RESUMO
Drug discovery and development is a lengthy and expensive process. Although no one, simple, single solution can significantly accelerate this process, steps can be taken to avoid unnecessary delays. Using the development of antiviral therapies as a model, we describe options for acceleration that cover target selection, assay development and high-throughput screening, hit confirmation, lead identification and development, animal model evaluations, toxicity studies, regulatory issues, and the general drug discovery and development infrastructure. Together, these steps could result in accelerated timelines for bringing antiviral therapies to market so they can treat emerging infections and reduce human suffering.
Assuntos
Antivirais/administração & dosagem , Sistemas de Liberação de Medicamentos/tendências , Descoberta de Drogas/tendências , Vírus/efeitos dos fármacos , Animais , Antivirais/química , Química Farmacêutica/métodos , Química Farmacêutica/tendências , Sistemas de Liberação de Medicamentos/métodos , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/tendências , Humanos , Viroses/tratamento farmacológico , Viroses/patologia , Vírus/crescimento & desenvolvimentoRESUMO
RATIONALE: Premature termination codons (PTCs) in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF). Several agents are known to suppress PTCs but are poorly efficacious or toxic. OBJECTIVES: To determine whether there are clinically available agents that elicit translational readthrough and improve CFTR function sufficient to confer therapeutic benefit to patients with CF with PTCs. METHODS: Two independent screens, firefly luciferase and CFTR-mediated transepithelial chloride conductance assay, were performed on a library of 1,600 clinically approved compounds using fisher rat thyroid cells stably transfected with stop codons. Select agents were further evaluated using secondary screening assays including short circuit current analysis on primary cells from patients with CF. In addition, the effect of CFTR modulators (ivacaftor) was tested in combination with the most efficacious agents. MEASUREMENTS AND MAIN RESULTS: From the primary screen, 48 agents were selected as potentially active. Following confirmatory tests in the transepithelial chloride conductance assay and prioritizing agents based on favorable pharmacologic properties, eight agents were advanced for secondary screening. Ivacaftor significantly increased short circuit current following forskolin stimulation in cells treated with pyranoradine tetraphosphate, potassium p-aminobenzoate, and escin as compared with vehicle control. Escin, an herbal agent, consistently induced readthrough activity as demonstrated by enhanced CFTR expression and function in vitro. CONCLUSIONS: Clinically approved drugs identified as potential readthrough agents, in combination with ivacaftor, may induce nonsense suppression to restore therapeutic levels of CFTR function. One or more agents may be suitable to advance to human testing.
Assuntos
Códon sem Sentido/efeitos dos fármacos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Descoberta de Drogas/métodos , Animais , Linhagem Celular , Códon sem Sentido/genética , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Luciferases/metabolismo , Ratos Endogâmicos F344 , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Four years after the beginning of the COVID-19 pandemic, it is important to reflect on the events that have occurred during that time and the knowledge that has been gained. The response to the pandemic was rapid and highly resourced; it was also built upon a foundation of decades of federally funded basic and applied research. Laboratories in government, pharmaceutical, academic, and non-profit institutions all played roles in advancing pre-2020 discoveries to produce clinical treatments. This perspective provides a summary of how the development of high-throughput screening methods in a biosafety level 3 (BSL-3) environment at Southern Research Institute (SR) contributed to pandemic response efforts. The challenges encountered are described, including those of a technical nature as well as those of working under the pressures of an unpredictable virus and pandemic.
Assuntos
COVID-19 , Ensaios de Triagem em Larga Escala , Pandemias , SARS-CoV-2 , Humanos , COVID-19/epidemiologia , COVID-19/virologia , SARS-CoV-2/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Antivirais/uso terapêutico , Antivirais/farmacologiaRESUMO
Metastatic dissemination from the primary tumor is a complex process that requires crosstalk between tumor cells and the surrounding milieu and involves the interplay between numerous cellular-signaling programs. Epithelial-mesenchymal transition (EMT) remains at the forefront of orchestrating a shift in numerous cellular programs, such as stemness, drug resistance, and apoptosis that allow for successful metastasis. Till date, there is limited success in therapeutically targeting EMT. Utilizing a high throughput screen of FDA-approved compounds, we uncovered a novel role of the topoisomerase inhibitor, Teniposide, in reversing EMT. Here, we demonstrate Teniposide as a potent modulator of the EMT program, specifically through an IRF7-NMI mediated response. Furthermore, Teniposide significantly reduces the expression of the key EMT transcriptional regulator, Zinc Finger E-Box Binding Homeobox 2 (ZEB2). ZEB2 downregulation by Teniposide inhibited RNA polymerase I (Pol I) activity and rRNA biogenesis. Importantly, Teniposide treatment markedly reduced pulmonary colonization of breast cancer cells. We have uncovered a novel role of Teniposide, which when used at a very low concentration, mitigates mesenchymal-like invasive phenotype. Overall, its ability to target EMT and rRNA biogenesis makes Teniposide a viable candidate to be repurposed as a therapeutic option to restrict breast cancer metastases.
Assuntos
Neoplasias da Mama , Regulação para Baixo , Transição Epitelial-Mesenquimal , RNA Polimerase I , Teniposídeo , Homeobox 2 de Ligação a E-box com Dedos de Zinco , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Feminino , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , RNA Polimerase I/metabolismo , Teniposídeo/farmacologia , Animais , Camundongos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
Alzheimer's disease (AD) is the leading cause of dementia and lacks highly effective treatments. Tau-based therapies hold promise. Tau reduction prevents amyloid-ß-induced dysfunction in preclinical models of AD and also prevents amyloid-ß-independent dysfunction in diverse disease models, especially those with network hyperexcitability, suggesting that strategies exploiting the mechanisms underlying Tau reduction may extend beyond AD. Tau binds several SH3 domain-containing proteins implicated in AD via its central proline-rich domain. We previously used a peptide inhibitor to demonstrate that blocking Tau interactions with SH3 domain-containing proteins ameliorates amyloid-ß-induced dysfunction. Here, we identify a top hit from high-throughput screening for small molecules that inhibit Tau-FynSH3 interactions and describe its optimization with medicinal chemistry. The resulting lead compound is a potent cell-permeable Tau-SH3 interaction inhibitor that binds Tau and prevents amyloid-ß-induced dysfunction, including network hyperexcitability. These data support the potential of using small molecule Tau-SH3 interaction inhibitors as a novel therapeutic approach to AD.
Assuntos
Doença de Alzheimer , Proteínas tau , Humanos , Proteínas tau/metabolismo , Peptídeos beta-Amiloides/toxicidade , Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Ensaios de Triagem em Larga EscalaRESUMO
Neuromedin U (NMU), through its cognate receptor NMUR2 in the central nervous system, regulates several important physiological functions, including energy balance, stress response, and nociception. By random screening of our corporate compound collection with a ligand binding assay, we discovered (R)-5'-(phenylaminocarbonylamino)spiro[1-azabicyclo[2.2.2]octane-3,2'(3'H)-furo[2,3-b]pyridine] (R-PSOP), a highly potent and selective NMUR2 antagonist. R-PSOP is a nonpeptidic small-molecule with the chemical composition C(20)N(4)O(2)H(22). In competition binding experiments, this compound was found to bind to NMUR2 with high affinity; the K(i) values were determined to be 52 and 32 nM for the human and rat NMUR2, respectively. Moreover, in functional assays measuring phosphoinositide turnover or intracellular calcium mobilization, R-PSOP strongly inhibited the responses stimulated by peptide agonists NMU-25, NMU-23, and NMU-8 in human embryonic kidney 293 cells expressing NMUR2. From Schild analyses, the functional K(b) values for R-PSOP were determined to be 92 and 155 nM at human and rat NMUR2, respectively. Highly selective for NMUR2, R-PSOP exhibited low affinity to the other subtype of NMU receptor, NMUR1, with a K(i) value >10 microM. R-PSOP in vivo attenuated NMU-23-evoked nociceptive responses in a rat spinal reflex preparation. To our knowledge, this is the first antagonist ever reported for NMU receptors. This compound could serve as a valuable tool for further understanding the physiological and pathophysiological roles of NMU system, while providing a chemical starting point that may lead to development of new therapeutics for treatment of eating disorders, obesity, pain, and stress-related disorders.
Assuntos
Neuropeptídeos/metabolismo , Neuropeptídeos/farmacologia , Receptores de Neurotransmissores/antagonistas & inibidores , Receptores de Neurotransmissores/metabolismo , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Masculino , Neuropeptídeos/química , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores de Neurotransmissores/agonistasRESUMO
Antagonist and partial agonist modulators of the dopamine D3 receptor (D3R) have emerged as promising therapeutics for the treatment of substance abuse and neuropsychiatric disorders. However, development of druglike lead compounds with selectivity for the D3 receptor has been challenging because of the high sequence homology between the D3R and the dopamine D2 receptor (D2R). In this effort, we synthesized a series of acylaminobutylpiperazines incorporating aza-aromatic units and evaluated their binding and functional activities at the D3 and D2 receptors. Docking studies and results from evaluations against a set of chimeric and mutant receptors suggest that interactions at the extracellular end of TM7 contribute to the D3R versus D2R selectivity of these ligands. Molecular insights from this study could potentially enable rational design of potent and selective D3R ligands.
Assuntos
Antagonistas de Dopamina/farmacologia , Piperazinas/química , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/metabolismo , Animais , Arrestinas/metabolismo , Células CHO/efeitos dos fármacos , Técnicas de Química Sintética , Cricetulus , Antagonistas de Dopamina/química , Antagonistas dos Receptores de Dopamina D2/química , Antagonistas dos Receptores de Dopamina D2/farmacologia , Desenho de Fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutação , Piperazinas/síntese química , Piperazinas/metabolismo , Receptores de Dopamina D2/química , Receptores de Dopamina D2/genética , Receptores de Dopamina D3/química , Receptores de Dopamina D3/genética , Relação Estrutura-Atividade , beta-ArrestinasRESUMO
A recently identified novel Galphaolf variant, XLGalphaolf, is shown to functionally couple to the human adenosine A2A receptor (A2AR). In Sf9 cells expressing A2AR, beta1, and gamma2, co-expression of XLGalphaolf increased NECA-induced [35S]GTPgammaS binding from approximately 130% to 300% of basal levels. Pharmacological characteristics of A2AR ligands on these cells were evaluated by using [3H]ZM241385- and [35S]GTPgammaS- binding assays. The rank order of the equilibrium binding constants (Kd or Ki) of adenosine receptor ligands were [3H]ZM241385 approximately CGS15943 < MRS1220 < < CV1808 approximately NECA < CGS21680 approximately adenosine < IBMECA < HEMADO approximately CPA approximately CCPA. The rank order of EC50 values for agonists were CV1808 approximately NECA < adenosine approximately CGS26180 < IBMECA < HEMADO approximately CPA approximately CCPA. This pharmacology is consistent with the literature for A2AR and suggests that Sf9 cells co-expressing A2AR, beta1, gamma2, and XLGalphaolf could serve as a heterologous expression system for A2AR drug screening.