the multiply humanized strains will be superior to the single-gene and multiple-gene-copy transgenic models available to date. These mouse models will represent state-of-the-art tools for investigating mechanisms of COVID-19 pathogenesis and immunity and developing vaccines and drugs., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: The only therapies currently available for cutaneous neurofibromas (cNF) are procedural. The goals of the Therapies Development Working Group were to (1) summarize currently available treatment options for cNF, (2) define key considerations for drug discovery and development generally, and specifically for cNF, and (3) outline recommendations for the successful development of medical therapies for cNF., Methods: The subgroup reviewed published and unpublished data on procedural, drug/device, and medical treatment approaches utilized for cNFs via literature search. The team defined disease- and patient-specific factors to consider for therapies development in a series of consensus meetings., Results: The team identified 5 approaches entailing procedural and drug/device methods currently under study. There have been 4 clinical studies exploring various interventional therapies, from which outcomes were highly variable. The team identified 4 key factors to prioritize during the development of products for the treatment for cNF: safety, anatomic distribution of cNF, numbers of tumors to be treated, and route of administration., Conclusions: The number, size, and distribution of cNF is highly variable among patients with NF1 and it is possible that different phenotypes will require different drug development paths. The nonfatal nature of the disease and relatively limited patient numbers suggest that for any product to have a higher likelihood of acceptance, it will have to (1) demonstrate an effect that is clinically meaningful, (2) have a safety profile conducive to long-term dosing, and (3) have a low manufacturing cost., (© 2018 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Objective: Several clinical trials targeting cutaneous neurofibromas (cNF) have been conducted; however, none has resulted in meaningful changes to care. The Clinical Trial Design and Development subgroup's goals were to (1) define key considerations in the design of clinical trials for cNF, (2) summarize existing data in relation to these considerations, and (3) provide consensus recommendations about key elements of trial design to accelerate the clinical development of therapies for cNF., Methods: The subgroup, with experts from genetics, dermatology, neurology, oncology, and basic science, spanning academia, government research, and regulatory programs, and industry, reviewed published and unpublished data on clinical trials for cNF and other diseases in the skin. Discussions of these data resulted in formulation of a list of priority issues to address in order to develop efficient and effective clinical trials for cNF., Results: The subgroup identified 2 natural history studies of cNF, 4 priority outcome measures, and 6 patient-reported outcome tools for potential use in efficacy trials of cNF. Time to initiate intervention, patient eligibility, mechanism of action, route of administration, safety monitoring, and regulatory agency interactions were identified as key factors to consider when designing clinical trials for cNF., Conclusions: Alignment on endpoints and methods for the measurement and quantification of cNF represent a priority for therapeutic development for cNF. Advances in technological methods and outcome tools utilized in other skin diseases may be applicable to cNF studies. Patient age is an important factor guiding trial design and clinical development path., (© 2018 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Background: Reliance on just one drug to treat the prevalent tropical disease, schistosomiasis, spurs the search for new drugs and drug targets. Inhibitors of human cyclic nucleotide phosphodiesterases (huPDEs), including PDE4, are under development as novel drugs to treat a range of chronic indications including asthma, chronic obstructive pulmonary disease and Alzheimer's disease. One class of huPDE4 inhibitors that has yielded marketed drugs is the benzoxaboroles (Anacor Pharmaceuticals)., Methodology/principal Findings: A phenotypic screen involving Schistosoma mansoni and 1,085 benzoxaboroles identified a subset of huPDE4 inhibitors that induced parasite hypermotility and degeneration. To uncover the putative schistosome PDE4 target, we characterized four PDE4 sequences (SmPDE4A-D) in the parasite's genome and transcriptome, and cloned and recombinantly expressed the catalytic domain of SmPDE4A. Among a set of benzoxaboroles and catechol inhibitors that differentially inhibit huPDE4, a relationship between the inhibition of SmPDE4A, and parasite hypermotility and degeneration, was measured. To validate SmPDE4A as the benzoxaborole molecular target, we first generated Caenorhabditis elegans lines that express a cDNA for smpde4a on a pde4(ce268) mutant (hypermotile) background: the smpde4a transgene restored mutant worm motility to that of the wild type. We then showed that benzoxaborole inhibitors of SmPDE4A that induce hypermotility in the schistosome also elicit a hypermotile response in the C. elegans lines that express the smpde4a transgene, thereby confirming SmPDE4A as the relevant target., Conclusions/significance: The orthogonal chemical, biological and genetic strategies employed identify SmPDE4A's contribution to parasite motility and degeneration, and its potential as a drug target. Transgenic C. elegans is highlighted as a potential screening tool to optimize small molecule chemistries to flatworm molecular drug targets.

Crisaborole topical ointment, 2% (formerly known as AN2728) is a benzoxaborole, nonsteroidal, topical, anti-inflammatory phosphodiesterase 4 (PDE4) inhibitor investigational compound that recently completed phase 3 studies for the treatment of mild to moderate atopic dermatitis (AD). The unique configuration of boron within the crisaborole molecule enables selective targeting and inhibition of PDE4, an enzyme that converts the intracellular second messenger 3'5'-cyclic adenosine monophosphate (cAMP) into the active metabolite adenosine monophosphate (AMP). By inhibiting PDE4 and thus increasing levels of cAMP, crisaborole controls inflammation. The use of boron chemistry enabled synthesis of a low-molecular-weight compound (251 daltons), thereby facilitating effective penetration of crisaborole through human skin. In vitro experiments showed that crisaborole inhibits cytokine production from peripheral blood mononuclear cells in a pattern similar to other PDE4 inhibitors and distinct from corticosteroids. Crisaborole also displayed topical anti-inflammatory activity in a skin inflammation model. Once crisaborole reaches systemic circulation after topical application, it is metabolized to inactive metabolites. This limits systemic exposure to crisaborole and systemic PDE4 inhibition. In phase 1 and 2 clinical studies, crisaborole ointment, 2% was generally well tolerated and improved AD disease severity scores, pruritus, and all other AD signs and symptoms. Two large, randomized, controlled, phase 3, pivotal clinical trials assessing the efficacy and safety of crisaborole topical ointment, 2% in children, adolescents, and adults with mild to moderate AD were recently completed with positive results.

Benzoxaboroles are a novel class of drug-like compounds that have been rich sources of novel inhibitors for various enzymes and of new drugs. While examining benzoxaborole activity in phenotypic screens, our attention was attracted by the (aminomethylphenoxy)benzoxaborole family, which potently inhibited Toll-like receptor-stimulated cytokine secretion from leukocytes. After considering their structure-activity relationships and the central role of kinases in leukocyte biology, we performed a kinome-wide screen to investigate the members of the (aminomethylphenoxy)benzoxaborole family. This technique identified Rho-activated kinase (ROCK) as a target. We showed competitive behavior, with respect to ATP, and then determined the ROCK2-drug cocrystal structure. The drug occupies the ATP site in which the oxaborole moiety provides hydrogen bond donors and acceptors to the hinge, and the aminomethyl group interacts with the magnesium/ATP-interacting aspartic acid common to protein kinases. The series exhibits excellent selectivity against most of the kinome, with greater than 15-fold selectivity against the next best member of the AGC protein kinase subfamily. Medicinal chemistry efforts with structure-based design resulted in a compound with a Ki of 170 nM. Cellular studies revealed strong enzyme inhibition rank correlation with suppression of intracellular phosphorylation of a ROCK substrate. The biochemical potencies of these compounds also translated to functional activity, causing smooth muscle relaxation in rat aorta and guinea pig trachea. The series exhibited oral availability and one member reduced rat blood pressure, consistent with ROCK's role in smooth muscle contraction. Thus, the benzoxaborole moiety represents a novel hinge-binding kinase scaffold that may have potential for therapeutic use.

Structure-activity relationships of 6-(benzoylamino)benzoxaborole analogs were investigated for the inhibition of TNF-α, IL-1β, and IL-6 from lipopolysaccharide stimulated peripheral blood mononuclear cells. Compound 1q showed potent activity against all three cytokines with IC50 values between 0.19 and 0.50μM, inhibited LPS-induced TNF-α and IL-6 elevation in mice and improved collagen-induced arthritis in mice. Compound 1q (AN4161) is considered to be a promising lead for novel anti-inflammatory agent with an excellent pharmacokinetic profile., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A series of novel 6-(aminomethylphenoxy)benzoxaborole analogs was synthesized for the investigation of the structure-activity relationship of the inhibition of TNF-alpha, IL-1beta, and IL-6, from lipopolysaccharide stimulated peripheral blood mononuclear cells. Compounds 9d and 9e showed potent activity against all three cytokines with IC50 values between 33 and 83nM. Chloro substituted analog 9e (AN3485) is considered to be a promising lead for novel anti-inflammatory agent with a favorable pharmacokinetic profile., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Pro-inflammatory cytokines play a critical role in the development of autoimmune and inflammatory diseases. Targeting the cytokine environment has proven efficient for averting inflammation. In this study, we reported that 6-[4-(aminomethyl)-2-chlorophenoxyl]benzo[c][1,2]oxaborol-1(3H)-ol (AN3485), a benzoxaborole analog, inhibited TLR2-, TLR3-, TLR4-, and TLR5-mediated TNF-α, IL-1β, and IL-6 release from human PBMCs and isolated monocytes with IC(50) values ranging from 18 to 580 nM, and the inhibition was mediated at the transcriptional level. Topical administration of AN3485 significantly reduced PMA-induced contact dermatitis and oxazolone-induced delayed-type hypersensitivity in mice, indicating its capability of penetrating skin and potential topical application in skin inflammation. Oral administration of AN3485 showed dose-dependent suppression of LPS-induced TNF-α and IL-6 production in mice with an ED(90) of 30 mg/kg. Oral AN3485, 35 mg/kg, twice a day, suppressed collagen-induced arthritis in mice over a 20-day period. The potent anti-inflammatory activity in in vitro and in vivo disease models makes AN3485 an attractive therapeutic lead for a variety of cutaneous and systemic inflammatory diseases.

There is an increasing interest in in vivo metabolite identification in early drug discovery in order to (i) give a more complete picture of metabolic profile in investigational animal models, (ii) propose phase I and phase II metabolites using the same pharmacokinetic/toxicokinetic study samples, (iii) expose metabolically labile groups where chemical modifications could improve stability, and (iv) enable early safety assessment of metabolites. In the early discovery stage of our anti-inflammatory program, one novel benzoxaborole, AN6414, exhibiting both PDE4 enzyme and TNFα inhibition activities, became our primary candidate for further investigation. The traditional metabolite identifications usually require high dosed samples with long data scans and analysis. In this study, we conducted quick and more selective core-structure related precursor scans followed by daughter ion scans and identified a total of 10 major phase I and phase II metabolites using rat plasma samples from a toxicokinetic study at an oral dosing of 30 mg/kg. Plasma samples were treated with solid phase extraction (SPE) prior to LC/MS/MS. An AB SCIEX API 4000 QTRAP mass spectrometer coupled with a Shimadzu LC system was used for LC/MS/MS analysis. We found the major metabolites of AN6414 to be oxidative deboronation, protodeboronation, oxidation products and their sulfate-conjugated species. This analysis drove analoging efforts which improved the pharmacokinetic profile, namely, lowering clearance and increasing exposure relative to AN6414. Toxicity predictions by the software program DEREK suggest the identified potential metabolites to be safe., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We have used boron-based molecules to create novel, competitive, reversible inhibitors of phosphodiesterase 4 (PDE4). The co-crystal structure reveals a binding configuration which is unique compared to classical catechol PDE4 inhibitors, with boron binding to the activated water in the bimetal center. These phenoxybenzoxaboroles can be optimized to generate submicromolar potency enzyme inhibitors, which inhibit TNF-α, IL-2, IFN-γ, IL-5 and IL-10 activities in vitro and show safety and efficacy for topical treatment of human psoriasis. They provide a valuable new route for creating novel potent anti-PDE4 inhibitors., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

We report the discovery of novel boron-containing molecules, exemplified by N-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)-2-trifluoromethylbenzamide (AN3520) and 4-fluoro-N-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)-2-trifluoromethylbenzamide (SCYX-6759), as potent compounds against Trypanosoma brucei in vitro, including the two subspecies responsible for human disease T. b. rhodesiense and T. b. gambiense. These oxaborole carboxamides cured stage 1 (hemolymphatic) trypanosomiasis infection in mice when administered orally at 2.5 to 10 mg/kg of body weight for 4 consecutive days. In stage 2 disease (central nervous system [CNS] involvement), mice infected with T. b. brucei were cured when AN3520 or SCYX-6759 were administered intraperitoneally or orally (50 mg/kg) twice daily for 7 days. Oxaborole-treated animals did not exhibit gross signs of compound-related acute or subchronic toxicity. Metabolism and pharmacokinetic studies in several species, including nonhuman primates, demonstrate that both SCYX-6759 and AN3520 are low-clearance compounds. Both compounds were well absorbed following oral dosing in multiple species and also demonstrated the ability to cross the blood-brain barrier with no evidence of interaction with the P-glycoprotein transporter. Overall, SCYX-6759 demonstrated superior pharmacokinetics, and this was reflected in better efficacy against stage 2 disease in the mouse model. On the whole, oxaboroles demonstrate potent activity against all T. brucei subspecies, excellent physicochemical profiles, in vitro metabolic stability, a low potential for CYP450 inhibition, a lack of active efflux by the P-glycoprotein transporter, and high permeability. These properties strongly suggest that these novel chemical entities are suitable leads for the development of new and effective orally administered treatments for human African trypanosomiasis.

We have used a supervised classification approach to systematically mine a large microarray database derived from livers of compound-treated rats. Thirty-four distinct signatures (classifiers) for pharmacological and toxicological end points can be identified. Just 200 genes are sufficient to classify these end points. Signatures were enriched in xenobiotic and immune response genes and contain un-annotated genes, indicating that not all key genes in the liver xenobiotic responses have been characterized. Many signatures with equal classification capabilities but with no gene in common can be derived for the same phenotypic end point. The analysis of the union of all genes present in these signatures can reveal the underlying biology of that end point as illustrated here using liver fibrosis signatures. Our approach using the whole genome and a diverse set of compounds allows a comprehensive view of most pharmacological and toxicological questions and is applicable to other situations such as disease and development.

Toxicogenomics using a reference database can provide a better understanding and prediction of toxicity, largely by creating biomarkers that tie gene expression to actual pathology events. During the course of building a toxicogenomic database, an observation was made that a number of non-steroidal anti-inflammatory compounds (NSAIDs) at supra-pharmacologic doses induced an acute phase response (APR) and displayed hepatic gene expression patterns similar to that of intravenous lipopolysaccharide (LPS). Since NSAIDs are known to cause injury along the gastrointestinal tract, it has been suggested that NSAIDs increase intestinal permeability, allowing LPS and/or bacteria into the systemic circulation and stimulating an APR detectable in the liver. A short term study was subsequently conducted examining the effects of aspirin, indomethacin, ibuprofen, and rofecoxib to rats and a variety of endpoints were examined that included serum levels of inflammatory cytokines, histologic evaluation, and hepatic gene expression. Both indomethacin and ibuprofen injured the gastrointestinal tract, induced an APR, and increased serum levels of LPS, while rofecoxib and aspirin did not affect the GI tract or induce an APR. In treatments that eventually showed a systemic inflammatory response, hepatic expression of many inflammatory genes was noted as early as 6 hours after treatment well before alterations in traditional clinical pathology markers were detected. This finding led to the creation of a hepatic gene expression biomarker of APR that was effectively shown to be an early identifier of imminent inflammatory injury. In terms of the relative gastrointestinal safety and the NSAIDs studied, an important safety distinction can be made between the presumptive efficacious dose and the APR-inducing dose for indomethacin (1-2-fold), ibuprofen (5-fold), and rofecoxib (approximately 250-fold). Our data support the notion that NSAID-induced intestinal injury results in leakage of commensural bacteria and/or LPS into the circulation, provoking a systemic inflammatory response and that hepatic gene expression-based biomarkers can be used as early and sensitive biomarkers of APR onset.

The comparability and reliability of data generated using microarray technology would be enhanced by use of a common set of standards that allow accuracy, reproducibility and dynamic range assessments on multiple formats. We designed and tested a complex biological reagent for performance measurements on three commercial oligonucleotide array formats that differ in probe design and signal measurement methodology. The reagent is a set of two mixtures with different proportions of RNA for each of four rat tissues (brain, liver, kidney and testes). The design provides four known ratio measurements of >200 reference probes, which were chosen for their tissue-selectivity, dynamic range coverage and alignment to the same exemplar transcript sequence across all three platforms. The data generated from testing three biological replicates of the reagent at eight laboratories on three array formats provides a benchmark set for both laboratory and data processing performance assessments. Close agreement with target ratios adjusted for sample complexity was achieved on all platforms and low variance was observed among platforms, replicates and sites. The mixed tissue design produces a reagent with known gene expression changes within a complex sample and can serve as a paradigm for performance standards for microarrays that target other species.

Successful drug discovery requires accurate decision making in order to advance the best candidates from initial lead identification to final approval. Chemogenomics, the use of genomic tools in pharmacology and toxicology, offers a promising enhancement to traditional methods of target identification/validation, lead identification, efficacy evaluation, and toxicity assessment. To realize the value of chemogenomics information, a contextual database is needed to relate the physiological outcomes induced by diverse compounds to the gene expression patterns measured in the same animals. Massively parallel gene expression characterization coupled with traditional assessments of drug candidates provides additional, important mechanistic information, and therefore a means to increase the accuracy of critical decisions. A large-scale chemogenomics database developed from in vivo treated rats provides the context and supporting data to enhance and accelerate accurate interpretation of mechanisms of toxicity and pharmacology of chemicals and drugs. To date, approximately 600 different compounds, including more than 400 FDA approved drugs, 60 drugs approved in Europe and Japan, 25 withdrawn drugs, and 100 toxicants, have been profiled in up to 7 different tissues of rats (representing over 3200 different drug-dose-time-tissue combinations). Accomplishing this task required evaluating and improving a number of in vivo and microarray protocols, including over 80 rigorous quality control steps. The utility of pairing clinical pathology assessments with gene expression data is illustrated using three anti-neoplastic drugs: carmustine, methotrexate, and thioguanine, which had similar effects on the blood compartment, but diverse effects on hepatotoxicity. We will demonstrate that gene expression events monitored in the liver can be used to predict pathological events occurring in that tissue as well as in hematopoietic tissues.

A large gene expression database has been produced that characterizes the gene expression and physiological effects of hundreds of approved and withdrawn drugs, toxicants, and biochemical standards in various organs of live rats. In order to derive useful biological knowledge from this large database, a variety of supervised classification algorithms were compared using a 597-microarray subset of the data. Our studies show that several types of linear classifiers based on Support Vector Machines (SVMs) and Logistic Regression can be used to derive readily interpretable drug signatures with high classification performance. Both methods can be tuned to produce classifiers of drug treatments in the form of short, weighted gene lists which upon analysis reveal that some of the signature genes have a positive contribution (act as "rewards" for the class-of-interest) while others have a negative contribution (act as "penalties") to the classification decision. The combination of reward and penalty genes enhances performance by keeping the number of false positive treatments low. The results of these algorithms are combined with feature selection techniques that further reduce the length of the drug signatures, an important step towards the development of useful diagnostic biomarkers and low-cost assays. Multiple signatures with no genes in common can be generated for the same classification end-point. Comparison of these gene lists identifies biological processes characteristic of a given class.

One application of genomics in drug safety assessment is the identification of biomarkers to predict compound toxicity before it is detected using traditional approaches, such as histopathology. However, many genomic approaches have failed to demonstrate superiority to traditional methods, have not been appropriately validated on external samples, or have been derived using small data sets, thus raising concerns of their general applicability. Using kidney gene expression profiles from male SD rats treated with 64 nephrotoxic or non-nephrotoxic compound treatments, a gene signature consisting of only 35 genes was derived to predict the future development of renal tubular degeneration weeks before it appears histologically following short-term test compound administration. By comparison, histopathology or clinical chemistry fails to predict the future development of tubular degeneration, thus demonstrating the enhanced sensitivity of gene expression relative to traditional approaches. In addition, the performance of the signature was validated on 21 independent compound treatments structurally distinct from the training set. The signature correctly predicted the ability of test compounds to induce tubular degeneration 76% of the time, far better than traditional approaches. This study demonstrates that genomic data can be more sensitive than traditional methods for the early prediction of compound-induced pathology in the kidney.

Multicellular organisms must have means of preserving their genomic integrity or face catastrophic consequences such as uncontrolled cell proliferation or massive cell death. One response is a modification of nuclear proteins by the addition and removal of polymers of ADP-ribose that modulate the properties of DNA-binding proteins involved in DNA repair and metabolism. These ADP-ribose units are added by poly(ADP-ribose) polymerase (PARP) and removed by poly(ADP-ribose) glycohydrolase. Although budding yeast Saccharomyces cerevisiae does not possess proteins with significant sequence similarity to the human PARP family of proteins, we identified novel small molecule inhibitors against two family members, PARP1 and PARP2, using a cell-based assay in yeast. The assay was based on the reversal of growth inhibition caused by the heterologous expression of either PARP1 or PARP2. Validation of the assay was achieved by showing that the growth inhibition was relieved by a mutation in a single residue in the catalytic site of PARP1 or PARP2 or exposure of yeast to a known PARP1 inhibitor, 6(5H)-phenanthridinone. In separate experiments, when a putative protein regulator of PARP activity, human poly(ADP-ribose) glycohydrolase, was coexpressed with PARP1 or PARP2, yeast growth was restored. Finally, the inhibitors identified by screening the yeast assay are active in a mammalian PARP biochemical assay and inhibit PARP1 and PARP2 activity in yeast cell extracts. Thus, our data reflect the strength of using yeast to identify small molecule inhibitors of therapeutically relevant gene families, including those that are not found in yeast, such as PARP. The resultant inhibitors have two critical uses (a) as leads for drug development and (b) as tools to dissect cellular function.

-Bradykinin, a major vasodilator peptide, plays an important role in the local regulation of blood pressure, blood flow, and vascular permeability; however, the cellular distribution of the major bradykinin B(2) receptor in the cardiovascular system is not precisely known. Immunoblot analysis with an anti-peptide antibody to the bradykinin B(2) receptor or chemical cross-linkage with [(125)I]Tyr(0)-bradykinin revealed a band of 69+/-3 kDa at varying intensity in the homogenates of the endothelium and tunica media of the rat aorta and endocardium. Immunostaining showed that the B(2) receptor is abundant in the endothelial linings of the aorta, other elastic arteries, muscular arteries, capillaries, venules, and large veins, where it localizes preferentially to the luminal face of the endothelial cells. In marked contrast, small arterioles (ie, the principal blood-pressure regulating vessels) of the mesenterium, heart, urinary bladder, brain, salivary gland, and kidney had a different staining pattern in which B(2) receptor was prominent in the perivascular smooth muscle cells of the tunica media. A similar distribution pattern was found in mouse as well as in human tissues, indicating that the particular distribution pattern of the B(2) receptor in arterioles is not a species-specific phenomenon. During development, the distribution of B(2) receptor in the heart changes; for example, in the heart of newborn rats, the B(2) receptor was abundant in the myocardium, whereas in the adult heart, the receptor was present in the endocardium of atria, atrioventricular valves, and ventricles but not in the myocardium. Thus, B(2) receptors are localized differentially in different parts of the cardiovascular system: the arterioles have smooth muscle-localized B(2) receptors, and large elastic vessels have endothelium-localized receptors.

Monocyte chemoattracant-1 (MCP-1) stimulates leukocyte chemotaxis to inflammatory sites, such as rheumatoid arthritis, atherosclerosis, and asthma, by use of the MCP-1 receptor, CCR2, a member of the G-protein-coupled seven-transmembrane receptor superfamily. These studies identified a family of antagonists, spiropiperidines. One of the more potent compounds blocks MCP-1 binding to CCR2 with a K(d) of 60 nm, but it is unable to block binding to CXCR1, CCR1, or CCR3. These compounds were effective inhibitors of chemotaxis toward MCP-1 but were very poor inhibitors of CCR1-mediated chemotaxis. The compounds are effective blockers of MCP-1-driven inhibition of adenylate cyclase and MCP-1- and MCP-3-driven cytosolic calcium influx; the compounds are not agonists for these pathways. We showed that glutamate 291 (Glu(291)) of CCR2 is a critical residue for high affinity binding and that this residue contributes little to MCP-1 binding to CCR2. The basic nitrogen present in the spiropiperidine compounds may be the interaction partner for Glu(291), because the basicity of this nitrogen was essential for affinity; furthermore, a different class of antagonists, a class that does not have a basic nitrogen (2-carboxypyrroles), were not affected by mutations of Glu(291). In addition to the CCR2 receptor, spiropiperidine compounds have affinity for several biogenic amine receptors. Receptor models indicate that the acidic residue, Glu(291), from transmembrane-7 of CCR2 is in a position similar to the acidic residue contributed from transmembrane-3 of biogenic amine receptors, which may account for the shared affinity of spiropiperidines for these two receptor classes. The models suggest that the acid-base pair, Glu(291) to piperidine nitrogen, anchors the spiropiperidine compound within the transmembrane ovoid bundle. This binding site may overlap with the space required by MCP-1 during binding and signaling; thus the small molecule ligands act as antagonists. An acidic residue in transmembrane region 7 is found in most chemokine receptors and is rare in other serpentine receptors. The model of the binding site may suggest ways to make new small molecule chemokine receptor antagonists, and it may rationalize the design of more potent and selective antagonists.

The CC chemokine, monocyte chemotactic protein, 1 (MCP-1) functions as a major chemoattractant for T-cells and monocytes by interacting with the seven-transmembrane G protein-coupled receptor CCR2. To identify which residues of MCP-1 contribute to signaling though CCR2, we mutated all the surface-exposed residues to alanine and other amino acids and made some selective large changes at the amino terminus. We then characterized the impact of these mutations on three postreceptor pathways involving inhibition of cAMP synthesis, stimulation of cytosolic calcium influx, and chemotaxis. The results highlight several important features of the signaling process and the correlation between binding and signaling: The amino terminus of MCP-1 is essential as truncation of residues 2-8 ([1+9-76]hMCP-1) results in a protein that cannot stimulate chemotaxis. However, the exact peptide sequence may be unimportant as individual alanine mutations or simultaneous replacement of residues 3-6 with alanine had little effect. Y13 is also important and must be a large nonpolar residue for chemotaxis to occur. Interestingly, both Y13 and [1+9-76]hMCP-1 are high-affinity binders and thus affinity of these mutants is not correlated with ability to promote chemotaxis. For the other surface residues there is a strong correlation between binding affinity and agonist potency in all three signaling pathways. Perhaps the most interesting observation is that although Y13A and [1+9-76]hMCP are antagonists of chemotaxis, they are agonists of pathways involving inhibition of cAMP synthesis and, in the case of Y13A, calcium influx. These results demonstrate that these two well-known signaling events are not sufficient to drive chemotaxis. Furthermore, it suggests that specific molecular features of MCP-1 induce different conformations in CCR2 that are coupled to separate postreceptor pathways. Therefore, by judicious design of antagonists, it should be possible to trap CCR2 in conformational states that are unable to stimulate all of the pathways required for chemotaxis.

The CC chemokine, MCP-1, has been identified as a major chemoattractant for T cells and monocytes, and plays a significant role in the pathology of inflammatory diseases. To identify the regions of MCP-1 that contact its receptor, CCR2, we substituted all surface-exposed residues with alanine. Some residues were also mutated to other amino acids to identify the importance of charge, hydrophobicity, or aromaticity at specific positions. The binding affinity of each mutant for CCR2 was assayed with THP-1 and CCR2-transfected CHL cells. The majority of point mutations had no effect. Residues at the N-terminus of the protein, known to be crucial for signaling, contribute less than a factor of 10 to the binding affinity. However, two clusters of primarily basic residues (R24, K35, K38, K49, and Y13), separated by a 35 A hydrophobic groove, reduced the level of binding by 15-100-fold. A peptide fragment encompassing residues 13-35 recapitulated some of the mutational data derived from the intact protein. It exhibited modest binding as a linear peptide and dramatically improved affinity when the region which adopts a single turn of a 3(10)-helix in the protein, which includes R24, was constrained by a disulfide bond. Additional constraints at the ends of the peptide, corresponding to the disulfide between the first and third cysteines in MCP-1, yielded further improvements in affinity. Together, these data suggest a model in which a large surface area of MCP-1 contacts the receptor, and the accumulation of a number of weak interactions results in the 35 pM affinity observed for the wild-type (WT) protein. The receptor binding site of MCP-1 also is significantly different from the binding sites of RANTES and IL-8, providing insight into the issue of receptor specificity. It was previously shown that the N-terminus of CCR2 is critical for binding MCP-1 [Monteclaro, F. S., and Charo, I. F. (1996) J. Biol. Chem. 271, 19084-92; Monteclaro, F. S., and Charo, I. F. (1997) J. Biol. Chem. 272, 23186-90]. Point mutations of six acidic residues in this region of the receptor were made to test their role in ligand binding. This identified D25 and D27 of the DYDY motif as being important. On the basis of our data, we propose a model in which the receptor N-terminus lies along the hydrophobic groove in an extended fashion, placing the DYDY motif near the basic cluster involving R24 and K49 of MCP-1. This in turn orients the signaling residues (Y13 and the N-terminus) for productive interaction with the receptor.

Four cyclic-nucleotide phosphodiesterase (PDE) genes belonging to the PDE4 family (PDE4A, 4B, 4C and 4D) have been identified. All four isogenes, including several deletions and alterations of the amino, carboxyl and central catalytic domains, were expressed in insect cells. Lysates were characterised for enzyme activity by using the Km for substrate and the EC50 for activation by the cofactor Mg2+. The catalytic domain alone appears to be sufficient for the normal enzymatic function of PDE4 proteins. Substrate affinity varied by less than 2-fold between catalytic-domain forms of the PDE4A, 4B and 4D isogenes and the long forms (PDE4A5, PDE4B1 and PDE4D3). The affinity for Mg2+ varied by less than 4-fold between long and catalytic-domain forms of PDE4A and 4B. The catalytic-domain form of PDE4D, however, had a 12-fold lower affinity for Mg2+ that was restored by including a portion of the amino-terminal domain, upstream conserved region-2 (UCR2). This result suggests that the Mg2+-binding site of PDE4D involves the UCR2 region. Inhibition of the PDE4 proteins by synthetic compounds is apparently affected differently by the domains. For PDE4B, the catalytic domain is sufficient for interactions with the inhibitors studied: IBMX, trequinsin, rolipram, TVX 2706, RP 73401 and RS-25344. For PDE4D the catalytic-domain form is less sensitive than the long form to inhibition by RS-25344, rolipram and TVX 2706, by 1463-, 11-and 12-fold, respectively. Addition of UCR2 to the catalytic-domain form of PDE4D restored all the lost sensitivities. The catalytic-domain form of PDE4A showed a reduced inhibitor affinity with RS-25344 and TVX 2706 by 77- and 90-fold, respectively. Both catalytic-domain and long forms of PDE4 isogenes interacted with equal affinity with the non-specific inhibitors IBMX and trequinsin, as well as the very potent PDE4-specific inhibitor RP 73401. Other potent and specific PDE4 inhibitors, such as rolipram, RS-25344 or TVX 2706, appear to utilize non-catalytic domain interactions with PDE4D and 4A to supplement those within the catalytic domains. These observations suggest a different relation between amino and catalytic domains in PDE4D relative to PDE4B. We therefore propose a model to illustrate these isogene-specific PDE4 domain interactions with substrate, inhibitors and the co-factor Mg2+. The model for PDE4D is also discussed in relation to changes in the activation curve for Mg2+ and sensitivity to RS-25344 that accompany phosphorylation of the long form by protein kinase A.