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Artificial intelligence is revolutionizing protein structure prediction, providing unprecedented opportunities for drug design. To assess the potential impact on ligand discovery, we compared virtual screens using protein structures generated by the AlphaFold machine learning method and traditional homology modeling. More than 16 million compounds were docked to models of the trace amine-associated receptor 1 (TAAR1), a G protein-coupled receptor of unknown structure and target for treating neuropsychiatric disorders. Sets of 30 and 32 highly ranked compounds from the AlphaFold and homology model screens, respectively, were experimentally evaluated. Of these, 25 were TAAR1 agonists with potencies ranging from 12 to 0.03 µM. The AlphaFold screen yielded a more than twofold higher hit rate (60%) than the homology model and discovered the most potent agonists. A TAAR1 agonist with a promising selectivity profile and drug-like properties showed physiological and antipsychotic-like effects in wild-type but not in TAAR1 knockout mice. These results demonstrate that AlphaFold structures can accelerate drug discovery.
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Descubrimiento de Drogas , Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Animales , Ratones , Humanos , Ratones Noqueados , Psicotrópicos/farmacología , Psicotrópicos/química , Simulación del Acoplamiento Molecular , LigandosRESUMEN
Here, we describe the identification of an antibiotic class acting via LpxH, a clinically unexploited target in lipopolysaccharide synthesis. The lipopolysaccharide synthesis pathway is essential in most Gram-negative bacteria and there is no analogous pathway in humans. Based on a series of phenotypic screens, we identified a hit targeting this pathway that had activity on efflux-defective strains of Escherichia coli. We recognized common structural elements between this hit and a previously published inhibitor, also with activity against efflux-deficient bacteria. With the help of X-ray structures, this information was used to design inhibitors with activity on efflux-proficient, wild-type strains. Optimization of properties such as solubility, metabolic stability and serum protein binding resulted in compounds having potent in vivo efficacy against bloodstream infections caused by the critical Gram-negative pathogens E. coli and Klebsiella pneumoniae. Other favorable properties of the series include a lack of pre-existing resistance in clinical isolates, and no loss of activity against strains expressing extended-spectrum-ß-lactamase, metallo-ß-lactamase, or carbapenemase-resistance genes. Further development of this class of antibiotics could make an important contribution to the ongoing struggle against antibiotic resistance.
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Antibacterianos , Lipopolisacáridos , Humanos , Antibacterianos/química , Escherichia coli/metabolismo , Bacterias Gramnegativas/metabolismo , beta-Lactamasas/genética , Pruebas de Sensibilidad MicrobianaRESUMEN
Data on drug transfer into human breast milk are sparse. This study aimed to quantify concentrations of cetirizine and levocetirizine in breast milk and to estimate drug exposure to infants. Breastfeeding women at least 8 weeks postpartum and using cetirizine or its pure (R)-enantiomer levocetirizine were eligible to participate. Breast milk samples were collected at six predefined times during a dose interval (0, 2, 4, 8, 12 and 24 h after drug intake) at steady state. Infant drug exposure was estimated by calculating the absolute infant dose (AID) and the weight-adjusted relative infant dose (RID). In total, 32 women were eligible for final inclusion, 31 women using cetirizine and one woman using levocetirizine. Means of the individual maximum and average cetirizine milk concentrations were 41.0 and 16.8 µg/L, respectively. Maximum concentrations occurred on average 2.4 h after intake, and the mean half-life in milk was 7.0 h. Estimated AID and RID for cetirizine in a day were 2.5 µg/kg and 1.9%, respectively. The corresponding values for levocetirizine were 1.1 µg/kg and 1.9%. No severe adverse events were reported. Our findings demonstrate that the transfer of cetirizine and levocetirizine into breast milk is low and compatible with breastfeeding.
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Lactancia Materna , Cetirizina , Lactante , Humanos , Femenino , Cetirizina/efectos adversos , Leche Humana , LactanciaRESUMEN
The majority of women have health problems that require medication after giving birth. Complications such as allergies, postpartum depression, and diabetes are often treated with drugs such as cetirizine, venlafaxine, and metformin, respectively. These treatments are considered safe during lactation, but information of the transfer of drugs to breast milk and possible effects on the infant is scarce. Therefore, this needs to be systematically investigated in larger populations. To enable the determination of drug transfer, we here describe the validation of two rapid, sensitive, and high-throughput analysis methods for 1) simultaneous quantification of cetirizine, venlafaxine, and O-desmethylvenlafaxine in human breast milk, and 2) metformin in human breast milk and plasma. In both methods, a simple protein precipitation protocol with acetonitrile and benchtop-centrifugation was used prior to compound analysis with liquid-chromatography tandem mass spectrometry. The methods had linear ranges between 0.39 - 194.5 ng/mL for cetirizine, 0.28 - 138.7 ng/mL for venlafaxine, 0.26 - 131.7 ng/mL for O-desmethylvenlafaxine, in milk, and 0.65 - 193.7 ng/mL for metformin in both milk and plasma. Intra-run and inter-run precision and accuracy were ≤ 9% for cetirizine, venlafaxine, and O-desmethylvenlafaxine in milk, and ≤ 7% for metformin in milk and plasma. Cetirizine was measured to median milk concentrations of 13 ng/mL (range: 0.65 - 65 ng/mL) in 228 donor samples from breast-feeding women.
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Metformina , Espectrometría de Masas en Tándem , Cetirizina , Cromatografía Líquida de Alta Presión/métodos , Succinato de Desvenlafaxina , Femenino , Humanos , Lactante , Leche Humana , Embarazo , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/métodos , Clorhidrato de VenlafaxinaAsunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Infecciones Bacterianas/tratamiento farmacológico , Desarrollo de Medicamentos , Descubrimiento de Drogas , Asociación entre el Sector Público-Privado/organización & administración , Animales , Infecciones Bacterianas/microbiología , HumanosRESUMEN
This issue of the Journal of Pharmaceutical Sciences is dedicated to Professor Per Artursson and the groundbreaking contributions he has made and continues to make in the Pharmaceutical Sciences. Per is one of the most cited researchers in his field, with more than 30,000 citations and an h-index of 95 as of September 2020. Importantly, these citations are distributed over the numerous fields he has explored, clearly showing the high impact the research has had on the discipline. We provide a short portrait of Per, with emphasis on his personality, driving forces and the inspirational sources that shaped his career as a world-leading scientist in the field. He is a curious scientist who deftly moves between disciplines and has continued to innovate, expand boundaries, and profoundly impact the pharmaceutical sciences throughout his career. He has developed new tools and provided insights that have significantly contributed to today's molecular and mechanistic approaches to research in the fields of intestinal absorption, cellular disposition, and exposure-efficacy relationships of pharmaceutical drugs. We want to celebrate these important contributions in this special issue of the Journal of Pharmaceutical Sciences in Per's honor.
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Investigación Farmacéutica , Farmacia , Historia del Siglo XX , Humanos , MentoresRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
The clinical impact of drug-drug interactions based on time-dependent inhibition of cytochrome P450 (CYP) 3A4 has often been overpredicted, likely due to use of improper inhibitor concentration estimates at the enzyme. Here, we investigated if use of cytosolic unbound inhibitor concentrations could improve predictions of time-dependent drug-drug interactions. First, we assessed the inhibitory effects of ten time-dependent CYP3A inhibitors on midazolam 1'-hydroxylation in human liver microsomes. Then, using a novel method, we determined the cytosolic bioavailability of the inhibitors in human hepatocytes, and used the obtained values to calculate their concentrations at the active site of the enzyme, i.e. the cytosolic unbound concentrations. Finally, we combined the data in mechanistic static predictions, by considering different combinations of inhibitor concentrations in intestine and liver, including hepatic concentrations corrected for cytosolic bioavailability. The results were then compared to clinical data. Compared to no correction, correction for cytosolic bioavailability resulted in higher accuracy and precision, generally in line with those obtained by more demanding modelling. The best predictions were obtained when the inhibition of hepatic CYP3A was based on unbound maximal inhibitor concentrations corrected for cytosolic bioavailability. Our findings suggest that cytosolic unbound inhibitor concentrations improves predictions of time-dependent drug-drug interactions for CYP3A.
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Citosol/química , Interacciones Farmacológicas/fisiología , Preparaciones Farmacéuticas/metabolismo , Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/metabolismo , Inhibidores del Citocromo P-450 CYP3A/química , Inhibidores del Citocromo P-450 CYP3A/metabolismo , Citosol/metabolismo , Humanos , Cetólidos/química , Cetólidos/metabolismo , Cinética , Microsomas Hepáticos/química , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Preparaciones Farmacéuticas/química , Piperazinas/química , Piperazinas/metabolismo , Triazoles/química , Triazoles/metabolismoRESUMEN
The onset of inflammation is associated with reactive oxygen species and oxidative damage to macromolecules like 7,8-dihydro-8-oxoguanine (8-oxoG) in DNA. Because 8-oxoguanine DNA glycosylase 1 (OGG1) binds 8-oxoG and because Ogg1-deficient mice are resistant to acute and systemic inflammation, we hypothesized that OGG1 inhibition may represent a strategy for the prevention and treatment of inflammation. We developed TH5487, a selective active-site inhibitor of OGG1, which hampers OGG1 binding to and repair of 8-oxoG and which is well tolerated by mice. TH5487 prevents tumor necrosis factor-α-induced OGG1-DNA interactions at guanine-rich promoters of proinflammatory genes. This, in turn, decreases DNA occupancy of nuclear factor κB and proinflammatory gene expression, resulting in decreased immune cell recruitment to mouse lungs. Thus, we present a proof of concept that targeting oxidative DNA repair can alleviate inflammatory conditions in vivo.
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Antiinflamatorios no Esteroideos/farmacología , Bencimidazoles/farmacología , ADN Glicosilasas/antagonistas & inhibidores , Inhibidores Enzimáticos/uso terapéutico , Expresión Génica/efectos de los fármacos , Inflamación/tratamiento farmacológico , Piperidinas/farmacología , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Bencimidazoles/uso terapéutico , ADN Glicosilasas/metabolismo , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Técnicas de Inactivación de Genes , Guanina/análogos & derivados , Guanina/antagonistas & inhibidores , Guanina/metabolismo , Células HEK293 , Humanos , Inflamación/genética , Células Jurkat , Ratones , Ratones Mutantes , FN-kappa B/genética , FN-kappa B/metabolismo , Piperidinas/uso terapéutico , Regiones Promotoras Genéticas , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Rift Valley fever virus (RVFV) is a mosquito-borne hemorrhagic fever virus affecting both humans and animals with severe morbidity and mortality and is classified as a potential bioterror agent due to the possible aerosol transmission. At present there is no human vaccine or antiviral therapy available. Thus, there is a great need to develop new antivirals for treatment of RVFV infections. Benzavir-2 was previously identified as potent inhibitor of human adenovirus, herpes simplex virus type 1, and type 2. Here we assess the anti-RVFV activity of benzavir-2 together with four structural analogs and determine pre-clinical pharmacokinetic parameters of benzavir-2. In vitro, benzavir-2 efficiently inhibited RVFV infection, viral RNA production and production of progeny viruses. In vitro, benzavir-2 displayed satisfactory solubility, good permeability and metabolic stability. In mice, benzavir-2 displayed oral bioavailability with adequate maximum serum concentration. Oral administration of benzavir-2 formulated in peanut butter pellets gave high systemic exposure without any observed toxicity in mice. To summarize, our data demonstrated potent anti-RVFV activity of benzavir-2 in vitro together with a promising pre-clinical pharmacokinetic profile. This data support further exploration of the antiviral activity of benzavir-2 in in vivo efficacy models that may lead to further drug development for human use.
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Antivirales/farmacología , Antivirales/farmacocinética , Benzoatos/farmacología , Benzoatos/farmacocinética , Virus de la Fiebre del Valle del Rift/fisiología , Células A549 , Administración Oral , Animales , Antivirales/administración & dosificación , Antivirales/química , Benzoatos/administración & dosificación , Benzoatos/química , Disponibilidad Biológica , Femenino , Humanos , Ratones Endogámicos BALB C , ARN Viral/genética , Fiebre del Valle del Rift/tratamiento farmacológico , Fiebre del Valle del Rift/prevención & control , Fiebre del Valle del Rift/virología , Virus de la Fiebre del Valle del Rift/efectos de los fármacosRESUMEN
With a diverse network of substrates, NUDIX hydrolases have emerged as a key family of nucleotide-metabolizing enzymes. NUDT5 (also called NUDIX5) has been implicated in ADP-ribose and 8-oxo-guanine metabolism and was recently identified as a rheostat of hormone-dependent gene regulation and proliferation in breast cancer cells. Here, we further elucidate the physiological relevance of known NUDT5 substrates and underscore the biological requirement for NUDT5 in gene regulation and proliferation of breast cancer cells. We confirm the involvement of NUDT5 in ADP-ribose metabolism and dissociate a relationship to oxidized nucleotide sanitation. Furthermore, we identify potent NUDT5 inhibitors, which are optimized to promote maximal NUDT5 cellular target engagement by CETSA. Lead compound, TH5427, blocks progestin-dependent, PAR-derived nuclear ATP synthesis and subsequent chromatin remodeling, gene regulation and proliferation in breast cancer cells. We herein present TH5427 as a promising, targeted inhibitor that can be used to further study NUDT5 activity and ADP-ribose metabolism.
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Inhibidores Enzimáticos/farmacología , Progestinas/metabolismo , Pirofosfatasas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Adenosina Difosfato Ribosa/metabolismo , Adenosina Trifosfato/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Femenino , Células HL-60 , Humanos , Estructura Molecular , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , Interferencia de ARN , Especificidad por SustratoRESUMEN
The dCTP pyrophosphatase 1 (dCTPase) is a nucleotide pool "housekeeping" enzyme responsible for the catabolism of canonical and noncanonical nucleoside triphosphates (dNTPs) and has been associated with cancer progression and cancer cell stemness. We have identified a series of piperazin-1-ylpyridazines as a new class of potent dCTPase inhibitors. Lead compounds increase dCTPase thermal and protease stability, display outstanding selectivity over related enzymes and synergize with a cytidine analogue against leukemic cells. This new class of dCTPase inhibitors lays the first stone toward the development of drug-like probes for the dCTPase enzyme.
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Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Piperazinas/química , Piperazinas/farmacología , Piridazinas/química , Piridazinas/farmacología , Pirofosfatasas/antagonistas & inhibidores , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Leucemia/tratamiento farmacológico , Leucemia/enzimología , Simulación del Acoplamiento Molecular , Pirofosfatasas/metabolismoRESUMEN
The dCTP pyrophosphatase 1 (dCTPase) is involved in the regulation of the cellular dNTP pool and has been linked to cancer progression. Here we report on the discovery of a series of 3,6-disubstituted triazolothiadiazoles as potent dCTPase inhibitors. Compounds 16 and 18 display good correlation between enzymatic inhibition and target engagement, together with efficacy in a cellular synergy model, deeming them as a promising starting point for hit-to-lead development.
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Inhibidores Enzimáticos/farmacología , Pirofosfatasas/antagonistas & inhibidores , Tiadiazoles/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , Simulación del Acoplamiento MolecularRESUMEN
In this study, we provide insight into the metabolic profile of a series of piperazin-1-ylpyridazines suffering from rapid in vitro intrinsic clearance in a metabolic stability assay using liver microsomes (e.g. compound 1 MLM/HLM t1/2 = 2/3 min). Aided by empirical metabolite identification and computational predictive models, we designed the structural modifications required to improve in vitro intrinsic clearance by more than 50-fold (e.g. compound 29 MLM/HLM t1/2 = 113/105 min).
RESUMEN
The dCTPase pyrophosphatase 1 (dCTPase) regulates the intracellular nucleotide pool through hydrolytic degradation of canonical and noncanonical nucleotide triphosphates (dNTPs). dCTPase is highly expressed in multiple carcinomas and is associated with cancer cell stemness. Here we report on the development of the first potent and selective dCTPase inhibitors that enhance the cytotoxic effect of cytidine analogues in leukemia cells. Boronate 30 displays a promising in vitro ADME profile, including plasma and mouse microsomal half-lives, aqueous solubility, cell permeability and CYP inhibition, deeming it a suitable compound for in vivo studies.
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Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Pirofosfatasas/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Células HL-60 , Humanos , Ligandos , Estructura Molecular , Pirofosfatasas/metabolismo , Relación Estructura-ActividadRESUMEN
BACKGROUND: The disposition of a drug is dependent on interactions between the body and the drug, its molecular properties and the physical and biological barriers presented in the body. In order for a drug to have a desired pharmacological effect it has to have the right properties to be able to reach the target site in sufficient concentration. This review details how drug metabolism and pharmacokinetics (DMPK) and physicochemical deliveries played an important role in data interpretation and compound optimization at AstraZeneca R&D in Södertälje, Sweden. METHODS: A selection of assays central in the evaluation of the DMPK properties of new chemical entities is presented, with guidance and consideration on assay outcome interpretation. Early in projects, solubility, LogD, permeability and metabolic stability were measured to support effective optimization of DMPK properties. Changes made to facilitate high throughput, efficient bioanalysis and the handling of large amounts of samples are described. Already early in drug discovery, we used an integrated approach for the prediction of the fate of drugs in human (early dose to man) based on data obtained from in vitro experiments. The early dose to man was refined with project progression, which triggered more intricate assays and experiments. At later stages, preclinical in vivo pharmacokinetic (PK) data was integrated with pharmacodynamics (PD) to allow predictions of required dose, dose intervals and exposure profile to achieve the desired effect in man. RESULTS AND CONCLUSIONS: A well-defined work flow of DMPK activities from early lead identification up to the selection of a candidate drug was developed. This resulted in a cost effective and efficient optimization of chemical series, and facilitated informed decision making throughout project progress.
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Descubrimiento de Drogas , Preparaciones Farmacéuticas/metabolismo , Farmacocinética , Animales , Proteínas Sanguíneas/metabolismo , Industria Farmacéutica , Interacciones Farmacológicas , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Glutatión/metabolismo , Humanos , Permeabilidad , Preparaciones Farmacéuticas/química , Unión ProteicaRESUMEN
Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bind in the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.
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Enzimas Reparadoras del ADN/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Nucleótidos/metabolismo , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Animales , Dominio Catalítico , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cristalización , Daño del ADN , Enzimas Reparadoras del ADN/química , Enzimas Reparadoras del ADN/metabolismo , Nucleótidos de Desoxiguanina/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Femenino , Humanos , Masculino , Ratones , Modelos Moleculares , Conformación Molecular , Terapia Molecular Dirigida , Neoplasias/patología , Oxidación-Reducción/efectos de los fármacos , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/metabolismo , Pirimidinas/química , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Pirofosfatasas/antagonistas & inhibidores , Reproducibilidad de los Resultados , Ensayos Antitumor por Modelo de Xenoinjerto , Hidrolasas NudixRESUMEN
PURPOSE: Static and dynamic (PBPK) prediction models were applied to estimate the drug-drug interaction (DDI) risk of AZD2066. The predictions were compared to the results of an in vivo cocktail study. Various in vivo measures for tolbutamide as a probe agent for cytochrome P450 2C9 (CYP2C9) were also compared. METHODS: In vitro inhibition data for AZD2066 were obtained using human liver microsomes and CYP-specific probe substrates. DDI prediction was performed using PBPK modelling with the SimCYP simulator™ or static model. The cocktail study was an open label, baseline, controlled interaction study with 15 healthy volunteers receiving multiple doses of AD2066 for 12 days. A cocktail of single doses of 100 mg caffeine (CYP1A2 probe), 500 mg tolbutamide (CYP2C9 probe), 20 mg omeprazole (CYP2C19 probe) and 7.5 mg midazolam (CYP3A probe) was simultaneously applied at baseline and during the administration of AZD2066. Bupropion as a CYP2B6 probe (150 mg) and 100 mg metoprolol (CYP2D6 probe) were administered on separate days. The pharmacokinetic parameters for the probe drugs and their metabolites in plasma and urinary recovery were determined. RESULTS: In vitro AZD2066 inhibited CYP1A2, CYP2B6, CYP2C9, CYP2C19 and CYP2D6. The static model predicted in vivo interaction with predicted AUC ratio values of >1.1 for all CYP (except CYP3A4). The PBPK simulations predicted no risk for clinical relevant interactions. The cocktail study showed no interaction for the CYP2B6 and CYP2C19 enzymes, a possible weak inhibition of CYP1A2, CYP2C9 and CYP3A4 activities and a slight inhibition (29 %) of CYP2D6 activity. The tolbutamide phenotyping metrics indicated that there were significant correlations between CLform and AUCTOL, CL, Aemet and LnTOL24h. The MRAe in urine showed no correlation to CLform. CONCLUSIONS: DDI prediction using the static approach based on total concentration indicated that AZD20066 has a potential risk for inhibition. However, no DDI risk could be predicted when a more in vivo-like dynamic prediction method with the PBPK with SimCYP™ software based on early human PK data was used and more parameters (i.e. free fraction in plasma, no DDI risk) were taken into account. The clinical cocktail study showed no or low risks for clinical relevant DDI interactions. Our findings are in line with the hypothesis that the dynamic prediction method predicts DDI in vivo in humans better than the static model based on total plasma concentrations.