RESUMO
The Plasmodium falciparum proteasome is a potential antimalarial drug target. We have identified a series of amino-amide boronates that are potent and specific inhibitors of the P. falciparum 20S proteasome (Pf20S) ß5 active site and that exhibit fast-acting antimalarial activity. They selectively inhibit the growth of P. falciparum compared with a human cell line and exhibit high potency against field isolates of P. falciparum and Plasmodium vivax They have a low propensity for development of resistance and possess liver stage and transmission-blocking activity. Exemplar compounds, MPI-5 and MPI-13, show potent activity against P. falciparum infections in a SCID mouse model with an oral dosing regimen that is well tolerated. We show that MPI-5 binds more strongly to Pf20S than to human constitutive 20S (Hs20Sc). Comparison of the cryo-electron microscopy (EM) structures of Pf20S and Hs20Sc in complex with MPI-5 and Pf20S in complex with the clinically used anti-cancer agent, bortezomib, reveal differences in binding modes that help to explain the selectivity. Together, this work provides insights into the 20S proteasome in P. falciparum, underpinning the design of potent and selective antimalarial proteasome inhibitors.
Assuntos
Compostos de Boro/farmacologia , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/química , Inibidores de Proteassoma/farmacologia , Administração Oral , Animais , Compostos de Boro/administração & dosagem , Compostos de Boro/química , Domínio Catalítico , Humanos , Malária Falciparum/enzimologia , Malária Falciparum/parasitologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Moleculares , Plasmodium falciparum/enzimologia , Inibidores de Proteassoma/administração & dosagem , Inibidores de Proteassoma/químicaRESUMO
The proteasome inhibitor bortezomib is associated with the development of peripheral neuropathy in patients, but the mechanism by which bortezomib can induce peripheral neuropathy is not fully understood. One study suggested that off-target inhibition of proteases other than the proteasome, particularly HtraA2/Omi, may be the underlying mechanism of the neuropathy. The same study also concluded that carfilzomib, a second proteasome inhibitor that is associated with less peripheral neuropathy in patients than bortezomib, showed no inhibition of HtrA2/Omi. The goal of the work described here was to determine whether either proteasome inhibitors truly affected HtrA2/Omi activity. A variety of methods were used to test the effects of both bortezomib and carfilzomib on HtrA2/Omi activity that included in vitro recombinant enzyme assays, and studies with the human neuroblastoma SH-SY5Y cell line and HtrA2/Omi-knockout mouse embryonic fibroblasts. The compound ucf-101 was used to assess the effects of specific HtrA2/Omi inhibition. In contrast to previously published data, our results clearly demonstrated that neither bortezomib nor carfilzomib inhibited HtrA2/Omi activity in recombinant enzyme assays at concentrations up to 100 µM, while the specific inhibitor ucf-101 did inhibit the enzyme. The proteasome inhibitors did not inhibit HtrA2/Omi activity in either SH-SY5Y cells or mouse embryonic fibroblasts, as determined by expression of the HtrA2/Omi substrates eIF4G1 and UCH-L1. Based on our biochemical and cell-based assays, we conclude that neither bortezomib nor carfilzomib inhibited HtrA2/Omi activity. Therefore, it is unlikely that bortezomib associated peripheral neuropathy is a direct result of off-target inhibition of HtrA2/Omi.
RESUMO
In non-clinical studies, the proteasome inhibitor ixazomib inhibits cell growth in a broad panel of solid tumor cell lines in vitro. In contrast, antitumor activity in xenograft tumors is model-dependent, with some solid tumors showing no response to ixazomib. In this study we examined factors responsible for ixazomib sensitivity or resistance using mouse xenograft models. A survey of 14 non-small cell lung cancer (NSCLC) and 6 colon xenografts showed a striking relationship between ixazomib activity and KRAS genotype; tumors with wild-type (WT) KRAS were more sensitive to ixazomib than tumors harboring KRAS activating mutations. To confirm the association between KRAS genotype and ixazomib sensitivity, we used SW48 isogenic colon cancer cell lines. Either KRAS-G13D or KRAS-G12V mutations were introduced into KRAS-WT SW48 cells to generate cells that stably express activated KRAS. SW48 KRAS WT tumors, but neither SW48-KRAS-G13D tumors nor SW48-KRAS-G12V tumors, were sensitive to ixazomib in vivo. Since activated KRAS is known to be associated with metabolic reprogramming, we compared metabolite profiling of SW48-WT and SW48-KRAS-G13D tumors treated with or without ixazomib. Prior to treatment there were significant metabolic differences between SW48 WT and SW48-KRAS-G13D tumors, reflecting higher oxidative stress and glucose utilization in the KRAS-G13D tumors. Ixazomib treatment resulted in significant metabolic regulation, and some of these changes were specific to KRAS WT tumors. Depletion of free amino acid pools and activation of GCN2-eIF2α-pathways were observed both in tumor types. However, changes in lipid beta oxidation were observed in only the KRAS WT tumors. The non-clinical data presented here show a correlation between KRAS genotype and ixazomib sensitivity in NSCLC and colon xenografts and provide new evidence of regulation of key metabolic pathways by proteasome inhibition.
Assuntos
Compostos de Boro/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Glicina/análogos & derivados , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteassoma/uso terapêutico , Proteínas Proto-Oncogênicas p21(ras)/genética , Aminoácidos/metabolismo , Animais , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Ácidos Graxos/metabolismo , Transportador de Glucose Tipo 4/biossíntese , Glicina/uso terapêutico , Células HCT116 , Humanos , Neoplasias Pulmonares/metabolismo , Metaboloma/fisiologia , Camundongos , Oxirredução/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: The clinical success of the first-in-class proteasome inhibitor bortezomib (VELCADE) has validated the proteasome as a therapeutic target for treating human cancers. MLN9708 is an investigational proteasome inhibitor that, compared with bortezomib, has improved pharmacokinetics, pharmacodynamics, and antitumor activity in preclinical studies. Here, we focused on evaluating the in vivo activity of MLN2238 (the biologically active form of MLN9708) in a variety of mouse models of hematologic malignancies, including tumor xenograft models derived from a human lymphoma cell line and primary human lymphoma tissue, and genetically engineered mouse (GEM) models of plasma cell malignancies (PCM). EXPERIMENTAL DESIGN: Both cell line-derived OCI-Ly10 and primary human lymphoma-derived PHTX22L xenograft models of diffuse large B-cell lymphoma were used to evaluate the pharmacodynamics and antitumor effects of MLN2238 and bortezomib. The iMyc(Cα)/Bcl-X(L) GEM model was used to assess their effects on de novo PCM and overall survival. The newly developed DP54-Luc-disseminated model of iMyc(Cα)/Bcl-X(L) was used to determine antitumor activity and effects on osteolytic bone disease. RESULTS: MLN2238 has an improved pharmacodynamic profile and antitumor activity compared with bortezomib in both OCI-Ly10 and PHTX22L models. Although both MLN2238 and bortezomib prolonged overall survival, reduced splenomegaly, and attenuated IgG2a levels in the iMyc(Cα)/Bcl-X(L) GEM model, only MLN2238 alleviated osteolytic bone disease in the DP54-Luc model. CONCLUSIONS: Our results clearly showed the antitumor activity of MLN2238 in a variety of mouse models of B-cell lymphoma and PCM, supporting its clinical development. MLN9708 is being evaluated in multiple phase I and I/II trials.
Assuntos
Antineoplásicos/farmacologia , Compostos de Boro/farmacologia , Glicina/análogos & derivados , Linfoma de Células B/tratamento farmacológico , Neoplasias de Plasmócitos/tratamento farmacológico , Animais , Antineoplásicos/farmacocinética , Compostos de Boro/administração & dosagem , Compostos de Boro/farmacocinética , Ácidos Borônicos/farmacocinética , Ácidos Borônicos/farmacologia , Bortezomib , Linhagem Celular Tumoral , Glicina/administração & dosagem , Glicina/farmacocinética , Glicina/farmacologia , Humanos , Linfoma de Células B/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Neoplasias de Plasmócitos/metabolismo , Osteólise/tratamento farmacológico , Osteólise/etiologia , Inibidores de Proteases/farmacologia , Inibidores de Proteassoma , Pirazinas/farmacocinética , Pirazinas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: To investigate whether clinically relevant levels of epigallocatechin gallate (EGCG, a component of green tea) or vitamin C (ascorbic acid) could antagonize bortezomib antitumor activity in CWR22 human prostate xenograft tumors. METHODS: The pharmacokinetics (PK) of EGCG and ascorbic acid were determined in immunocompromised mice and compared with concentrations measured in human PK studies of dietary supplements. Antitumor activity of bortezomib in combination with EGCG or ascorbic acid was determined using several dosing regimens to evaluate different target plasma concentrations of EGCG and ascorbic acid. RESULTS: Bortezomib dosed twice-weekly at 0.8 mg/kg IV demonstrated tumor growth inhibition (TGI) of 53.9-58.9%. However, when combined with EGCG such that the plasma concentrations of EGCG were >200 µM at the time of bortezomib dosing, all antitumor activity was abrogated (TGI = -17.7%). A lower concentration of EGCG (11-16 µM), which is severalfold higher than measured clinically in humans taking EGCG supplements (0.6-3 µM), was not antagonistic to bortezomib (TGI 63.5%). Pharmacodynamic studies of proteasome inhibition reflected these findings. Ascorbic acid (40 and 500 mg/kg PO daily) was evaluated under a similar study design and did not antagonize bortezomib antitumor activity (TGI 57.2 and 72.2%). CONCLUSIONS: No antagonism of bortezomib is seen in preclinical in vivo experiments, where EGCG or ascorbic acid plasma concentrations are commensurate with dietary or supplemental intake. The data suggest that patients receiving bortezomib treatment do not need to avoid normal dietary consumption of green tea, vitamin C-containing foods, or EGCG or vitamin C dietary supplements.
Assuntos
Antineoplásicos/farmacologia , Ácido Ascórbico/farmacologia , Ácidos Borônicos/farmacologia , Catequina/análogos & derivados , Pirazinas/farmacologia , Chá/química , Animais , Antineoplásicos/administração & dosagem , Ácido Ascórbico/administração & dosagem , Ácido Ascórbico/farmacocinética , Ácidos Borônicos/administração & dosagem , Bortezomib , Catequina/administração & dosagem , Catequina/farmacologia , Cromatografia Líquida , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Masculino , Camundongos , Camundongos SCID , Neoplasias da Próstata/patologia , Pirazinas/administração & dosagem , Espectrometria de Massas em Tandem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The mammalian 26S proteasome is a 2500 kDa multi-catalytic complex involved in intracellular protein degradation. We describe the synthesis and properties of a novel series of non-covalent di-peptide inhibitors of the proteasome based [corrected] on a capped tri-peptide that was first identified by high-throughput screening of a library of approx. 350000 compounds for inhibitors of the ubiquitin-proteasome system in cells. We show that these compounds are entirely selective for the beta5 (chymotrypsin-like) site over the beta1 (caspase-like) and beta2 (trypsin-like) sites of the 20S core particle of the proteasome, and over a panel of less closely related proteases. Compound optimization, guided by X-ray crystallography of the liganded 20S core particle, confirmed their non-covalent binding mode and provided a structural basis for their enhanced in vitro and cellular potencies. We demonstrate that such compounds show low nanomolar IC50 values for the human 20S beta5 site in vitro, and that pharmacological inhibition of this site in cells is sufficient to potently inhibit the degradation of a tetra-ubiquitin-luciferase reporter, activation of NFkappaB (nuclear factor kappaB) in response to TNF-alpha (tumour necrosis factor-alpha) and the proliferation of cancer cells. Finally, we identified capped di-peptides that show differential selectivity for the beta5 site of the constitutively expressed proteasome and immunoproteasome in vitro and in B-cell lymphomas. Collectively, these studies describe the synthesis, activity and binding mode of a new series of non-covalent proteasome inhibitors with unprecedented potency and selectivity for the beta5 site, and which can discriminate between the constitutive proteasome and immunoproteasome in vitro and in cells.
Assuntos
Oligopeptídeos/farmacologia , Inibidores de Proteases/farmacologia , Inibidores de Proteassoma , Sequência de Aminoácidos , Sítios de Ligação , Ácidos Borônicos/farmacologia , Bortezomib , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Células HCT116 , Células HT29 , Humanos , Cinética , Luciferases/genética , Luciferases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , NF-kappa B/genética , NF-kappa B/metabolismo , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Pirazinas/farmacologia , Interferência de RNA , Homologia de Sequência de Aminoácidos , Ubiquitina/genética , Ubiquitina/metabolismoRESUMO
Multiple pathways have been proposed to explain how proteasome inhibition induces cell death, but mechanisms remain unclear. To approach this issue, we performed a genome-wide siRNA screen to evaluate the genetic determinants that confer sensitivity to bortezomib (Velcade (R); PS-341). This screen identified 100 genes whose knockdown affected lethality to bortezomib and to a structurally diverse set of other proteasome inhibitors. A comparison of three cell lines revealed that 39 of 100 genes were commonly linked to cell death. We causally linked bortezomib-induced cell death to the accumulation of ASF1B, Myc, ODC1, Noxa, BNIP3, Gadd45alpha, p-SMC1A, SREBF1, and p53. Our results suggest that proteasome inhibition promotes cell death primarily by dysregulating Myc and polyamines, interfering with protein translation, and disrupting essential DNA damage repair pathways, leading to programmed cell death.
Assuntos
Antineoplásicos/farmacologia , Ácidos Borônicos/farmacologia , Morte Celular/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Inibidores de Proteassoma , Pirazinas/farmacologia , RNA Interferente Pequeno/genética , Bortezomib , Morte Celular/genética , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Dano ao DNA , Técnicas de Silenciamento de Genes , Células HCT116 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas Proto-Oncogênicas c-myc/genética , Ribossomos/efeitos dos fármacos , Serina-Treonina Quinases TOR , TransfecçãoRESUMO
The proteasome was validated as an oncology target following the clinical success of VELCADE (bortezomib) for injection for the treatment of multiple myeloma and recurring mantle cell lymphoma. Consequently, several groups are pursuing the development of additional small-molecule proteasome inhibitors for both hematologic and solid tumor indications. Here, we describe MLN9708, a selective, orally bioavailable, second-generation proteasome inhibitor that is in phase I clinical development. MLN9708 has a shorter proteasome dissociation half-life and improved pharmacokinetics, pharmacodynamics, and antitumor activity compared with bortezomib. MLN9708 has a larger blood volume distribution at steady state, and analysis of 20S proteasome inhibition and markers of the unfolded protein response confirmed that MLN9708 has greater pharmacodynamic effects in tissues than bortezomib. MLN9708 showed activity in both solid tumor and hematologic preclinical xenograft models, and we found a correlation between greater pharmacodynamic responses and improved antitumor activity. Moreover, antitumor activity was shown via multiple dosing routes, including oral gavage. Taken together, these data support the clinical development of MLN9708 for both hematologic and solid tumor indications.
Assuntos
Compostos de Boro/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Glicina/análogos & derivados , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Inibidores de Proteassoma , Animais , Compostos de Boro/farmacocinética , Ácidos Borônicos/farmacologia , Bortezomib , Inibidores de Cisteína Proteinase/farmacocinética , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Glicina/farmacocinética , Glicina/farmacologia , Células HCT116 , Células HT29 , Humanos , Linfoma/tratamento farmacológico , Linfoma/enzimologia , Camundongos , Camundongos SCID , Complexo de Endopeptidases do Proteassoma/sangue , Pirazinas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Human angiotensin-converting enzyme-related carboxypeptidase (ACE2) is a zinc metalloprotease whose closest homolog is angiotensin I-converting enzyme. To begin to elucidate the physiological role of ACE2, ACE2 was purified, and its catalytic activity was characterized. ACE2 proteolytic activity has a pH optimum of 6.5 and is enhanced by monovalent anions, which is consistent with the activity of ACE. ACE2 activity is increased approximately 10-fold by Cl(-) and F(-) but is unaffected by Br(-). ACE2 was screened for hydrolytic activity against a panel of 126 biological peptides, using liquid chromatography-mass spectrometry detection. Eleven of the peptides were hydrolyzed by ACE2, and in each case, the proteolytic activity resulted in removal of the C-terminal residue only. ACE2 hydrolyzes three of the peptides with high catalytic efficiency: angiotensin II () (k(cat)/K(m) = 1.9 x 10(6) m(-1) s(-1)), apelin-13 (k(cat)/K(m) = 2.1 x 10(6) m(-1) s(-1)), and dynorphin A 1-13 (k(cat)/K(m) = 3.1 x 10(6) m(-1) s(-1)). The ACE2 catalytic efficiency is 400-fold higher with angiotensin II () as a substrate than with angiotensin I (). ACE2 also efficiently hydrolyzes des-Arg(9)-bradykinin (k(cat)/K(m) = 1.3 x 10(5) m(-1) s(-1)), but it does not hydrolyze bradykinin. An alignment of the ACE2 peptide substrates reveals a consensus sequence of: Pro-X((1-3 residues))-Pro-Hydrophobic, where hydrolysis occurs between proline and the hydrophobic amino acid.