RESUMEN
Antibody-drug conjugates (ADCs) have become an important therapeutic modality for oncology, with three approved by the FDA and over 60 others in clinical trials. Despite the progress, improvements in ADC therapeutic index are desired. Peptide-based ADC linkers that are cleaved by lysosomal proteases have shown sufficient stability in serum and effective payload-release in targeted cells. If the linker can be preferentially hydrolyzed by tumor-specific proteases, safety margin may improve. However, the use of peptide-based linkers limits our ability to modulate protease specificity. Here we report the structure-guided discovery of novel, nonpeptidic ADC linkers. We show that a cyclobutane-1,1-dicarboxamide-containing linker is hydrolyzed predominantly by cathepsin B while the valine-citrulline dipeptide linker is not. ADCs bearing the nonpeptidic linker are as efficacious and stable in vivo as those with the dipeptide linker. Our results strongly support the application of the peptidomimetic linker and present new opportunities for improving the selectivity of ADCs.
Asunto(s)
Catepsina B/metabolismo , Descubrimiento de Drogas , Inmunoconjugados/química , Inmunoconjugados/metabolismo , Peptidomiméticos/química , Peptidomiméticos/metabolismo , Humanos , Espacio Intracelular/metabolismo , Especificidad por SustratoRESUMEN
Three rationally designed pyrrolobenzodiazepine (PBD) drug-linkers have been synthesized via intermediate 19 for use in antibody-drug conjugates (ADCs). They lack a cleavable trigger in the linker and consist of a maleimide for cysteine antibody conjugation, a hydrophilic spacer, and either an alkyne (6), triazole (7), or piperazine (8) link to the PBD. In vitro IC50 values were 11-48 ng/mL in HER2 3+ SK-BR-3 and KPL-4 (7 inactive) for the anti-HER2 ADCs (HER2 0 MCF7, all inactive) and 0.10-1.73 µg/mL (7 inactive) in CD22 3+ BJAB and WSU-DLCL2 for anti-CD22 ADCs (CD22 0 Jurkat, all inactive at low doses). In vivo antitumor efficacy for the anti-HER2 ADCs in Founder 5 was observed with tumor stasis at 0.5-1 mg/kg, 1 mg/kg, and 3-6 mg/kg for 6, 8, and 7, respectively. Tumor stasis at 2 mg/kg was observed for anti-CD22 6 in WSU-DLCL2. In summary, noncleavable PBD-ADCs exhibit potent activity, particularly in HER2 models.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/uso terapéutico , Benzodiazepinas/química , Benzodiazepinas/uso terapéutico , Inmunoconjugados/química , Inmunoconjugados/uso terapéutico , Neoplasias/tratamiento farmacológico , Pirroles/química , Pirroles/uso terapéutico , Animales , Antineoplásicos/farmacología , Benzodiazepinas/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Dimerización , Femenino , Humanos , Inmunoconjugados/farmacología , Ratones , Modelos Moleculares , Pirroles/farmacología , Receptor ErbB-2/antagonistas & inhibidores , Lectina 2 Similar a Ig de Unión al Ácido Siálico/antagonistas & inhibidoresRESUMEN
A novel disulfide linker was designed to enable a direct connection between cytotoxic pyrrolobenzodiazepine (PBD) drugs and the cysteine on a targeting antibody for use in antibody-drug conjugates (ADCs). ADCs composed of a cysteine-engineered antibody were armed with a PBD using a self-immolative disulfide linker. Both the chemical linker and the antibody site were optimized for this new bioconjugation strategy to provide a highly stable and efficacious ADC. This novel disulfide ADC was compared with a conjugate containing the same PBD drug, but attached to the antibody via a peptide linker. Both ADCs had similar efficacy in mice bearing human tumor xenografts. Safety studies in rats revealed that the disulfide-linked ADC had a higher MTD than the peptide-linked ADC. Overall, these data suggest that the novel self-immolative disulfide linker represents a valuable way to construct ADCs with equivalent efficacy and improved safety. Mol Cancer Ther; 16(5); 871-8. ©2017 AACR.
Asunto(s)
Anticuerpos/administración & dosificación , Benzodiazepinas/administración & dosificación , Inmunoconjugados/administración & dosificación , Neoplasias/tratamiento farmacológico , Pirroles/administración & dosificación , Animales , Anticuerpos/química , Anticuerpos/inmunología , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Antineoplásicos/inmunología , Benzodiazepinas/química , Benzodiazepinas/inmunología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Disulfuros/química , Disulfuros/inmunología , Humanos , Inmunoconjugados/química , Ratones , Neoplasias/inmunología , Neoplasias/patología , Pirroles/química , Pirroles/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Potent, trans-2-(pyridin-3-yl)cyclopropanecarboxamide-containing inhibitors of the human nicotinamide phosphoribosyltransferase (NAMPT) enzyme were identified using fragment-based screening and structure-based design techniques. Multiple crystal structures were obtained of initial fragment leads, and this structural information was utilized to improve the biochemical and cell-based potency of the associated molecules. Many of the optimized compounds exhibited nanomolar antiproliferative activities against human tumor lines in in vitro cell culture experiments. In a key example, a fragment lead (13, KD = 51 µM) was elaborated into a potent NAMPT inhibitor (39, NAMPT IC50 = 0.0051 µM, A2780 cell culture IC50 = 0.000 49 µM) which demonstrated encouraging in vivo efficacy in an HT-1080 mouse xenograft tumor model.
Asunto(s)
Amidas/síntesis química , Antineoplásicos/síntesis química , Ciclopropanos/síntesis química , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Piridinas/síntesis química , Sulfonas/síntesis química , Amidas/química , Amidas/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Cristalografía por Rayos X , Ciclopropanos/química , Ciclopropanos/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Xenoinjertos , Humanos , Ratones , Ratones Desnudos , Modelos Moleculares , Trasplante de Neoplasias , Conformación Proteica , Piridinas/química , Piridinas/farmacología , Estereoisomerismo , Relación Estructura-Actividad , Sulfonas/química , Sulfonas/farmacologíaRESUMEN
Crystal structures of several urea- and thiourea-derived compounds in complex with the nicotinamide phosphoribosyltransferase (Nampt) protein were utilized to design a potent amide-containing inhibitor bearing an aza-indole moiety (7, Nampt BC IC50 = 9.0 nM, A2780 cell proliferation IC50 = 10 nM). The Nampt-7 cocrystal structure was subsequently obtained and enabled the design of additional amide-containing inhibitors which incorporated various other fused 6,5-heterocyclic moieties and biaryl sulfone or sulfonamide motifs. Additional modifications of these molecules afforded many potent biaryl sulfone-containing Nampt inhibitors which also exhibited favorable in vitro ADME properties (microsomal and hepatocyte stability, MDCK permeability, plasma protein binding). An optimized compound (58) was a potent inhibitor of multiple cancer cell lines (IC50 <10 nM vs U251, HT1080, PC3, MiaPaCa2, and HCT116 lines), displayed acceptable mouse PK properties (F = 41%, CL = 52.4 mL/min/kg), and exhibited robust efficacy in a U251 mouse xenograft model.
Asunto(s)
Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Animales , Inhibidores Enzimáticos/farmacocinética , Espectroscopía de Resonancia Magnética , Ratones , Ratones Desnudos , Modelos Moleculares , Estructura Molecular , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Potent, reversible inhibition of the cytochrome P450 CYP2C9 isoform was observed in a series of urea-containing nicotinamide phosphoribosyltransferase (NAMPT) inhibitors. This unwanted property was successfully removed from the described inhibitors through a combination of structure-based design and medicinal chemistry activities. An optimized compound which did not inhibit CYP2C9 exhibited potent anti-NAMPT activity (17; BC NAMPT IC50=3 nM; A2780 antiproliferative IC50=70 nM), good mouse PK properties, and was efficacious in an A2780 mouse xenograft model. The crystal structure of this compound in complex with the NAMPT protein is also described.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Urea/análogos & derivados , Urea/farmacología , Animales , Hidrocarburo de Aril Hidroxilasas/química , Hidrocarburo de Aril Hidroxilasas/metabolismo , Citocromo P-450 CYP2C9 , Humanos , Ratones , Ratones Endogámicos BALB C , Nicotinamida Fosforribosiltransferasa/química , Nicotinamida Fosforribosiltransferasa/metabolismo , Urea/síntesis químicaRESUMEN
Nicotinamide phosphoribosyltransferase (Nampt) is a promising anticancer target. Virtual screening identified a thiourea analogue, compound 5, as a novel highly potent Nampt inhibitor. Guided by the cocrystal structure of 5, SAR exploration revealed that the corresponding urea compound 7 exhibited similar potency with an improved solubility profile. These studies also indicated that a 3-pyridyl group was the preferred substituent at one inhibitor terminus and also identified a urea moiety as the optimal linker to the remainder of the inhibitor structure. Further SAR optimization of the other inhibitor terminus ultimately yielded compound 50 as a urea-containing Nampt inhibitor which exhibited excellent biochemical and cellular potency (enzyme IC50 = 0.007 µM; A2780 IC50 = 0.032 µM). Compound 50 also showed excellent in vivo antitumor efficacy when dosed orally in an A2780 ovarian tumor xenograft model (TGI of 97% was observed on day 17).
Asunto(s)
Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Urea/química , Urea/farmacología , Humanos , Concentración 50 Inhibidora , Nicotinamida Fosforribosiltransferasa/química , Conformación Proteica , Relación Estructura-ActividadRESUMEN
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of familial Parkinson's disease (PD). Although biochemical studies have shown that certain PD mutations confer elevated kinase activity in vitro on LRRK2, there are no methods available to directly monitor LRRK2 kinase activity in vivo. We demonstrate that LRRK2 autophosphorylation on Ser(1292) occurs in vivo and is enhanced by several familial PD mutations including N1437H, R1441G/C, G2019S, and I2020T. Combining two PD mutations together further increases Ser(1292) autophosphorylation. Mutation of Ser(1292) to alanine (S1292A) ameliorates the effects of LRRK2 PD mutations on neurite outgrowth in cultured rat embryonic primary neurons. Using cell-based and pharmacodynamic assays with phosphorylated Ser(1292) as the readout, we developed a brain-penetrating LRRK2 kinase inhibitor that blocks Ser(1292) autophosphorylation in vivo and attenuates the cellular consequences of LRRK2 PD mutations in vitro. These data suggest that Ser(1292) autophosphorylation may be a useful indicator of LRRK2 kinase activity in vivo and may contribute to the cellular effects of certain PD mutations.
Asunto(s)
Mutación/genética , Enfermedad de Parkinson/enzimología , Enfermedad de Parkinson/patología , Fosfoserina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Sitios de Unión , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Encéfalo/patología , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Ratones , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Proteínas Mutantes/metabolismo , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Enfermedad de Parkinson/genética , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Transporte de Proteínas/efectos de los fármacos , RatasRESUMEN
There is a high demand for potent, selective, and brain-penetrant small molecule inhibitors of leucine-rich repeat kinase 2 (LRRK2) to test whether inhibition of LRRK2 kinase activity is a potentially viable treatment option for Parkinson's disease patients. Herein we disclose the use of property and structure-based drug design for the optimization of highly ligand efficient aminopyrimidine lead compounds. High throughput in vivo rodent cassette pharmacokinetic studies enabled rapid validation of in vitro-in vivo correlations. Guided by this data, optimal design parameters were established. Effective incorporation of these guidelines into our molecular design process resulted in the discovery of small molecule inhibitors such as GNE-7915 (18) and 19, which possess an ideal balance of LRRK2 cellular potency, broad kinase selectivity, metabolic stability, and brain penetration across multiple species. Advancement of GNE-7915 into rodent and higher species toxicity studies enabled risk assessment for early development.
Asunto(s)
Encéfalo/metabolismo , Morfolinas/farmacología , Enfermedad de Parkinson/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirimidinas/farmacología , Animales , Diseño de Fármacos , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Macaca fascicularis , Ratones , Ratones Transgénicos , Modelos Moleculares , Morfolinas/síntesis química , Morfolinas/farmacocinética , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacocinética , Pirimidinas/síntesis química , Pirimidinas/farmacocinética , Ratas , Bibliotecas de Moléculas Pequeñas , Distribución TisularRESUMEN
Mutations in the genetic sequence of leucine-rich repeat kinase 2 (LRRK2) have been linked to increased LRRK2 activity and risk for the development of Parkinson's disease (PD). Potent and selective small molecules capable of inhibiting the kinase activity of LRRK2 will be important tools for establishing a link between the kinase activity of LRRK2 and PD. In the absence of LRRK2 kinase domain crystal structures, a LRRK2 homology model was developed that provided robust guidance in the hit-to-lead optimization of small molecule LRRK2 inhibitors. Through a combination of molecular modeling, sequence analysis, and matched molecular pair (MMP) activity cliff analysis, a potent and selective lead inhibitor was discovered. The selectivity of this compound could be understood using the LRRK2 homology model, and application of this learning to a series of 2,4-diaminopyrimidine inhibitors in a scaffold hopping exercise led to the identification of highly potent and selective LRRK2 inhibitors that were also brain penetrable.
Asunto(s)
Modelos Moleculares , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Biología Computacional , Humanos , Janus Quinasa 2/antagonistas & inhibidores , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Morfolinas/química , Morfolinas/farmacocinética , Morfolinas/farmacología , Permeabilidad , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Piridinas/química , Piridinas/farmacocinética , Piridinas/farmacología , Pirimidinas/química , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Tiazoles/química , Tiazoles/farmacocinética , Tiazoles/farmacologíaRESUMEN
Recent clinical data provided proof-of-concept for selective B-Raf inhibitors in treatment of B-Raf(V600E) mutant melanoma. Pyrazolopyridine-type B-Raf inhibitors previously described by the authors are potent and selective but exhibit low solubility requiring the use of amorphous dispersion-based formulation for achieving efficacious drug exposures. Through structure-based design, we discovered a new class of highly potent aminopyrimidine-based B-Raf inhibitors with improved solubility and pharmacokinetic profiles. The hinge binding moiety possesses a basic center imparting high solubility at gastric pH, addressing the dissolution limitation observed with our previous series. In our search for an optimal linker-hinge binding moiety system, amide-linked thieno[3,2-d]pyrimidine analogues 32 and 35 (G945), molecules with desirable physicochemical properties, emerged as lead compounds with strong efficacy in a B-Raf(V600E) mutant mouse xenograft model. Synthesis, SAR, lead selection, and evaluation of key compounds in animal studies will be described.
Asunto(s)
Aminopiridinas/síntesis química , Antineoplásicos/síntesis química , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Aminopiridinas/farmacocinética , Aminopiridinas/farmacología , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Línea Celular Tumoral , Cristalografía por Rayos X , Ensayos de Selección de Medicamentos Antitumorales , Ratones , Modelos Moleculares , Estructura Molecular , Trasplante de Neoplasias , Quinazolinas/síntesis química , Quinazolinas/farmacocinética , Quinazolinas/farmacología , Ratas , Solubilidad , Relación Estructura-Actividad , Tiofenos/síntesis química , Tiofenos/farmacocinética , Tiofenos/farmacología , Trasplante HeterólogoRESUMEN
The V600E mutation of B-Raf kinase results in constitutive activation of the MAPK signaling pathway and is present in approximately 7% of all cancers. Using structure-based design, a novel series of pyrazolopyridine inhibitors of B-Raf(V600E) was developed. Optimization led to the identification of 3-methoxy pyrazolopyridines 17 and 19, potent, selective, and orally bioavailable agents that inhibited tumor growth in a mouse xenograft model driven by B-Raf(V600E) with no effect on body weight. On the basis of their in vivo efficacy and preliminary safety profiles, 17 and 19 were selected for further preclinical evaluation.