RESUMEN
Live vaccines are ideal for inducing immunity but suffer from the need to attenuate their pathogenicity or replication to preclude the possibility of escape. Unnatural amino acids (UAAs) provide a strategy to engineer stringent auxotrophies, yielding conditionally replication incompetent live bacteria with excellent safety profiles. Here, we engineer Pseudomonas aeruginosa to maintain auxotrophy for the UAA p-benzoyl-L-phenylalanine (BzF) through its incorporation into the essential protein DnaN. In vivo evolution using an Escherichia coli-based two-hybrid selection system enabled engineering of a mutant DnaN homodimeric interface completely dependent on a BzF-specific interaction. This engineered strain, Pa Vaccine, exhibits undetectable escape frequency (<10-11) and shows excellent safety in naïve mice. Animals vaccinated via intranasal or intraperitoneal routes are protected from lethal challenge with pathogenic P. aeruginosa PA14. These results establish UAA-auxotrophic bacteria as promising candidates for bacterial vaccine therapy and outline a platform for expanding this technology to diverse bacterial pathogens.
Asunto(s)
Infecciones por Pseudomonas , Vacunas contra la Infección por Pseudomonas , Pseudomonas aeruginosa , Animales , Pseudomonas aeruginosa/inmunología , Pseudomonas aeruginosa/genética , Infecciones por Pseudomonas/prevención & control , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/microbiología , Ratones , Femenino , Vacunas contra la Infección por Pseudomonas/inmunología , Vacunas contra la Infección por Pseudomonas/genética , Aminoácidos , Fenilalanina/análogos & derivados , Escherichia coli/inmunología , Escherichia coli/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/genética , Ratones Endogámicos BALB CRESUMEN
In efforts towards eliminating malaria, a discovery program was initiated to identify a novel antimalarial using KAF156 as a starting point. Following the most recent TCP/TPP guidelines, we have identified mCMQ069 with a predicted single oral dose for treatment (â¼40-106 mg) and one-month chemoprevention (â¼96-216 mg). We have improved unbound MPC and predicted human clearance by 18-fold and 10-fold respectively when compared to KAF156.
RESUMEN
Recent malaria drug discovery approaches have been extensively focused on the development of oral, smallmolecule inhibitors for disease treatment whereas parenteral routes of administration have been avoided due to limitations in deploying a shelf-stable injectable even though it could be dosed less frequently. However, an updated target candidate profile from Medicines for Malaria Venture (MMV) and stakeholders have advocated for long-acting injectable chemopreventive agents as an important interventive tool to improve malaria prevention. Here, we present strategies for the development of a long-acting, intramuscular, injectable atovaquone prophylactic therapy. We have generated three prodrug approaches that are contrasted by their differential physiochemical properties and pharmacokinetic profiles: mCBK068, a docosahexaenoic acid ester of atovaquone formulated in sesame oil, mCKX352, a heptanoic acid ester of atovaquone formulated as a solution in sesame oil, and mCBE161, an acetic acid ester of atovaquone formulated as an aqueous suspension. As a result, from a single 20 mg/kg intramuscular injection, mCKX352 and mCBE161 maintain blood plasma exposure of atovaquone above the minimal efficacious concentration for >70 days and >30 days, respectively, in cynomolgus monkeys. The differences in plasma exposure are reflective of the prodrug strategy, which imparts altered chemical properties that ultimately influence aqueous solubility and depot release kinetics. On the strength of the pharmacokinetic and safety profiles, mCBE161 is being advanced as a first-in-class clinical candidate for first-in-human trials.
RESUMEN
Insufficient telomerase activity, stemming from low telomerase reverse transcriptase (TERT) gene transcription, contributes to telomere dysfunction and aging pathologies. Besides its traditional function in telomere synthesis, TERT acts as a transcriptional co-regulator of genes pivotal in aging and age-associated diseases. Here, we report the identification of a TERT activator compound (TAC) that upregulates TERT transcription via the MEK/ERK/AP-1 cascade. In primary human cells and naturally aged mice, TAC-induced elevation of TERT levels promotes telomere synthesis, blunts tissue aging hallmarks with reduced cellular senescence and inflammatory cytokines, and silences p16INK4a expression via upregulation of DNMT3B-mediated promoter hypermethylation. In the brain, TAC alleviates neuroinflammation, increases neurotrophic factors, stimulates adult neurogenesis, and preserves cognitive function without evident toxicity, including cancer risk. Together, these findings underscore TERT's critical role in aging processes and provide preclinical proof of concept for physiological TERT activation as a strategy to mitigate multiple aging hallmarks and associated pathologies.
Asunto(s)
Envejecimiento , Metilación de ADN , Telomerasa , Telomerasa/metabolismo , Telomerasa/genética , Humanos , Animales , Ratones , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , Senescencia Celular , Regiones Promotoras Genéticas , ADN Metiltransferasa 3B , Encéfalo/metabolismo , Telómero/metabolismo , Ratones Endogámicos C57BL , Masculino , Factor de Transcripción AP-1/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , NeurogénesisRESUMEN
Type 2 alveolar epithelial cells (AEC2s) are stem cells in the adult lung that contribute to lower airway repair. Agents that promote the selective expansion of these cells might stimulate regeneration of the compromised alveolar epithelium, an etiology-defining event in several pulmonary diseases. From a high-content imaging screen of the drug repurposing library ReFRAME, we identified that dipeptidyl peptidase 4 (DPP4) inhibitors, widely used type 2 diabetes medications, selectively expand AEC2s and are broadly efficacious in several mouse models of lung damage. Mechanism of action studies revealed that the protease DPP4, in addition to processing incretin hormones, degrades IGF-1 and IL-6, essential regulators of AEC2 expansion whose levels are increased in the luminal compartment of the lung in response to drug treatment. To selectively target DPP4 in the lung with sufficient drug exposure, we developed NZ-97, a locally delivered, lung persistent DPP4 inhibitor that broadly promotes efficacy in mouse lung damage models with minimal peripheral exposure and good tolerability. This work reveals DPP4 as a central regulator of AEC2 expansion and affords a promising therapeutic approach to broadly stimulate regenerative repair in pulmonary disease.
Asunto(s)
Células Epiteliales Alveolares , Diabetes Mellitus Tipo 2 , Animales , Ratones , Células Epiteliales Alveolares/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Pulmón/metabolismo , Modelos Animales de EnfermedadRESUMEN
There remains a need to develop novel SARS-CoV-2 therapeutic options that improve upon existing therapies by an increased robustness of response, fewer safety liabilities, and global-ready accessibility. Functionally critical viral main protease (Mpro, 3CLpro) of SARS-CoV-2 is an attractive target due to its homology within the coronaviral family, and lack thereof toward human proteases. In this disclosure, we outline the advent of a novel SARS-CoV-2 3CLpro inhibitor, CMX990, bearing an unprecedented trifluoromethoxymethyl ketone warhead. Compared with the marketed drug nirmatrelvir (combination with ritonavir = Paxlovid), CMX990 has distinctly differentiated potency (â¼5× more potent in primary cells) and human in vitro clearance (>4× better microsomal clearance and >10× better hepatocyte clearance), with good in vitro-to-in vivo correlation. Based on its compelling preclinical profile and projected once or twice a day dosing supporting unboosted oral therapy in humans, CMX990 advanced to a Phase 1 clinical trial as an oral drug candidate for SARS-CoV-2.
Asunto(s)
COVID-19 , Humanos , SARS-CoV-2 , Diferenciación Celular , Revelación , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/uso terapéutico , Antivirales/farmacologíaRESUMEN
Chronic cutaneous wounds remain a persistent unmet medical need that decreases life expectancy and quality of life. Here, we report that topical application of PY-60, a small-molecule activator of the transcriptional coactivator Yes-associated protein (YAP), promotes regenerative repair of cutaneous wounds in pig and human models. Pharmacological YAP activation enacts a reversible pro-proliferative transcriptional program in keratinocytes and dermal cells that results in accelerated re-epithelization and regranulation of the wound bed. These results demonstrate that transient topical administration of a YAP activating agent may represent a generalizable therapeutic approach to treating cutaneous wounds.
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Calidad de Vida , Cicatrización de Heridas , Humanos , Animales , Porcinos , Cicatrización de Heridas/fisiología , Piel/lesiones , Queratinocitos/metabolismo , Administración CutáneaRESUMEN
KEAP1 (Kelch-like ECH-associated protein), a cytoplasmic repressor of the oxidative stress responsive transcription factor Nuclear factor erythroid 2-related factor 2 (NRF2), senses the presence of electrophilic agents by modification of its sensor cysteine residues. In addition to xenobiotics, several reactive metabolites have been shown to covalently modify key cysteines on KEAP1, although the full repertoire of these molecules and their respective modifications remain undefined. Here, we report the discovery of sAKZ692, a small molecule identified by high-throughput screening that stimulates NRF2 transcriptional activity in cells by inhibiting the glycolytic enzyme pyruvate kinase. sAKZ692 treatment promotes the buildup of glyceraldehyde 3-phosphate, a metabolite which leads to S-lactate modification of cysteine sensor residues of KEAP1, resulting in NRF2-dependent transcription. This work identifies a posttranslational modification of cysteine derived from a reactive central carbon metabolite and helps further define the complex relationship between metabolism and the oxidative stress-sensing machinery of the cell.
Asunto(s)
Cisteína , Factor 2 Relacionado con NF-E2 , Proteína 1 Asociada A ECH Tipo Kelch/química , Factor 2 Relacionado con NF-E2/metabolismo , Cisteína/metabolismo , Transducción de Señal , Estrés OxidativoRESUMEN
Epithelial-mesenchymal transition (EMT) endows stem cell-like properties to cancer cells. Targeting this process represents a potential therapeutic approach to overcome cancer metastasis and chemotherapy resistance. FiVe1 was identified from an EMT-based synthetic lethality screen and was found to inhibit the stem cell-like properties and proliferation of not only cancer cells undergoing EMT, but also more broadly in mesenchymal cancers that include therapeutically intractable soft tissue sarcomas. FiVe1 functions by directly binding to the type III intermediate filament protein vimentin (VIM) in a mode that induces hyperphosphorylation of Ser56, which results in selective disruption of mitosis and induced multinucleation in transformed VIM-expressing mesenchymal cancer cell types. Cell-based potency (IC50 = 1.6 µM, HT-1080 fibrosarcoma), poor solubility (<1 µM) and low oral bioavailability limits the direct application of FiVe1 as an in vivo probe or therapeutic agent. To overcome these drawbacks, we performed structure-activity relationship (SAR) studies and synthesized a set of 35 new compounds, consisting of diverse modifications of the FiVe1 scaffold. Among these compounds, 4e showed a marked improvement in potency (IC50 = 44 nM, 35-fold improvement, HT-1080) and cell type selectivity (19-fold improvement), when compared to FiVe1. Improvements in the potency of 4e, in terms of overall cytotoxicity, directly correlate with VIM Ser56 phosphorylation status and the oral bioavailability and pharmacokinetic profiles of 4e in mouse are superior to FiVe1. Successful optimization also resulted in potent and selective derivatives 11a, 11j and 11k, which exhibited superior pharmacological profiles, in terms of metabolic stability and aqueous solubility. Collectively, these optimization efforts have resulted in the development of promising FiVe1 analogs with potential applications in the treatment of mesenchymal cancers, as well as in the study of VIM-related biology.
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Transición Epitelial-Mesenquimal , Sarcoma , Animales , Línea Celular Tumoral , Ratones , Mitosis , Fosforilación , Vimentina/genéticaRESUMEN
There is a growing appreciation for the idea that bacterial utilization of host-derived lipids, including cholesterol, supports Mycobacterium tuberculosis (Mtb) pathogenesis. This has generated interest in identifying novel antibiotics that can disrupt cholesterol utilization by Mtb in vivo. Here we identify a novel small molecule agonist (V-59) of the Mtb adenylyl cyclase Rv1625c, which stimulates 3', 5'-cyclic adenosine monophosphate (cAMP) synthesis and inhibits cholesterol utilization by Mtb. Similarly, using a complementary genetic approach that induces bacterial cAMP synthesis independent of Rv1625c, we demonstrate that inducing cAMP synthesis is sufficient to inhibit cholesterol utilization in Mtb. Although the physiological roles of individual adenylyl cyclase enzymes in Mtb are largely unknown, here we demonstrate that the transmembrane region of Rv1625c is required during cholesterol metabolism. Finally, the pharmacokinetic properties of Rv1625c agonists have been optimized, producing an orally-available Rv1625c agonist that impairs Mtb pathogenesis in infected mice. Collectively, this work demonstrates a role for Rv1625c and cAMP signaling in controlling cholesterol metabolism in Mtb and establishes that cAMP signaling can be pharmacologically manipulated for the development of new antibiotic strategies.
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Adenilil Ciclasas/metabolismo , Colesterol/metabolismo , AMP Cíclico/metabolismo , Mycobacterium tuberculosis/genética , Animales , Proteínas Bacterianas/metabolismo , Ratones Endogámicos BALB C , Transducción de Señal/fisiología , Activación Transcripcional/fisiologíaRESUMEN
Despite the development of next-generation antiandrogens, metastatic castration-resistant prostate cancer (mCRPC) remains incurable. Here, we describe a unique semisynthetic bispecific antibody that uses site-specific unnatural amino acid conjugation to combine the potency of a T cell-recruiting anti-CD3 antibody with the specificity of an imaging ligand (DUPA) for prostate-specific membrane antigen. This format enabled optimization of structure and function to produce a candidate (CCW702) with specific, potent in vitro cytotoxicity and improved stability compared with a bispecific single-chain variable fragment format. In vivo, CCW702 eliminated C4-2 xenografts with as few as three weekly subcutaneous doses and prevented growth of PCSD1 patient-derived xenograft tumors in mice. In cynomolgus monkeys, CCW702 was well tolerated up to 34.1 mg/kg per dose, with near-complete subcutaneous bioavailability and a PK profile supporting testing of a weekly dosing regimen in patients. CCW702 is being evaluated in a first in-human clinical trial for men with mCRPC who had progressed on prior therapies (NCT04077021).
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Anticuerpos Biespecíficos , Neoplasias de la Próstata Resistentes a la Castración , Animales , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Complejo CD3/uso terapéutico , Línea Celular Tumoral , Ensayos Clínicos como Asunto , Humanos , Ligandos , Masculino , Ratones , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Linfocitos TRESUMEN
The ongoing pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), necessitates strategies to identify prophylactic and therapeutic drug candidates for rapid clinical deployment. Here, we describe a screening pipeline for the discovery of efficacious SARS-CoV-2 inhibitors. We screen a best-in-class drug repurposing library, ReFRAME, against two high-throughput, high-content imaging infection assays: one using HeLa cells expressing SARS-CoV-2 receptor ACE2 and the other using lung epithelial Calu-3 cells. From nearly 12,000 compounds, we identify 49 (in HeLa-ACE2) and 41 (in Calu-3) compounds capable of selectively inhibiting SARS-CoV-2 replication. Notably, most screen hits are cell-line specific, likely due to different virus entry mechanisms or host cell-specific sensitivities to modulators. Among these promising hits, the antivirals nelfinavir and the parent of prodrug MK-4482 possess desirable in vitro activity, pharmacokinetic and human safety profiles, and both reduce SARS-CoV-2 replication in an orthogonal human differentiated primary cell model. Furthermore, MK-4482 effectively blocks SARS-CoV-2 infection in a hamster model. Overall, we identify direct-acting antivirals as the most promising compounds for drug repurposing, additional compounds that may have value in combination therapies, and tool compounds for identification of viral host cell targets.
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Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , Reposicionamiento de Medicamentos/métodos , Pandemias , SARS-CoV-2 , Animales , COVID-19/prevención & control , COVID-19/virología , Línea Celular , Citidina/administración & dosificación , Citidina/análogos & derivados , Citidina/farmacología , Bases de Datos Farmacéuticas , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Células HeLa , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Hidroxilaminas/administración & dosificación , Hidroxilaminas/farmacología , Mesocricetus , Nelfinavir/farmacología , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/fisiología , Replicación Viral/efectos de los fármacosRESUMEN
Gram-positive bacteria assemble a multilayered cell wall that provides tensile strength to the cell. The cell wall is composed of glycan strands cross-linked by nonribosomally synthesized peptide stems. Herein, we modify the peptide stems of the Gram-positive bacterium Bacillus subtilis with noncanonical electrophilic d-amino acids, which when in proximity to adjacent stem peptides form novel covalent 5,3-cross-links. Approximately 20% of canonical cell-wall cross-links can be replaced with synthetic cross-links. While a low level of synthetic cross-link formation does not affect B. subtilis growth and phenotype, at higher levels cell growth is perturbed and bacteria elongate. A comparison of the accumulation of synthetic cross-links over time in Gram-negative and Gram-positive bacteria highlights key differences between them. The ability to perturb cell-wall architecture with synthetic building blocks provides a novel approach to studying the adaptability, elasticity, and porosity of bacterial cell walls.
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Pared Celular/química , Bacilos Grampositivos/química , Peptidoglicano/química , Aminoácidos/química , Aminoácidos/metabolismo , Bacillus subtilis/química , Bacillus subtilis/citología , Bacillus subtilis/crecimiento & desarrollo , Bacillus subtilis/metabolismo , Pared Celular/metabolismo , Bacterias Gramnegativas/química , Bacterias Gramnegativas/citología , Bacterias Gramnegativas/metabolismo , Bacilos Grampositivos/citología , Bacilos Grampositivos/crecimiento & desarrollo , Bacilos Grampositivos/metabolismo , Peptidoglicano/metabolismo , Peptidil Transferasas/genética , Peptidil Transferasas/metabolismo , FenotipoRESUMEN
The transcriptional coactivator Yes-associated protein 1 (YAP) orchestrates a proproliferative transcriptional program that controls the fate of somatic stem cells and the regenerative responses of certain tissues. As such, agents that activate YAP may hold therapeutic potential in disease states exacerbated by insufficient proliferative repair. Here we report the discovery of a small molecule, termed PY-60, which robustly activates YAP transcriptional activity in vitro and promotes YAP-dependent expansion of epidermal keratinocytes in mouse following topical drug administration. Chemical proteomics revealed the relevant target of PY-60 to be annexin A2 (ANXA2), a protein that directly associates with YAP at the cell membrane in response to increased cell density. PY-60 treatment liberates ANXA2 from the membrane, ultimately promoting a phosphatase-bound, nonphosphorylated and transcriptionally active form of YAP. This work reveals ANXA2 as a previously undescribed, druggable component of the Hippo pathway and suggests a mechanistic rationale to promote regenerative repair in disease.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Anexina A2/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Transcripción/metabolismo , Administración Tópica , Células Madre Adultas/efectos de los fármacos , Células Madre Adultas/metabolismo , Animales , Anexina A2/metabolismo , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ratones , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Bibliotecas de Moléculas Pequeñas/química , Proteínas Señalizadoras YAPRESUMEN
The transcription factor NRF2 controls resistance to oxidative insult and is thus a key therapeutic target for treating a number of disease states associated with oxidative stress and aging. We previously reported CBR-470-1, a bis-sulfone which activates NRF2 by increasing the levels of methylglyoxal, a metabolite that covalently modifies NRF2 repressor KEAP1. Here, we report the design, synthesis, and structure activity relationship of a series of bis-sulfones derived from this unexplored chemical template. We identify analogs with sub-micromolar potencies, 7f and 7g, as well as establish that efficacious NRF2 activation can be achieved by non-toxic analogs 7c, 7e, and 9, a key limitation with CBR-470-1. Further efforts to identify non-covalent NRF2 activators of this kind will likely provide new insight into revealing the role of central metabolism in cellular signaling.
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Antioxidantes/farmacología , Descubrimiento de Drogas , Tiofenos/farmacología , Antioxidantes/síntesis química , Antioxidantes/química , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Relación Estructura-Actividad , Tiofenos/síntesis química , Tiofenos/químicaRESUMEN
Recent technological advances have expanded the annotated protein coding content of mammalian genomes, as hundreds of previously unidentified, short open reading frame (ORF)-encoded peptides (SEPs) have now been found to be translated. Although several studies have identified important physiological roles for this emerging protein class, a general method to define their interactomes is lacking. Here, we demonstrate that genetic incorporation of the photo-crosslinking noncanonical amino acid AbK into SEP transgenes allows for the facile identification of SEP cellular interaction partners using affinity-based methods. From a survey of seven SEPs, we report the discovery of short ORF-encoded histone binding protein (SEHBP), a conserved microprotein that interacts with chromatin-associated proteins, localizes to discrete genomic loci, and induces a robust transcriptional program when overexpressed in human cells. This work affords a straightforward method to help define the physiological roles of SEPs and demonstrates its utility by identifying SEHBP as a short ORF-encoded transcription factor.
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Diazometano/metabolismo , Histonas/genética , Lisina/metabolismo , Sistemas de Lectura Abierta , Péptidos/genética , Transcripción Genética , Secuencia de Aminoácidos , Animales , Bovinos , Cromatina/química , Cromatina/metabolismo , Diazometano/análogos & derivados , Regulación de la Expresión Génica , Sitios Genéticos , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Células K562 , Lisina/análogos & derivados , Ratones , Pan troglodytes , Péptidos/metabolismo , Unión Proteica/efectos de la radiación , Mapeo de Interacción de Proteínas , Ratas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción Genética/efectos de la radiación , Transgenes , Rayos UltravioletaRESUMEN
The maturation of chemical synthesis during the 20th century has elevated the discipline from a largely empirical into a rational science. This ability to purposefully craft matter at the molecular level has put chemists in a privileged position to contribute to progress in neighboring natural sciences. Recently, we have witnessed another major advance in the field in which chemists use chemical and biological "synthetic" methods together to alter the structures and properties of biological macromolecules in ways heretofore unimagined. This interdisciplinary approach to synthesis has even allowed us to expand upon the defining characteristics of living organisms at the molecular level. In this perspective, we present a case study for the successful addition of new chemistries to the fundamental processes of the central dogma of molecular biology, exemplified by the expansion of the genetic code.