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1.
Nat Chem Biol ; 2024 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-38519575

RESUMO

Cotransins target the Sec61 translocon and inhibit the biogenesis of an undefined subset of secretory and membrane proteins. Remarkably, cotransin inhibition depends on the unique signal peptide (SP) of each Sec61 client, which is required for cotranslational translocation into the endoplasmic reticulum. It remains unknown how an SP's amino acid sequence and biophysical properties confer sensitivity to structurally distinct cotransins. Here we describe a fluorescence-based, pooled-cell screening platform to interrogate nearly all human SPs in parallel. We profiled two cotransins with distinct effects on cancer cells and discovered a small subset of SPs, including the oncoprotein human epidermal growth factor receptor 3 (HER3), with increased sensitivity to the more selective cotransin, KZR-9873. By comparing divergent mouse and human orthologs, we unveiled a position-dependent effect of arginine on SP sensitivity. Our multiplexed profiling platform reveals how cotransins can exploit subtle sequence differences to achieve SP discrimination.

2.
Front Immunol ; 14: 1043680, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36969170

RESUMO

Zetomipzomib (KZR-616) is a selective inhibitor of the immunoproteasome currently undergoing clinical investigation in autoimmune disorders. Here, we characterized KZR-616 in vitro and in vivo using multiplexed cytokine analysis, lymphocyte activation and differentiation, and differential gene expression analysis. KZR-616 blocked production of >30 pro-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs), polarization of T helper (Th) cells, and formation of plasmablasts. In the NZB/W F1 mouse model of lupus nephritis (LN), KZR-616 treatment resulted in complete resolution of proteinuria that was maintained at least 8 weeks after the cessation of dosing and was mediated in part by alterations in T and B cell activation, including reduced numbers of short and long-lived plasma cells. Gene expression analysis of human PBMCs and tissues from diseased mice revealed a consistent and broad response focused on inhibition of T, B, and plasma cell function and the Type I interferon pathway and promotion of hematopoietic cell lineages and tissue remodeling. In healthy volunteers, KZR-616 administration resulted in selective inhibition of the immunoproteasome and blockade of cytokine production following ex vivo stimulation. These data support the ongoing development of KZR-616 in autoimmune disorders such as systemic lupus erythematosus (SLE)/LN.


Assuntos
Leucócitos Mononucleares , Nefrite Lúpica , Humanos , Animais , Camundongos , Leucócitos Mononucleares/metabolismo , Citocinas/metabolismo , Imunidade
3.
Br J Haematol ; 198(1): 137-141, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35434798

RESUMO

Glucocorticoid (GC) resistance is a poor prognostic factor in T-cell acute lymphoblastic leukaemia (T-ALL). Interleukin-7 (IL-7) mediates GC resistance via GC-induced upregulation of IL-7 receptor (IL-7R) expression, leading to increased pro-survival signalling. IL-7R reaches the cell surface via the secretory pathway, so we hypothesized that inhibiting the translocation of IL-7R into the secretory pathway would overcome GC resistance. Sec61 is an endoplasmic reticulum (ER) channel that is required for insertion of polypeptides into the ER. Here, we demonstrate that KZR-445, a novel inhibitor of Sec61, potently attenuates the dexamethasone (DEX)-induced increase in cell surface IL-7R and overcomes IL-7-induced DEX resistance.


Assuntos
Leucemia-Linfoma Linfoblástico de Células T Precursoras , Canais de Translocação SEC , Citocinas/metabolismo , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Humanos , Interleucina-7 , Erros Inatos do Metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Receptores de Glucocorticoides/deficiência , Canais de Translocação SEC/metabolismo , Linfócitos T/metabolismo
4.
Drug Metab Dispos ; 49(9): 810-821, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34234005

RESUMO

KZR-616 is an irreversible tripeptide epoxyketone-based selective inhibitor of the human immunoproteasome. Inhibition of the immunoproteasome results in anti-inflammatory activity in vitro and based on promising therapeutic activity in animal models of rheumatoid arthritis and systemic lupus erythematosus KZR-616 is being developed for potential treatment of multiple autoimmune and inflammatory diseases. The presence of a ketoepoxide pharmacophore presents unique challenges in the study of drug metabolism during lead optimization and clinical candidate profiling. This study presents a thorough and systematic in vitro and cell-based enzymatic metabolism and kinetic investigation to identify the major enzymes involved in the metabolism and elimination of KZR-616. Upon exposure to liver microsomes in the absence of NADPH, KZR-616 and its analogs were converted to their inactive diol derivatives with varying degrees of stability. Diol formation was also shown to be the major metabolite in pharmacokinetic studies in monkeys and correlated with in vitro stability results for individual compounds. Further study in intact hepatocytes revealed that KZR-616 metabolism was sensitive to an inhibitor of microsomal epoxide hydrolase (mEH) but not inhibitors of cytochrome P450 (P450) or soluble epoxide hydrolase (sEH). Primary human hepatocytes were determined to be the most robust source of mEH activity for study in vitro. These findings also suggest that the exposure of KZR-616 in vivo is unlikely to be affected by coadministration of inhibitors or inducers of P450 and sEH. SIGNIFICANCE STATEMENT: This work presents a thorough and systematic investigation of metabolism and kinetics of KZR-616 and related analogs in in vitro and cell-based enzymatic systems. Information gained could be useful in assessing novel covalent proteasome inhibitors during lead compound optimization. These studies also demonstrate a robust source in vitro test system that correlated with in vivo pharmacokinetics for KZR-616 and two additional tripeptide epoxyketones.


Assuntos
Cisteína Endopeptidases/imunologia , Sistema Enzimático do Citocromo P-450/metabolismo , Epóxido Hidrolases/metabolismo , Morfolinas/farmacologia , Complexo de Endopeptidases do Proteassoma/imunologia , Proteínas/imunologia , Animais , Doenças Autoimunes/tratamento farmacológico , Células Cultivadas , Cisteína Endopeptidases/metabolismo , Epóxido Hidrolases/imunologia , Hepatócitos/metabolismo , Humanos , Inativação Metabólica , Inflamação/tratamento farmacológico , Macaca fascicularis , Inibidores de Proteassoma/farmacologia
5.
J Med Chem ; 61(24): 11127-11143, 2018 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-30380863

RESUMO

Selective immunoproteasome inhibition is a promising approach for treating autoimmune disorders, but optimal proteolytic active site subunit inhibition profiles remain unknown. We reveal here our design of peptide epoxyketone-based selective low molecular mass polypeptide-7 (LMP7) and multicatalytic endopeptidase complex subunit-1 (MECL-1) subunit inhibitors. Utilizing these and our previously disclosed low molecular mass polypeptide-2 (LMP2) inhibitor, we demonstrate a requirement of dual LMP7/LMP2 or LMP7/MECL-1 subunit inhibition profiles for potent cytokine expression inhibition and in vivo efficacy in an inflammatory disease model. These and additional findings toward optimized solubility led the design and selection of KZR-616 disclosed here and presently in clinical trials for treatment of rheumatic disease.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Artrite Reumatoide/tratamento farmacológico , Morfolinas/farmacologia , Inibidores de Proteassoma/farmacologia , Administração Intravenosa , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacocinética , Artrite Experimental/tratamento farmacológico , Linhagem Celular Tumoral , Cisteína Endopeptidases/metabolismo , Citocinas/metabolismo , Desenho de Fármacos , Feminino , Humanos , Camundongos Endogâmicos BALB C , Morfolinas/química , Morfolinas/farmacocinética , Oligopeptídeos/farmacologia , Complexo de Endopeptidases do Proteassoma/imunologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/química , Inibidores de Proteassoma/farmacocinética , Relação Estrutura-Atividade
6.
ACS Med Chem Lett ; 8(4): 413-417, 2017 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-28435528

RESUMO

Building upon the success of bortezomib (VELCADE) and carfilzomib (KYPROLIS), the design of a next generation of inhibitors targeting specific subunits within the immunoproteasome is of interest for the treatment of autoimmune disease. There are three catalytic subunits within the immunoproteasome (low molecular mass polypeptide-7, -2, and multicatalytic endopeptidase complex subunit-1; LMP7, LMP2, and MECL-1), and a campaign was undertaken to design a potent and selective LMP2 inhibitor with sufficient properties to allow for sustained inhibition in vivo. Screening a focused library of epoxyketones revealed a series of potent dipeptides that were optimized to provide the highly selective inhibitor KZR-504 (12).

7.
ACS Med Chem Lett ; 5(8): 894-9, 2014 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-25147610

RESUMO

Continued optimization of the N-substituent in the piperidinone series provided potent piperidinone-pyridine inhibitors 6, 7, 14, and 15 with improved pharmacokinetic properties in rats. Reducing structure complexity of the N-alkyl substituent led to the discovery of 23, a potent and simplified inhibitor of MDM2. Compound 23 exhibits excellent pharmacokinetic properties and substantial in vivo antitumor activity in the SJSA-1 osteosarcoma xenograft mouse model.

8.
J Med Chem ; 57(8): 3430-49, 2014 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-24641103

RESUMO

We describe the structural optimization of a lead compound 1 that exhibits dual inhibitory activities against FLT3 and CDK4. A series of pyrido[4',3':4,5]pyrrolo[2,3-d]pyrimidine derivatives was synthesized, and SAR analysis, using cell-based assays, led to the discovery of 28 (AMG 925), a potent and orally bioavailable dual inhibitor of CDK4 and FLT3, including many FLT3 mutants reported to date. Compound 28 inhibits the proliferation of a panel of human tumor cell lines including Colo205 (Rb(+)) and U937 (FLT3(WT)) and induced cell death in MOLM13 (FLT3(ITD)) and even in MOLM13 (FLT3(ITD, D835Y)), which exhibits resistance to a number of FLT3 inhibitors currently under clinical development. At well-tolerated doses, compound 28 leads to significant growth inhibition of MOLM13 xenografts in nude mice, and the activity correlates with inhibition of STAT5 and Rb phosphorylation.


Assuntos
Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Compostos Heterocíclicos com 3 Anéis/síntese química , Naftiridinas/síntese química , Inibidores de Proteínas Quinases/síntese química , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Animais , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Citocromo P-450 CYP3A , Inibidores do Citocromo P-450 CYP3A , Cães , Descoberta de Drogas , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Macaca fascicularis , Naftiridinas/farmacologia , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Ratos , Relação Estrutura-Atividade , Células U937 , Tirosina Quinase 3 Semelhante a fms/genética
9.
J Med Chem ; 57(6): 2472-88, 2014 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-24548297

RESUMO

We previously reported the discovery of AMG 232, a highly potent and selective piperidinone inhibitor of the MDM2-p53 interaction. Our continued search for potent and diverse analogues led to the discovery of novel morpholinone MDM2 inhibitors. This change to a morpholinone core has a significant impact on both potency and metabolic stability compared to the piperidinone series. Within this morpholinone series, AM-8735 emerged as an inhibitor with remarkable biochemical potency (HTRF IC50 = 0.4 nM) and cellular potency (SJSA-1 EdU IC50 = 25 nM), as well as pharmacokinetic properties. Compound 4 also shows excellent antitumor activity in the SJSA-1 osteosarcoma xenograft model with an ED50 of 41 mg/kg. Lead optimization toward the discovery of this inhibitor as well as key differences between the morpholinone and the piperidinone series will be described herein.


Assuntos
Acetatos/síntese química , Acetatos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Morfolinas/síntese química , Morfolinas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/química , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/química , Animais , Linhagem Celular Tumoral , Cristalografia por Raios X , Descoberta de Drogas , Humanos , Indicadores e Reagentes , Camundongos , Modelos Moleculares , Conformação Molecular , Morfolinas/farmacocinética , Ratos , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto
10.
J Med Chem ; 56(10): 4053-70, 2013 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-23597064

RESUMO

Structural analysis of both the MDM2-p53 protein-protein interaction and several small molecules bound to MDM2 led to the design and synthesis of tetrasubstituted morpholinone 10, an MDM2 inhibitor with a biochemical IC50 of 1.0 µM. The cocrystal structure of 10 with MDM2 inspired two independent optimization strategies and resulted in the discovery of morpholinones 16 and 27 possessing distinct binding modes. Both analogues were potent MDM2 inhibitors in biochemical and cellular assays, and morpholinone 27 (IC50 = 0.10 µM) also displayed suitable PK profile for in vivo animal experiments. A pharmacodynamic (PD) experiment in mice implanted with human SJSA-1 tumors showed p21(WAF1) mRNA induction (2.7-fold over vehicle) upon oral dosing of 27 at 300 mg/kg.


Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/antagonistas & inibidores , Animais , Dicroísmo Circular , Cristalografia , Cristalografia por Raios X , Desenho de Fármacos , Feminino , Humanos , Indicadores e Reagentes , Camundongos , Camundongos Nus , Modelos Moleculares , Morfolinas/síntese química , Morfolinas/farmacologia , Piperidinas/síntese química , Piperidinas/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Bioorg Med Chem Lett ; 20(12): 3618-22, 2010 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-20493695

RESUMO

The Hedgehog (Hh) signaling pathway regulates cell proliferation and differentiation in developing tissues, and abnormal activation of the Hh pathway has been linked to several tumor subsets. As a transducer of Hh signaling, the GPCR-like protein Smoothened (Smo) is a promising target for disruption of unregulated Hh signaling. A series of 1-amino-4-arylphthalazines was developed as potent and orally bioavailable inhibitors of Smo. A representative compound from this class demonstrated significant tumor volume reduction in a mouse medulloblastoma model.


Assuntos
Ftalazinas/química , Ftalazinas/farmacologia , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Administração Oral , Animais , Disponibilidade Biológica , Linhagem Celular Tumoral , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Desenho de Fármacos , Proteínas Hedgehog , Humanos , Meduloblastoma/tratamento farmacológico , Meduloblastoma/patologia , Camundongos , Ftalazinas/síntese química , Transdução de Sinais , Receptor Smoothened
12.
Nucleic Acids Res ; 31(4): 1282-91, 2003 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-12582248

RESUMO

Polyamides are a class of heterocyclic small molecules with the potential of controlling gene expression by binding to the minor groove of DNA in a sequence-specific manner. To evaluate the feasibility of this class of compounds as antiviral therapeutics, molecules were designed to essential sequence elements occurring numerous times in the HPV genome. This sequence element is bound by a virus-encoded transcription and replication factor E2, which binds to a 12 bp recognition site as a homodimeric protein. Here, we take advantage of polyamide:DNA and E2:DNA co-crystal structural information and advances in polyamide synthetic chemistry to design tandem hairpin polyamides that are capable of displacing the major groove-binding E2 homodimer from its DNA binding site. The binding of tandem hairpin polyamides and the E2 DNA binding protein to the DNA site is mutually exclusive even though the two ligands occupy opposite faces of the DNA double helix. We show with circular permutation studies that the tandem hairpin polyamide prevents the intrinsic bending of the E2 DNA site important for binding of the protein. Taken together, these results illustrate the feasibility of inhibiting the binding of homodimeric, major groove-binding transcription factors by altering the local DNA geometry using minor groove-binding tandem hairpin polyamides.


Assuntos
DNA Viral/metabolismo , Proteínas de Ligação a DNA , Nylons/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Sítios de Ligação/genética , DNA Viral/química , Modelos Biológicos , Conformação de Ácido Nucleico , Nylons/química , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Proteínas Oncogênicas Virais/química , Papillomaviridae/genética , Papillomaviridae/metabolismo , Ligação Proteica
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