RESUMO
Despite being crucial for energy generation in most forms of life, few if any microbial antibiotics specifically inhibit glycolysis. To develop a specific inhibitor of the glycolytic enzyme enolase 2 (ENO2) for the treatment of cancers with deletion of ENO1 (encoding enolase 1), we modeled the synthetic tool compound inhibitor phosphonoacetohydroxamate (PhAH) into the active site of human ENO2. A ring-stabilized analog of PhAH, in which the hydroxamic nitrogen is linked to Cα by an ethylene bridge, was predicted to increase binding affinity by stabilizing the inhibitor in a bound conformation. Unexpectedly, a structure-based search revealed that our hypothesized backbone-stabilized PhAH bears strong similarity to SF2312, a phosphonate antibiotic of unknown mode of action produced by the actinomycete Micromonospora, which is active under anaerobic conditions. Here, we present multiple lines of evidence, including a novel X-ray structure, that SF2312 is a highly potent, low-nanomolar inhibitor of enolase.
Assuntos
Inibidores Enzimáticos/farmacologia , Organofosfonatos/farmacologia , Fosfopiruvato Hidratase/antagonistas & inibidores , Pirrolidinonas/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estrutura Molecular , Organofosfonatos/química , Fosfopiruvato Hidratase/metabolismo , Pirrolidinonas/química , Relação Estrutura-AtividadeRESUMO
Lack of robust activation of Stimulator of Interferon Genes (STING) pathway and subsequent induction of type I IFN responses is considered a barrier to antitumor immunity in acute myeloid leukemia (AML). Using common human AML cell lines as in vitro tools to evaluate the efficacy of novel STING agonists, we found most AML lines to be poor producers of IFNs upon exposure to extremely potent agonists, suggesting cell-intrinsic suppression of STING signaling may occur. We observed unexpected patterns of response that did not correlate with levels of STING pathway components or of known enzymes associated with resistance. To identify a genetic basis for these observations, we cloned and sequenced STING from the cDNA of human AML cell lines and found both frequent mutations and deviations from normal RNA splicing. We identified two novel spliced isoforms of STING in these lines and validated their expression in primary human AML samples. When transduced into reporter cells, these novel STING isoforms exhibited complete insensitivity to agonist stimulation. These observations identify alternative splicing as a mechanism of STING pathway suppression and suggest that most AML silences the STING pathway through direct modification rather than through engagement of external inhibitory factors. SIGNIFICANCE: We find that AML acquires resistance to innate immune activation via the STING pathway through aberrant splicing of the STING transcript including two novel forms described herein that act as dominant negatives. These data broaden understanding of how cancers evolve STING resistance, and suggest that the AML tumor microenvironment, not the cancer cell, should be the target of therapeutic interventions to activate STING.
Assuntos
Interferon Tipo I , Leucemia Mieloide Aguda , Humanos , Isoformas de Proteínas/genética , Leucemia Mieloide Aguda/genética , Processamento Alternativo/genética , Interferon Tipo I/genética , Linhagem Celular , Microambiente TumoralRESUMO
BACKGROUND: Intratumoral injection of cyclic dinucleotide (CDN) agonists of the stimulator of interferon genes (STING) pathway engages innate immune activation and priming of adaptive immune effectors to foster local and distal tumor clearance. Despite proven therapeutic efficacy in preclinical models, a thorough understanding of how CDNs reprogram suppressive myeloid stroma in mouse and man is lacking. METHODS: Here, we perform deep transcript-level and protein-level profiling of myeloid-derived suppressor cells and M2 macrophages following stimulation with CDNs of ascending potency. Additionally, we leverage orthotopic Kras+/G12DTP53+/R172HPdx1-Cre (KPC) derived models of pancreatic adenocarcinoma (PDAC) to determine the capacity for locally administered CDNs to sensitize PDAC to immune checkpoint blockade. We use bioluminescent in vivo imaging and 30-parameter flow cytometry to profile growth kinetics and remodeling of the tumor stroma post-therapy. RESULTS: Highly potent synthetic STING agonists repolarize suppressive myeloid populations of human and murine origin in part through inhibition of Myc signaling, metabolic modulation, and antagonism of cell cycle. Surprisingly, high-potency synthetic agonists engage qualitatively unique pathways as compared with natural CDNs. Consistent with our mechanistic observations, we find that intratumoral injection of the highest activity STING agonist, IACS-8803, into orthotopic pancreatic adenocarcinoma lesions unmasks sensitivity to checkpoint blockade immunotherapy. Dimensionality reduction analyses of high parameter flow cytometry data reveals substantial contributions of both myeloid repolarization and T cell activation underlying the in vivo therapeutic benefit of this approach. CONCLUSIONS: This study defines the molecular basis of STING-mediated myeloid reprogramming, revealing previously unappreciated and qualitatively unique pathways engaged by CDNs of ascending potency during functional repolarization. Furthermore, we demonstrate the potential for high potency CDNs to overcome immunotherapy resistance in an orthotopic, multifocal model of PDAC.
Assuntos
Imunoterapia/métodos , Proteínas de Membrana/uso terapêutico , Células Supressoras Mieloides/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Humanos , Masculino , Proteínas de Membrana/farmacologia , CamundongosRESUMO
Src homology 2 (SH2) domain-containing phosphatase 2 (SHP2) plays a role in receptor tyrosine kinase (RTK), neurofibromin-1 (NF-1), and Kirsten rat sarcoma virus (KRAS) mutant-driven cancers, as well as in RTK-mediated resistance, making the identification of small-molecule therapeutics that interfere with its function of high interest. Our quest to identify potent, orally bioavailable, and safe SHP2 inhibitors led to the discovery of a promising series of pyrazolopyrimidinones that displayed excellent potency but had a suboptimal in vivo pharmacokinetic (PK) profile. Hypothesis-driven scaffold optimization led us to a series of pyrazolopyrazines with excellent PK properties across species but a narrow human Ether-à-go-go-Related Gene (hERG) window. Subsequent optimization of properties led to the discovery of the pyrimidinone series, in which multiple members possessed excellent potency, optimal in vivo PK across species, and no off-target activities including no hERG liability up to 100 µM. Importantly, compound 30 (IACS-15414) potently suppressed the mitogen-activated protein kinase (MAPK) pathway signaling and tumor growth in RTK-activated and KRASmut xenograft models in vivo.
Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Humanos , Camundongos , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Relação Estrutura-AtividadeRESUMO
Hepatitis C represents a serious worldwide health-care problem. Recently, we have disclosed a novel class of P2-P4 macrocyclic inhibitors of NS3/4A protease containing a carbamate functionality as capping group at the P3 N-terminus. Herein we report our work aimed at further depeptidizing the P3 region by replacement of the urethane function with a succinamide motif. This peptidomimetic approach has led to the discovery of novel P2-P4 macrocyclic inhibitors of HCV NS3/4A protease with sub-nanomolar enzyme affinities. In addition to being potent inhibitors of HCV subgenomic replication, optimized analogues within this series have also presented attractive PK properties and showed promising liver levels in rat following oral administration.
Assuntos
Antivirais/química , Inibidores de Proteases/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Administração Oral , Amidas/química , Animais , Antivirais/síntese química , Antivirais/farmacocinética , Sítios de Ligação , Linhagem Celular , Simulação por Computador , Ciclopropanos , Humanos , Indóis/química , Indóis/farmacocinética , Isoindóis , Lactamas Macrocíclicas , Leucina/análogos & derivados , Prolina/análogos & derivados , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacocinética , Ratos , Ratos Wistar , Succinatos , Sulfonamidas , Proteínas não Estruturais Virais/metabolismoRESUMO
Inhibition of glutaminase-1 (GLS-1) hampers the proliferation of tumor cells reliant on glutamine. Known glutaminase inhibitors have potential limitations, and in vivo exposures are potentially limited due to poor physicochemical properties. We initiated a GLS-1 inhibitor discovery program focused on optimizing physicochemical and pharmacokinetic properties, and have developed a new selective inhibitor, compound 27 (IPN60090), which is currently in phase 1 clinical trials. Compound 27 attains high oral exposures in preclinical species, with strong in vivo target engagement, and should robustly inhibit glutaminase in humans.
Assuntos
Inibidores Enzimáticos/química , Glutaminase/antagonistas & inibidores , Triazóis/farmacocinética , Administração Oral , Animais , Linhagem Celular Tumoral , Cães , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Glutaminase/genética , Glutaminase/metabolismo , Meia-Vida , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Concentração Inibidora 50 , Masculino , Camundongos , Microssomos/metabolismo , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Relação Estrutura-Atividade , Triazóis/química , Triazóis/metabolismoRESUMO
Src homology 2 domain-containing phosphatase (SHP2) is a phosphatase that mediates signaling downstream of multiple receptor tyrosine kinases (RTK) and is required for full activation of the MAPK pathway. SHP2 inhibition has demonstrated tumor growth inhibition in RTK-activated cancers in preclinical studies. The long-term effectiveness of tyrosine kinase inhibitors such as the EGFR inhibitor (EGFRi), osimertinib, in non-small cell lung cancer (NSCLC) is limited by acquired resistance. Multiple clinically identified mechanisms underlie resistance to osimertinib, including mutations in EGFR that preclude drug binding as well as EGFR-independent activation of the MAPK pathway through alternate RTK (RTK-bypass). It has also been noted that frequently a tumor from a single patient harbors more than one resistance mechanism, and the plasticity between multiple resistance mechanisms could restrict the effectiveness of therapies targeting a single node of the oncogenic signaling network. Here, we report the discovery of IACS-13909, a specific and potent allosteric inhibitor of SHP2, that suppresses signaling through the MAPK pathway. IACS-13909 potently impeded proliferation of tumors harboring a broad spectrum of activated RTKs as the oncogenic driver. In EGFR-mutant osimertinib-resistant NSCLC models with EGFR-dependent and EGFR-independent resistance mechanisms, IACS-13909, administered as a single agent or in combination with osimertinib, potently suppressed tumor cell proliferation in vitro and caused tumor regression in vivo. Together, our findings provide preclinical evidence for using a SHP2 inhibitor as a therapeutic strategy in acquired EGFRi-resistant NSCLC. SIGNIFICANCE: These findings highlight the discovery of IACS-13909 as a potent, selective inhibitor of SHP2 with drug-like properties, and targeting SHP2 may serve as a therapeutic strategy to overcome tumor resistance to osimertinib.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Experimentais/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Acrilamidas/farmacologia , Compostos de Anilina/farmacologia , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Mutação , Neoplasias Experimentais/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
This paper describes the synthesis of a new class of peptidomimetic cysteine protease inhibitors based on a 1,4-benzodiazepine scaffold and on an electrophilic vinyl sulfone moiety. The former was introduced internally to a peptide sequence that mimics the fragment D-Ser-Gly; the latter was built on the P1-P1' site and reacts as a classical "Michael acceptor", leading to an alkylated enzyme by irreversible addition of the thiol group of the active site cysteine. The introduction of the vinyl sulfone moiety has been accomplished by olefin cross-metathesis, a powerful tool for the formation of carbon-carbon double bonds. New compounds 2-3 have been proven to be potent and selective inhibitors of falcipain-2, a cysteine protease isolated from Plasmodium falciparum, displaying antiplasmodial activity.
Assuntos
Antimaláricos/síntese química , Benzodiazepinas/síntese química , Carbamatos/síntese química , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/síntese química , Peptídeos/química , Sulfonas/síntese química , Animais , Antimaláricos/química , Antimaláricos/farmacologia , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Carbamatos/química , Carbamatos/farmacologia , Catepsina B/antagonistas & inibidores , Catepsina B/química , Catepsina L , Catepsinas/antagonistas & inibidores , Catepsinas/química , Cisteína Endopeptidases/química , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacologia , Humanos , Cinética , Mimetismo Molecular , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Estereoisomerismo , Relação Estrutura-Atividade , Sulfonas/química , Sulfonas/farmacologiaRESUMO
Current treatment regimens for pancreatic ductal adenocarcinoma (PDAC) yield poor 5-year survival, emphasizing the critical need to identify druggable targets essential for PDAC maintenance. We developed an unbiased and in vivo target discovery approach to identify molecular vulnerabilities in low-passage and patient-derived PDAC xenografts or genetically engineered mouse model-derived allografts. Focusing on epigenetic regulators, we identified WDR5, a core member of the COMPASS histone H3 Lys4 (H3K4) MLL (1-4) methyltransferase complex, as a top tumor maintenance hit required across multiple human and mouse tumors. Mechanistically, WDR5 functions to sustain proper execution of DNA replication in PDAC cells, as previously suggested by replication stress studies involving MLL1, and c-Myc, also found to interact with WDR5. We indeed demonstrate that interaction with c-Myc is critical for this function. By showing that ATR inhibition mimicked the effects of WDR5 suppression, these data provide rationale to test ATR and WDR5 inhibitors for activity in this disease.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células , Dano ao DNA , Progressão da Doença , Epigênese Genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lentivirus/metabolismo , Camundongos , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Ligação Proteica , Subunidades Proteicas/metabolismo , RNA Interferente Pequeno/metabolismo , Estresse FisiológicoRESUMO
This paper describes the synthesis and biological evaluation of a new class of peptidomimetic falcipain-2 inhibitors based on a 1,4-benzodiazepine scaffold combined with various alpha,beta-unsaturated electrophilic functions such as vinyl-ketone, -amide, -ester, and -nitrile. The profile of reactivity of this class of derivatives has been evaluated and 4c, containing a vinyl ester warhead, proved to be a highly potent and selective falcipain-2 inhibitor.