RESUMEN
A novel capillary-based microfluidic strategy to accelerate the process of small-molecule-compound screening by room-temperature X-ray crystallography using protein crystals is reported. The ultra-thin microfluidic devices are composed of a UV-curable polymer, patterned by cleanroom photolithography, and have nine capillary channels per chip. The chip was designed for ease of sample manipulation, sample stability and minimal X-ray background. 3D-printed frames and cassettes conforming to SBS standards are used to house the capillary chips, providing additional mechanical stability and compatibility with automated liquid- and sample-handling robotics. These devices enable an innovative in situ crystal-soaking screening workflow, akin to high-throughput compound screening, such that quantitative electron density maps sufficient to determine weak binding events are efficiently obtained. This work paves the way for adopting a room-temperature microfluidics-based sample delivery method at synchrotron sources to facilitate high-throughput protein-crystallography-based screening of compounds at high concentration with the aim of discovering novel binding events in an automated manner.
RESUMEN
In breast cancer, estrogen receptor alpha (ERα) positive cancer accounts for approximately 74% of all diagnoses, and in these settings, it is a primary driver of cell proliferation. Treatment of ERα positive breast cancer has long relied on endocrine therapies such as selective estrogen receptor modulators, aromatase inhibitors, and selective estrogen receptor degraders (SERDs). The steroid-based anti-estrogen fulvestrant (5), the only approved SERD, is effective in patients who have not previously been treated with endocrine therapy as well as in patients who have progressed after receiving other endocrine therapies. Its efficacy, however, may be limited due to its poor physicochemical properties. We describe the design and synthesis of a series of potent benzothiophene-containing compounds that exhibit oral bioavailability and preclinical activity as SERDs. This article culminates in the identification of LSZ102 (10), a compound in clinical development for the treatment of ERα positive breast cancer.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Receptor alfa de Estrógeno/efectos de los fármacos , Moduladores Selectivos de los Receptores de Estrógeno/síntesis química , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Tiofenos/síntesis química , Tiofenos/farmacología , Animales , Antineoplásicos/farmacocinética , Disponibilidad Biológica , Diseño de Fármacos , Descubrimiento de Drogas , Femenino , Humanos , Células MCF-7 , Ratones , Ratones Desnudos , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Moduladores Selectivos de los Receptores de Estrógeno/farmacocinética , Tiofenos/química , Tiofenos/farmacocinética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tetrahydroisoquinoline 40 has been identified as a potent ERα antagonist and selective estrogen receptor degrader (SERD), exhibiting good oral bioavailability, antitumor efficacy, and SERD activity in vivo. We outline the discovery and chemical optimization of the THIQ scaffold leading to THIQ 40 and showcase the racemization of the scaffold, pharmacokinetic studies in preclinical species, and the in vivo efficacy of THIQ 40 in a MCF-7 human breast cancer xenograft model.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Mama/efectos de los fármacos , Receptor alfa de Estrógeno/antagonistas & inhibidores , Tetrahidroisoquinolinas/química , Tetrahidroisoquinolinas/uso terapéutico , Acrilatos/química , Acrilatos/farmacocinética , Acrilatos/farmacología , Acrilatos/uso terapéutico , Administración Oral , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Perros , Descubrimiento de Drogas , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Células MCF-7 , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Proteolisis/efectos de los fármacos , Tetrahidroisoquinolinas/farmacocinética , Tetrahidroisoquinolinas/farmacologíaRESUMEN
The TMPRSS2:ERG gene fusion is common in androgen receptor (AR) positive prostate cancers, yet its function remains poorly understood. From a screen for functionally relevant ERG interactors, we identify the arginine methyltransferase PRMT5. ERG recruits PRMT5 to AR-target genes, where PRMT5 methylates AR on arginine 761. This attenuates AR recruitment and transcription of genes expressed in differentiated prostate epithelium. The AR-inhibitory function of PRMT5 is restricted to TMPRSS2:ERG-positive prostate cancer cells. Mutation of this methylation site on AR results in a transcriptionally hyperactive AR, suggesting that the proliferative effects of ERG and PRMT5 are mediated through attenuating AR's ability to induce genes normally involved in lineage differentiation. This provides a rationale for targeting PRMT5 in TMPRSS2:ERG positive prostate cancers. Moreover, methylation of AR at arginine 761 highlights a mechanism for how the ERG oncogene may coax AR towards inducing proliferation versus differentiation.
Asunto(s)
Células Epiteliales/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas de Fusión Oncogénica/genética , Proteína-Arginina N-Metiltransferasas/genética , Receptores Androgénicos/genética , Serina Endopeptidasas/genética , Secuencia de Bases , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Células Epiteliales/patología , Humanos , Masculino , Metilación , Modelos Moleculares , Mutación , Proteínas de Fusión Oncogénica/metabolismo , Próstata/metabolismo , Próstata/patología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Secundaria de Proteína , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores Androgénicos/química , Receptores Androgénicos/metabolismo , Serina Endopeptidasas/metabolismo , Transducción de Señal , Regulador Transcripcional ERG/genética , Regulador Transcripcional ERG/metabolismoRESUMEN
The highly conserved 70 kDa heat shock proteins (Hsp70) play an integral role in proteostasis such that dysregulation has been implicated in numerous diseases. Elucidating the precise role of Hsp70 family members in the cellular context, however, has been hampered by the redundancy and intricate regulation of the chaperone network, and relatively few selective and potent tools. We have characterized a natural product, novolactone, that targets cytosolic and ER-localized isoforms of Hsp70 through a highly conserved covalent interaction at the interface between the substrate-binding and ATPase domains. Biochemical and structural analyses indicate that novolactone disrupts interdomain communication by allosterically inducing a conformational change in the Hsp70 protein to block ATP-induced substrate release and inhibit refolding activities. Thus, novolactone is a valuable tool for exploring the requirements of Hsp70 chaperones in diverse cellular contexts.
Asunto(s)
Abietanos/metabolismo , Productos Biológicos/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Abietanos/química , Adenosina Trifosfatasas/metabolismo , Regulación Alostérica , Sitios de Unión , Productos Biológicos/química , Línea Celular , Cristalografía por Rayos X , Retículo Endoplásmico/metabolismo , Genoma Fúngico , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/química , Humanos , Simulación de Dinámica Molecular , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/genética , Especificidad por SustratoRESUMEN
Tankyrase 1 and 2 have been shown to be redundant, druggable nodes in the Wnt pathway. As such, there has been intense interest in developing agents suitable for modulating the Wnt pathway in vivo by targeting this enzyme pair. By utilizing a combination of structure-based design and LipE-based structure efficiency relationships, the core of XAV939 was optimized into a more stable, more efficient, but less potent dihydropyran motif 7. This core was combined with elements of screening hits 2, 19, and 33 and resulted in highly potent, selective tankyrase inhibitors that are novel three pocket binders. NVP-TNKS656 (43) was identified as an orally active antagonist of Wnt pathway activity in the MMTV-Wnt1 mouse xenograft model. With an enthalpy-driven thermodynamic signature of binding, highly favorable physicochemical properties, and high lipophilic efficiency, NVP-TNKS656 is a novel tankyrase inhibitor that is well suited for further in vivo validation studies.
Asunto(s)
Acetamidas/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Pirimidinonas/farmacología , Tanquirasas/antagonistas & inhibidores , Acetamidas/administración & dosificación , Acetamidas/química , Administración Oral , Animales , Área Bajo la Curva , Disponibilidad Biológica , Inhibidores Enzimáticos/administración & dosificación , Ratones , Modelos Moleculares , Pirimidinonas/administración & dosificación , Pirimidinonas/química , Relación Estructura-ActividadRESUMEN
Tankyrases 1 and 2 are members of the poly(ADP-ribose) polymerase (PARP) family of enzymes that modulate Wnt pathway signaling. While amide- and lactam-based nicotinamide mimetics that inhibit tankyrase activity, such as XAV939, are well-known, herein we report the discovery and evaluation of a novel nicotinamide isostere that demonstrates selectivity over other PARP family members. We demonstrate the utilization of lipophilic efficiency-based structure-efficiency relationships (SER) to rapidly drive the evaluation of this series. These efforts led to a series of selective, cell-active compounds with solubility, physicochemical, and in vitro properties suitable for further optimization.
Asunto(s)
Aminas/farmacología , Tanquirasas/antagonistas & inhibidores , Triazoles/farmacología , Aminas/química , Animales , Ensayo de Inmunoadsorción Enzimática , Masculino , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Triazoles/químicaRESUMEN
Argyrins, produced by myxobacteria and actinomycetes, are cyclic octapeptides with antibacterial and antitumor activity. Here, we identify elongation factor G (EF-G) as the cellular target of argyrin B in bacteria, via resistant mutant selection and whole genome sequencing, biophysical binding studies and crystallography. Argyrin B binds a novel allosteric pocket in EF-G, distinct from the known EF-G inhibitor antibiotic fusidic acid, revealing a new mode of protein synthesis inhibition. In eukaryotic cells, argyrin B was found to target mitochondrial elongation factor G1 (EF-G1), the closest homologue of bacterial EF-G. By blocking mitochondrial translation, argyrin B depletes electron transport components and inhibits the growth of yeast and tumor cells. Further supporting direct inhibition of EF-G1, expression of an argyrin B-binding deficient EF-G1 L693Q variant partially rescued argyrin B-sensitivity in tumor cells. In summary, we show that argyrin B is an antibacterial and cytotoxic agent that inhibits the evolutionarily conserved target EF-G, blocking protein synthesis in bacteria and mitochondrial translation in yeast and mammalian cells.
Asunto(s)
Oligopéptidos/metabolismo , Factor G de Elongación Peptídica/metabolismo , Sitio Alostérico , Secuencia de Aminoácidos , Animales , Burkholderia/efectos de los fármacos , Línea Celular Tumoral , Secuencia Conservada , Cristalografía por Rayos X , Humanos , Mamíferos , Pruebas de Sensibilidad Microbiana , Proteínas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oligopéptidos/química , Oligopéptidos/farmacología , Factor G de Elongación Peptídica/antagonistas & inhibidores , Factor G de Elongación Peptídica/química , Unión Proteica/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The crystal structures of tankyrase 1 (TNKS1) in complex with two small-molecule inhibitors, PJ34 and XAV939, both at 2.0 Å resolution, are reported. The structure of TNKS1 in complex with PJ34 reveals two molecules of PJ34 bound in the NAD(+) donor pocket. One molecule is in the nicotinamide portion of the pocket, as previously observed in other PARP structures, while the second molecule is bound in the adenosine portion of the pocket. Additionally, unlike the unliganded crystallization system, the TNKS1-PJ34 crystallization system has the NAD(+) donor site accessible to bulk solvent in the crystal, which allows displacement soaking. The TNKS1-PJ34 crystallization system was used to determine the structure of TNKS1 in complex with XAV939. These structures provide a basis for the start of a structure-based drug-design campaign for TNKS1.
Asunto(s)
Inhibidores Enzimáticos/química , Compuestos Heterocíclicos con 3 Anillos/química , Fenantrenos/química , Tanquirasas/química , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Dominios y Motivos de Interacción de Proteínas , Tanquirasas/antagonistas & inhibidoresRESUMEN
The Wnt signaling pathway is critical to the regulation of key cellular processes. When deregulated, it has been shown to play a crucial role in the growth and progression of multiple human cancers. The identification of small molecule modulators of Wnt signaling has proven challenging, largely due to the relative paucity of druggable nodes in this pathway. Several recent publications have identified small molecule inhibitors of the Wnt pathway, and tankyrase (TNKS) inhibition has been demonstrated to antagonize Wnt signaling via axin stabilization. Herein, we report the early hit assessment of a series of compounds previously reported to antagonize Wnt signaling. We report the biophysical, computational characterization, structure-activity relationship, and physicochemical properties of a novel series of [1,2,4]triazol-3-ylsulfanylmethyl)-3-phenyl-[1,2,4]oxadiazole inhibitors of TNKS1 and 2. Furthermore, a cocrystal structure of compound 24 complexed to TNKS1 demonstrates an alternate binding mode for PARP family member proteins that does not involve interactions with the nicotinamide binding pocket.