RESUMEN
Polymerase ß (POLB), with dual functionality as a lyase and polymerase, plays a critical role in the base excision repair (BER) pathway to maintain genomic stability. POLB knockout and rescue studies in BRCA1/2-mutant cancer cell lines revealed that inhibition of lyase and polymerase activity is required for the synthetic lethal interaction observed with PARP inhibitors, highlighting POLB as a valuable therapeutic target. Traditional biochemical assays to screen for enzyme inhibitors focus on a single substrate to product relationship and limit the comprehensive analysis of enzymes such as POLB that utilize multiple substrates or catalyze a multi-step reaction. This report describes the first high-throughput mass spectrometry-based screen to measure the two distinct biochemical activities of POLB in a single assay using a duplexed self-assembled monolayer desorption ionization (SAMDI) mass spectrometry methodology. A multiplexed assay for POLB dual enzymatic activities was developed optimizing for kinetically balanced conditions and a collection of 200,000 diverse small molecules was screened in the duplexed format. Small molecule modulators identified in the screen were confirmed in a traditional fluorescence-based polymerase strand-displacement assay and an orthogonal label-free binding assay using SAMDI affinity selection mass spectrometry (ASMS). This work demonstrates the flexibility of high-throughput mass spectrometry approaches in drug discovery and highlights a novel application of SAMDI technology that opens new avenues for multiplexed high-throughput screening.
RESUMEN
It has been shown that PRMT5 inhibition by small molecules can selectively kill cancer cells with homozygous deletion of the MTAP gene if the inhibitors can leverage the consequence of MTAP deletion, namely, accumulation of the MTAP substrate MTA. Herein, we describe the discovery of TNG908, a potent inhibitor that binds the PRMT5·MTA complex, leading to 15-fold-selective killing of MTAP-deleted (MTAP-null) cells compared to MTAPintact (MTAP WT) cells. TNG908 shows selective antitumor activity when dosed orally in mouse xenograft models, and its physicochemical properties are amenable for crossing the blood-brain barrier (BBB), supporting clinical study for the treatment of both CNS and non-CNS tumors with MTAP loss.
Asunto(s)
Antineoplásicos , Proteína-Arginina N-Metiltransferasas , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/metabolismo , Humanos , Animales , Ratones , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Antineoplásicos/síntesis química , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacocinética , Línea Celular Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Neoplasias/tratamiento farmacológico , Encéfalo/metabolismo , Relación Estructura-ActividadRESUMEN
CRISPR Cas9-based screening is a powerful approach for identifying and characterizing novel drug targets. Here, we elucidate the synthetic lethal mechanism of deubiquitinating enzyme USP1 in cancers with underlying DNA damage vulnerabilities, specifically BRCA1/2 mutant tumors and a subset of BRCA1/2 wild-type (WT) tumors. In sensitive cells, pharmacologic inhibition of USP1 leads to decreased DNA synthesis concomitant with S-phase-specific DNA damage. Genome-wide CRISPR-Cas9 screens identify RAD18 and UBE2K, which promote PCNA mono- and polyubiquitination respectively, as mediators of USP1 dependency. The accumulation of mono- and polyubiquitinated PCNA following USP1 inhibition is associated with reduced PCNA protein levels. Ectopic expression of WT or ubiquitin-dead K164R PCNA reverses USP1 inhibitor sensitivity. Our results show, for the first time, that USP1 dependency hinges on the aberrant processing of mono- and polyubiquitinated PCNA. Moreover, this mechanism of USP1 dependency extends beyond BRCA1/2 mutant tumors to selected BRCA1/2 WT cancer cell lines enriched in ovarian and lung lineages. We further show PARP and USP1 inhibition are strongly synergistic in BRCA1/2 mutant tumors. We postulate USP1 dependency unveils a previously uncharacterized vulnerability linked to posttranslational modifications of PCNA. Taken together, USP1 inhibition may represent a novel therapeutic strategy for BRCA1/2 mutant tumors and a subset of BRCA1/2 WT tumors.
Asunto(s)
Neoplasias , Mutaciones Letales Sintéticas , Humanos , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ubiquitina/genética , Ubiquitinación , Daño del ADN , Neoplasias/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteasas Ubiquitina-Específicas/genética , Proteasas Ubiquitina-Específicas/metabolismoRESUMEN
Bispecific antibodies (Bispecifics) demonstrate exceptional clinical potential to address some of the most complex diseases. However, Bispecific production in a single cell often requires the correct pairing of multiple polypeptide chains for desired assembly. This is a considerable hurdle that hinders the development of many immunoglobulin G (IgG)-like bispecific formats. Our approach focuses on the rational engineering of charged residues to facilitate the chain pairing of distinct heavy chains (HC). Here, we deploy structure-guided protein design to engineer charge pair mutations (CPMs) placed in the CH3-CH3' interface of the fragment crystallizable (Fc) region of an antibody (Ab) to correctly steer heavy chain pairing. When used in combination with our stable effector functionless 2 (SEFL2.2) technology, we observed high pairing efficiency without significant losses in expression yields. Furthermore, we investigate the relationship between CPMs and the sequence diversity in the parental antibodies, proposing a rational strategy to deploy these engineering technologies.
RESUMEN
The (Z)-fluoro-olefin amide bioisosteric replacement is an effective tool for addressing various shortcomings of the parent amide. In an effort to fine tune ADME properties of BACE1 preclinical candidate AM-6494, a series of structurally distinct (Z)-fluoro-olefin containing analogs was developed that culminated in compound 15. Herein, we detail design considerations, synthetic challenges, structure activity relationship (SAR) studies, and in vivo properties of an advanced compound in this novel series of BACE1 inhibitors.
Asunto(s)
Alquenos/farmacología , Amidas/farmacología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Desarrollo de Medicamentos , Inhibidores Enzimáticos/farmacología , Hidrocarburos Fluorados/farmacología , Alquenos/síntesis química , Alquenos/química , Amidas/síntesis química , Amidas/química , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Células HEK293 , Humanos , Hidrocarburos Fluorados/síntesis química , Hidrocarburos Fluorados/química , Conformación Molecular , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
ß-Site amyloid precursor protein cleaving enzyme 1 (BACE1) is an aspartyl protease that plays a key role in the production of amyloid ß (Aß) in the brain and has been extensively pursued as a target for the treatment of Alzheimer's disease (AD). BACE2, an aspartyl protease that is structurally related to BACE1, has been recently reported to be involved in melanosome maturation and pigmentation. Herein, we describe the development of a series of cyclopropylthiazines as potent and orally efficacious BACE1 inhibitors. Lead optimization led to the identification of 20, a molecule with biochemical IC50 BACE2/BACE1 ratio of 47. Administration of 20 resulted in no skin/fur color change in a 13-day mouse hypopigmentation study and demonstrated robust and sustained reduction of CSF and brain Aß40 levels in rat and monkey pharmacodynamic models. On the basis of a compelling data package, 20 (AM-6494) was advanced to preclinical development.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ciclopropanos/farmacología , Inhibidores Enzimáticos/farmacología , Tiazinas/farmacología , Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/líquido cefalorraquídeo , Péptidos beta-Amiloides/metabolismo , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Ciclopropanos/química , Ciclopropanos/farmacocinética , Ciclopropanos/uso terapéutico , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/uso terapéutico , Humanos , Masculino , Ratones , Modelos Moleculares , Fragmentos de Péptidos/líquido cefalorraquídeo , Fragmentos de Péptidos/metabolismo , Ratas Sprague-Dawley , Tiazinas/química , Tiazinas/farmacocinética , Tiazinas/uso terapéuticoRESUMEN
KRASG12C has emerged as a promising target in the treatment of solid tumors. Covalent inhibitors targeting the mutant cysteine-12 residue have been shown to disrupt signaling by this long-"undruggable" target; however clinically viable inhibitors have yet to be identified. Here, we report efforts to exploit a cryptic pocket (H95/Y96/Q99) we identified in KRASG12C to identify inhibitors suitable for clinical development. Structure-based design efforts leading to the identification of a novel quinazolinone scaffold are described, along with optimization efforts that overcame a configurational stability issue arising from restricted rotation about an axially chiral biaryl bond. Biopharmaceutical optimization of the resulting leads culminated in the identification of AMG 510, a highly potent, selective, and well-tolerated KRASG12C inhibitor currently in phase I clinical trials (NCT03600883).
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Piperazinas/uso terapéutico , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Piridinas/uso terapéutico , Pirimidinas/uso terapéutico , Pirimidinonas/uso terapéutico , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Ensayos Clínicos como Asunto , Perros , Descubrimiento de Drogas , Humanos , Isomerismo , Células de Riñón Canino Madin Darby , Ratones Endogámicos BALB C , Ratones Desnudos , Mutación , Piperazinas/química , Piperazinas/farmacología , Proteínas Proto-Oncogénicas p21(ras)/genética , Piridinas/química , Piridinas/farmacocinética , Piridinas/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Pirimidinonas/química , Pirimidinonas/farmacocinética , Ratas , Relación Estructura-ActividadRESUMEN
The prosurvival BCL2 family member MCL1 is frequently dysregulated in cancer. To overcome the significant challenges associated with inhibition of MCL1 protein-protein interactions, we rigorously applied small-molecule conformational restriction, which culminated in the discovery of AMG 176, the first selective MCL1 inhibitor to be studied in humans. We demonstrate that MCL1 inhibition induces a rapid and committed step toward apoptosis in subsets of hematologic cancer cell lines, tumor xenograft models, and primary patient samples. With the use of a human MCL1 knock-in mouse, we demonstrate that MCL1 inhibition at active doses of AMG 176 is tolerated and correlates with clear pharmacodynamic effects, demonstrated by reductions in B cells, monocytes, and neutrophils. Furthermore, the combination of AMG 176 and venetoclax is synergistic in acute myeloid leukemia (AML) tumor models and in primary patient samples at tolerated doses. These results highlight the therapeutic promise of AMG 176 and the potential for combinations with other BH3 mimetics. SIGNIFICANCE: AMG 176 is a potent, selective, and orally bioavailable MCL1 inhibitor that induces a rapid commitment to apoptosis in models of hematologic malignancies. The synergistic combination of AMG 176 and venetoclax demonstrates robust activity in models of AML at tolerated doses, highlighting the promise of BH3-mimetic combinations in hematologic cancers.See related commentary by Leber et al., p. 1511.This article is highlighted in the In This Issue feature, p. 1494.
RESUMEN
As part of an ongoing effort at Amgen to develop a disease-modifying therapy for Alzheimer's disease, we have previously used the aminooxazoline xanthene (AOX) scaffold to generate potent and orally efficacious BACE1 inhibitors. While AOX-BACE1 inhibitors demonstrated acceptable cardiovascular safety margins, a retinal pathological finding in rat toxicological studies demanded further investigation. It has been widely postulated that such retinal toxicity might be related to off-target inhibition of Cathepsin D (CatD), a closely related aspartyl protease. We report the development of AOX-BACE1 inhibitors with improved selectivity against CatD by following a structure- and property-based approach. Our efforts culminated in the discovery of a picolinamide-substituted 3-aza-AOX-BACE1 inhibitor absent of retinal effects in an early screening rat toxicology study.
RESUMEN
Optimization of the potency and pharmacokinetic profile of 2,3,4-trisubstituted quinoline, 4, led to the discovery of two potent, selective, and orally bioavailable PI3Kδ inhibitors, 6a (AM-0687) and 7 (AM-1430). On the basis of their improved profile, these analogs were selected for in vivo pharmacodynamic (PD) and efficacy experiments in animal models of inflammation. The in vivo PD studies, which were carried out in a mouse pAKT inhibition animal model, confirmed the observed potency of 6a and 7 in biochemical and cellular assays. Efficacy experiments in a keyhole limpet hemocyanin model in rats demonstrated that administration of either 6a or 7 resulted in a strong dose-dependent reduction of IgG and IgM specific antibodies. The excellent in vitro and in vivo profiles of these analogs make them suitable for further development.
Asunto(s)
Descubrimiento de Drogas , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Quinolinas/farmacología , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Piridinas/síntesis química , Piridinas/química , Quinolinas/síntesis química , Quinolinas/química , Relación Estructura-ActividadRESUMEN
One of the challenges for targeting B-Raf(V600E) with small molecule inhibitors had been achieving adequate selectivity over the wild-type protein B-Raf(WT), as inhibition of the latter has been associated with hyperplasia in normal tissues. Recent studies suggest that B-Raf inhibitors inducing the 'DFG-in/αC-helix-out' conformation (Type IIB) likely will exhibit improved selectivity for B-Raf(V600E). To explore this hypothesis, we transformed Type IIA inhibitor (1) into a series of Type IIB inhibitors (sulfonamides and sulfamides 4-6) and examined the SAR. Three selectivity indices were introduced to facilitate the analyses: the B-Raf(V600E)/B-Raf(WT) biochemical ((b)S), cellular ((c)S) selectivity, and the phospho-ERK activation ((p)A). Our data indicates that α-branched sulfonamides and sulfamides show higher selectivities than the linear derivatives. We rationalized this finding based on analysis of structural information from the literature and provided evidence for a monomeric B-Raf-inhibitor complex previously hypothesized to be responsible for the desired B-Raf(V600E) selectivity.
Asunto(s)
Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Purinas/química , Purinas/farmacología , Piridinas/química , Piridinas/farmacología , Aminación , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Modelos Moleculares , Mutación Puntual , Conformación Proteica en Hélice alfa/efectos de los fármacos , Proteínas Proto-Oncogénicas B-raf/química , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Relación Estructura-ActividadRESUMEN
Fragment-based drug discovery (FBDD) has become a widely used tool in small-molecule drug discovery efforts. One of the most commonly used biophysical methods in detecting weak binding of fragments is nuclear magnetic resonance (NMR) spectroscopy. In particular, FBDD performed with (19)F NMR-based methods has been shown to provide several advantages over (1)H NMR using traditional magnetization-transfer and/or two-dimensional methods. Here, we demonstrate the utility and power of (19)F-based fragment screening by detailing the identification of a second-site fragment through (19)F NMR screening that binds to a specific pocket of the aspartic acid protease, ß-secretase (BACE-1). The identification of this second-site fragment allowed the undertaking of a fragment-linking approach, which ultimately yielded a molecule exhibiting a more than 360-fold increase in potency while maintaining reasonable ligand efficiency and gaining much improved selectivity over cathepsin-D (CatD). X-ray crystallographic studies of the molecules demonstrated that the linked fragments exhibited binding modes consistent with those predicted from the targeted screening approach, through-space NMR data, and molecular modeling.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Espectroscopía de Resonancia Magnética/métodos , Cristalografía por Rayos X , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Flúor , Modelos Moleculares , Resonancia por Plasmón de SuperficieRESUMEN
Deregulation of the receptor tyrosine kinase mesenchymal epithelial transition factor (MET) has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of compound 23 (AMG 337), which demonstrates nanomolar inhibition of MET kinase activity, desirable preclinical pharmacokinetics, significant inhibition of MET phosphorylation in mice, and robust tumor growth inhibition in a MET-dependent mouse efficacy model.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Piridonas/síntesis química , Piridonas/farmacología , Triazoles/síntesis química , Triazoles/farmacología , Animales , Antineoplásicos/farmacocinética , Cristalografía por Rayos X , Diseño de Fármacos , Descubrimiento de Drogas , Humanos , Ratones , Modelos Moleculares , Piridonas/farmacocinética , Relación Estructura-Actividad , Triazoles/farmacocinética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Lead optimization efforts resulted in the discovery of two potent, selective, and orally bioavailable PI3Kδ inhibitors, 1 (AM-8508) and 2 (AM-9635), with good pharmacokinetic properties. The compounds inhibit B cell receptor (BCR)-mediated AKT phosphorylation (pAKT) in PI3Kδ-dependent in vitro cell based assays. These compounds which share a benzimidazole bicycle are effective when administered in vivo at unbound concentrations consistent with their in vitro cell potency as a consequence of improved unbound drug concentration with lower unbound clearance. Furthermore, the compounds demonstrated efficacy in a Keyhole Limpet Hemocyanin (KLH) study in rats, where the blockade of PI3Kδ activity by inhibitors 1 and 2 led to effective inhibition of antigen-specific IgG and IgM formation after immunization with KLH.
Asunto(s)
Bencimidazoles/síntesis química , Bencimidazoles/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Animales , Linfocitos B/efectos de los fármacos , Cristalografía por Rayos X , Hemocianinas/efectos de los fármacos , Humanos , Inmunoglobulina G/efectos de los fármacos , Inmunoglobulina M/efectos de los fármacos , Ratones , Modelos Moleculares , Ratas , Relación Estructura-ActividadRESUMEN
Based on lead compound 1, which was discovered from a high-throughput screen, a series of PI3Kα/mTOR inhibitors were evaluated that contained an imidazo[1,2-a]pyridine as a core replacement for the benzimidazole contained in 1. By exploring various ring systems that occupy the affinity pocket, two fragments containing a methoxypyridine were identified that gave <100 nM potency toward PI3Kα in enzyme and cellular assays with moderate stability in rat and human liver microsomes. With the two methoxypyridine groups selected to occupy the affinity pocket, analogs were prepared with various fragments intended to occupy the ribose pocket of PI3Kα and mTOR. From these analogs, tertiary alcohol 18 was chosen for in vivo pharmacodynamic evaluation based on its potency in the PI3Kα cellular assay, microsomal stability, and in vivo pharmacokinetic properties. In a mouse liver pharmacodynamic assay, compound 18 showed 56% inhibition of HFG-induced AKT (Ser473) phosphorylation at a 30 mg/kg dose.
Asunto(s)
Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/química , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Estructura Molecular , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Ratas , Relación Estructura-Actividad , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
BACE1 inhibition to prevent Aß peptide formation is considered to be a potential route to a disease-modifying treatment for Alzheimer's disease. Previous efforts in our laboratory using a combined structure- and property-based approach have resulted in the identification of aminooxazoline xanthenes as potent BACE1 inhibitors. Herein, we report further optimization leading to the discovery of inhibitor 15 as an orally available and highly efficacious BACE1 inhibitor that robustly reduces CSF and brain Aß levels in both rats and nonhuman primates. In addition, compound 15 exhibited low activity on the hERG ion channel and was well tolerated in an integrated cardiovascular safety model.
RESUMEN
The overexpression of c-Met and/or hepatocyte growth factor (HGF), the amplification of the MET gene, and mutations in the c-Met kinase domain can activate signaling pathways that contribute to cancer progression by enabling tumor cell proliferation, survival, invasion, and metastasis. Herein, we report the discovery of 8-fluorotriazolopyridines as inhibitors of c-Met activity. Optimization of the 8-fluorotriazolopyridine scaffold through the combination of structure-based drug design, SAR studies, and metabolite identification provided potent (cellular IC50 < 10 nM), selective inhibitors of c-Met with desirable pharmacokinetic properties that demonstrate potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver pharmacodynamic model.