RESUMO
Infant acute myeloid leukemia (AML) is a heterogeneous disease, genetically distinct from its adult counterpart. Chromosomal translocations involving the KMT2A gene (MLL) are especially common in affected infants of less than 1 year of age, and are associated with a dismal prognosis. While these rearrangements are likely to arise in utero, the cell of origin has not been conclusively identified. This knowledge could lead to a better understanding of the biology of the disease and support the identification of new therapeutic vulnerabilities. Over the last few years, important progress in understanding the dynamics of fetal hematopoiesis has been made. Several reports have highlighted how hematopoietic stem cells (HSC) provide little contribution to fetal hematopoiesis, which is instead largely sustained by HSC-independent progenitors. Here, we used conditional Cre-Lox transgenic mouse models to engineer the Mll-Af9 translocation in defined subsets of embryonic hematopoietic progenitors. We show that embryonic hematopoiesis is generally permissive for Mll-Af9-induced leukemic transformation. Surprisingly, the selective introduction of Mll-Af9 in HSC-independent progenitors generated a transplantable myeloid leukemia, whereas it did not when introduced in embryonic HSC-derived cells. Ex vivo engineering of the Mll-Af9 rearrangement in HSC-independent progenitors using a CRISPR/Cas9-based approach resulted in the activation of an aberrant myeloid-biased self-renewal program. Overall, our results demonstrate that HSC-independent hematopoietic progenitors represent a permissive environment for Mll-Af9-induced leukemic transformation, and can likely act as cells of origin of infant AML.
RESUMO
Metastasis is responsible for most breast cancer-related deaths; however, identifying the cellular determinants of metastasis has remained challenging. Here, we identified a minority population of immature THY1+/VEGFA+ tumor epithelial cells in human breast tumor biopsies that display angiogenic features and are marked by the expression of the oncogene, LMO2. Higher abundance of LMO2+ basal cells correlated with tumor endothelial content and predicted poor distant recurrence-free survival in patients. Using MMTV-PyMT/Lmo2CreERT2 mice, we demonstrated that Lmo2 lineage-traced cells integrate into the vasculature and have a higher propensity to metastasize. LMO2 knockdown in human breast tumors reduced lung metastasis by impairing intravasation, leading to a reduced frequency of circulating tumor cells. Mechanistically, we find that LMO2 binds to STAT3 and is required for STAT3 activation by tumor necrosis factor-α and interleukin-6. Collectively, our study identifies a population of metastasis-initiating cells with angiogenic features and establishes the LMO2-STAT3 signaling axis as a therapeutic target in breast cancer metastasis.
Assuntos
Neoplasias da Mama , Neoplasias Pulmonares , Humanos , Camundongos , Animais , Feminino , Neoplasias da Mama/patologia , Neoplasias Pulmonares/metabolismo , Transdução de Sinais , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismoRESUMO
The transcription factor complex, consisting of LMO2, TAL1 or LYL1, and GATA2, plays an important role in capillary sprouting by regulating VEGFR2, DLL4, and angiopoietin 2 in tip cells. Overexpression of the basic helix-loop-helix transcription factor LYL1 in transgenic mice results in shortened tails. This phenotype is associated with vessel hyperbranching and a relative paucity of straight vessels due to DLL4 downregulation in tip cells by forming aberrant complex consisting of LMO2 and LYL1. Knockdown of LMO2 or TAL1 inhibits capillary sprouting in spheroid-based angiogenesis assays, which is associated with decreased angiopoietin 2 secretion. In the same assay using mixed TAL1- and LYL1-expressing endothelial cells, TAL1 was found to be primarily located in tip cells, while LYL1-expressing cells tended to occupy the stalk position in sprouts by upregulating VEGFR1 than TAL1. Thus, the interaction between LMO2 and TAL1 in tip cells plays a key role in angiogenic switch of sprouting angiogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Angiopoietina-2 , Proteínas com Domínio LIM/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células Endoteliais , Proteínas com Domínio LIM/genética , Camundongos , Proteínas de Neoplasias/genética , Neovascularização Fisiológica/genética , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genéticaRESUMO
The SARS-CoV-2 receptor angiotensin converting enzyme 2 (ACE2) was previously engineered into a high affinity tetravalent format (ACE2-Fc-TD) that is a potential decoy protein in SARS-CoV-2 infection.We report that this protein shows greatly enhanced binding to SARS-CoV-2 spike proteins of the SARS-CoV-2 variants of concern B.1.1.7 (alpha variant, originally isolated in the United Kingdom) and B.1.351 (beta variant, originally isolated in South Africa) with picomolar compared with nanomolar Kd values. In addition, ACE2-Fc-TD displays greater neutralization of SARS-CoV-2 pseudotype viruses compared to a dimeric ACE2-Fc, with enhanced activity on variant B.1.351. This tetrameric decoy protein would be a valuable addition to SARS-CoV-2 therapeutic approaches, especially where vaccination cannot be used but also should there be any future coronavirus pandemics.
Assuntos
Enzima de Conversão de Angiotensina 2/farmacologia , Antivirais/metabolismo , COVID-19/prevenção & controle , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/química , COVID-19/enzimologia , COVID-19/virologia , Linhagem Celular , Humanos , Cinética , Mutação , Ligação Proteica , Domínios Proteicos , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/metabolismo , Tratamento Farmacológico da COVID-19RESUMO
Infection by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes COVID-19 disease. Therapeutic antibodies are being developed that interact with the viral spike proteins to limit viral infection of epithelium. We have applied a method to dramatically improve the performance of anti-SARS-CoV-2 antibodies by enhancing avidity through multimerization using simple engineering to yield tetrameric antibodies. We have re-engineered six anti-SARS-CoV-2 antibodies using the human p53 tetramerization domain, including three clinical trials antibodies casirivimab, imdevimab and etesevimab. The method yields tetrameric antibodies, termed quads, that retain efficient binding to the SARS-CoV-2 spike protein, show up to two orders of magnitude enhancement in neutralization of pseudovirus infection and retain potent interaction with virus variant of concern spike proteins. The tetramerization method is simple, general and its application is a powerful methodological development for SARS-CoV-2 antibodies that are currently in pre-clinical and clinical investigation.
Assuntos
SARS-CoV-2/metabolismo , Anticorpos de Cadeia Única/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Reações Antígeno-Anticorpo , COVID-19/virologia , Ensaio de Imunoadsorção Enzimática , Células HEK293 , Humanos , Testes de Neutralização , Domínios Proteicos , Multimerização Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/uso terapêutico , SARS-CoV-2/imunologia , SARS-CoV-2/isolamento & purificação , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/uso terapêutico , Ressonância de Plasmônio de Superfície , Proteína Supressora de Tumor p53/química , Tratamento Farmacológico da COVID-19RESUMO
Approaches are needed for therapy of the severe acute respiratory syndrome from SARS-CoV-2 coronavirus (COVID-19). Interfering with the interaction of viral antigens with the angiotensin converting enzyme 2 (ACE-2) receptor is a promising strategy by blocking the infection of the coronaviruses into human cells. We have implemented a novel protein engineering technology to produce a super-potent tetravalent form of ACE2, coupled to the human immunoglobulin γ1 Fc region, using a self-assembling, tetramerization domain from p53 protein. This high molecular weight Quad protein (ACE2-Fc-TD) retains binding to the SARS-CoV-2 receptor binding spike protein and can form a complex with the spike protein plus anti-viral antibodies. The ACE2-Fc-TD acts as a powerful decoy protein that out-performs soluble monomeric and dimeric ACE2 proteins and blocks both SARS-CoV-2 pseudovirus and SARS-CoV-2 virus infection with greatly enhanced efficacy. The ACE2 tetrameric protein complex promise to be important for development as decoy therapeutic proteins against COVID-19. In contrast to monoclonal antibodies, ACE2 decoy is unlikely to be affected by mutations in SARS-CoV-2 that are beginning to appear in variant forms. In addition, ACE2 multimeric proteins will be available as therapeutic proteins should new coronaviruses appear in the future because these are likely to interact with ACE2 receptor.
Assuntos
Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/farmacologia , Antivirais/metabolismo , COVID-19/prevenção & controle , Engenharia de Proteínas/métodos , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Antivirais/química , COVID-19/enzimologia , COVID-19/virologia , Linhagem Celular , Desenho de Fármacos , Haplorrinos , Humanos , Ligação Proteica , Elementos Estruturais de Proteínas , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Tratamento Farmacológico da COVID-19RESUMO
Intracellular antibodies are tools that can be used directly for target validation by interfering with properties like protein-protein interactions. An alternative use of intracellular antibodies in drug discovery is developing small-molecule surrogates using antibody-derived (Abd) technology. We previously used this strategy with an in vitro competitive surface plasmon resonance method that relied on high-affinity antibody fragments to obtain RAS-binding compounds. We now describe a novel implementation of the Abd method with a cell-based intracellular antibody-guided screening method that we have applied to the chromosomal translocation protein LMO2. We have identified a chemical series of anti-LMO2 Abd compounds that bind at the same LMO2 location as the inhibitory anti-LMO2 intracellular antibody combining site. Intracellular antibodies could therefore be used in cell-based screens to identify chemical surrogates of their binding sites and potentially be applied to any challenging proteins, such as transcription factors that have been considered undruggable.
Assuntos
Anticorpos , Translocação Genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sítios de Ligação , Sítios de Ligação de Anticorpos , Humanos , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/genéticaRESUMO
Protein-based affinity reagents (like antibodies or alternative binding scaffolds) offer wide-ranging applications for basic research and therapeutic approaches. However, whereas small chemical molecules efficiently reach intracellular targets, the delivery of macromolecules into the cytosol of cells remains a major challenge; thus cytosolic applications of protein-based reagents are rather limited. Some pathogenic bacteria have evolved a conserved type III secretion system (T3SS) which allows the delivery of effector proteins into eukaryotic cells. Here, we enhance the T3SS of an avirulent strain of Salmonella typhimurium to reproducibly deliver multiple classes of recombinant proteins into eukaryotic cells. The efficacy of the system is probed with both DARPins and monobodies to functionally inhibit the paradigmatic and largely undruggable RAS signaling pathway. Thus, we develop a bacterial secretion system for potent cytosolic delivery of therapeutic macromolecules.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Citosol/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Células HCT116 , Células HeLa , Humanos , Proteínas de Neoplasias/genética , Proteínas Recombinantes/genética , Salmonella typhimurium/genética , Salmonella typhimurium/crescimento & desenvolvimento , Sistemas de Secreção Tipo III/genéticaRESUMO
In drug development programmes, multiple assays are needed for the determination of protein-compound interactions and evaluation of potential use in assays with protein-protein interactions. In this protocol we describe the waterLOGSY NMR method for confirming protein-ligand binding events.
RESUMO
RHO (Ras HOmologous) GTPases are molecular switches that activate, in their state bound to Guanosine triphosphate (GTP), key signaling pathways, which involve actin cytoskeleton dynamics. Previously, we selected the nanobody RH12, from a synthetic phage display library, which binds the GTP-bound active conformation of RHOA (Ras Homologous family member A). However, when expressed as an intracellular antibody, its blocking effect on RHO signaling led to a loss of actin fibers, which in turn affected cell shape and cell survival. Here, in order to engineer an intracellular biosensor of RHOA-GTP activation, we screened the same phage nanobody library and identified another RHO-GTP selective intracellular nanobody, but with no apparent toxicity. The recombinant RH57 nanobody displays high affinity towards GTP-bound RHOA/B/C subgroup of small GTPases in vitro. Intracellular expression of the RH57 allowed selective co-precipitation with the GTP-bound state of the endogenous RHOA subfamily. When expressed as a fluorescent fusion protein, the chromobody GFP-RH57 was localized to the inner plasma membrane upon stimulation of the activation of endogenous RHO. Finally, the RH57 nanobody was used to establish a BRET-based biosensor (Bioluminescence Resonance Energy Transfer) of RHO activation. The dynamic range of the BRET signal could potentially offer new opportunities to develop cell-based screening of RHOA subfamily activation modulators.
RESUMO
Inhibiting the RAS oncogenic protein has largely been through targeting the switch regions that interact with signalling effector proteins. Here, we report designed ankyrin repeat proteins (DARPins) macromolecules that specifically inhibit the KRAS isoform by binding to an allosteric site encompassing the region around KRAS-specific residue histidine 95 at the helix α3/loop 7/helix α4 interface. We show that these DARPins specifically inhibit KRAS/effector interactions and the dependent downstream signalling pathways in cancer cells. Binding by the DARPins at that region influences KRAS/effector interactions in different ways, including KRAS nucleotide exchange and inhibiting KRAS dimerization at the plasma membrane. These results highlight the importance of targeting the α3/loop 7/α4 interface, a previously untargeted site in RAS, for specifically inhibiting KRAS function.
Assuntos
Sítio Alostérico/efeitos dos fármacos , Antineoplásicos/farmacologia , Desenho de Fármacos , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Repetição de Anquirina , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Histidina/metabolismo , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias/genética , Neoplasias/patologia , Biblioteca de Peptídeos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Many tumour causing proteins, such as those expressed after chromosomal translocations or from point mutations, are intracellular and are not enzymes per se amenable to conventional drug targeting. We previously demonstrated an approach (Antibody-antigen Interaction Dependent Apoptosis (AIDA)) whereby a single anti-ß-galactosidase intracellular single chain Fv antibody fragment, fused to inactive procaspase-3, induced auto-activation of caspase-3 after binding to the tetrameric ß-galactosidase protein. We now demonstrate that co-expressing an anti-RAS heavy chain single VH domain, that binds to mutant RAS several thousand times more strongly than to wild type RAS, with a complementary light chain VL domain, caused programmed cell death (PCD) in mutant RAS expressing cells when each variable region is fused to procaspase-3. The effect requires binding of both anti-RAS variable region fragments and is RAS-specific, producing a tri-molecular complex that auto-activates the caspase pathway leading to cell death. AIDA can be generally applicable for any target protein inside cells by involving appropriate pairs of antigen-specific intracellular antibodies.
Assuntos
Antígenos de Neoplasias/imunologia , Antineoplásicos Imunológicos , Caspase 3 , Neoplasias/tratamento farmacológico , Proteínas Recombinantes de Fusão , Anticorpos de Cadeia Única , Antineoplásicos Imunológicos/imunologia , Antineoplásicos Imunológicos/farmacologia , Caspase 3/imunologia , Caspase 3/farmacologia , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Linhagem Celular Tumoral , Humanos , Neoplasias/imunologia , Neoplasias/patologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacologiaRESUMO
The surfaceome is critical because surface proteins provide a gateway for internal signals and transfer of molecules into cells, and surfaceome differences can influence therapy response. We have used a surfaceome analysis method, based on comparing RNA-seq data between normal and abnormal cells (Surfaceome DataBase Mining or Surfaceome DBM), to identify sets of upregulated cell surface protein mRNAs in an LMO2-mediated T-ALL mouse model and corroborated by protein detection using antibodies. In this model the leukemia initiating cells (LICs) comprise pre-leukaemic, differentiation inhibited thymocytes allowing us to provide a profile of the LIC surfaceome in which GPR56, CD53 and CD59a are co-expressed with CD25. Implementation of cell surface interaction assays demonstrates fluid interaction of surface proteins and CD25 is only internalized when co-localized with other proteins. The Surfaceome DBM approach to analyse cancer cell surfaceomes is a way to find targetable surface biomarkers for clinical conditions where RNA-seq data from normal and abnormal cell are available.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biomarcadores Tumorais/metabolismo , Proteínas com Domínio LIM/metabolismo , Leucemia Linfoide/genética , Proteínas Proto-Oncogênicas/metabolismo , Transcriptoma , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Biomarcadores Tumorais/genética , Antígenos CD59/genética , Antígenos CD59/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Proteínas com Domínio LIM/genética , Leucemia Linfoide/metabolismo , Leucemia Linfoide/patologia , Camundongos , Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas/genética , RNA-Seq , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Tetraspanina 25/genética , Tetraspanina 25/metabolismoRESUMO
Protein-protein interactions (PPIs) are principle biological processes that control normal cell growth, differentiation, and homeostasis but are also crucial in diseases such as malignancy, neuropathy, and infection. Despite the importance of PPIs in biology, this target class has been very challenging to convert to therapeutics. In the last decade, much progress has been made in the inhibition of PPIs involved in diseases, but many remain difficult such as RAS-effector interactions in cancers. We describe here a protocol for using Bioluminescence Resonance Energy Transfer 2 (BRET2)-based RAS biosensors to detect and characterize RAS PPI inhibition by macromolecules and small molecules. This method could be extended to any other small GTPases or any other PPIs of interest. © 2019 by John Wiley & Sons, Inc.
Assuntos
Transferência de Energia , Medições Luminescentes/métodos , Proteínas ras/análise , Técnicas Biossensoriais , Células HEK293 , Humanos , Immunoblotting , Engenharia de ProteínasRESUMO
Cellular membranes are, in general, impermeable to macromolecules (herein referred to as macrodrugs, e.g., recombinant protein, expression plasmids, or mRNA), which is a major barrier for clinical translation of macrodrug-based therapies. Encapsulation of macromolecules in lipid nanoparticles (LNPs) can protect the therapeutic agent during transport through the body and facilitate the intracellular delivery via a fusion-based pathway. Furthermore, designing LNPs responsive to stimuli can make their delivery more localized, thus limiting the side effects. However, the principles and criteria for designing such nanoparticles remain unclear. We show that the thermodynamic state of the lipid membrane of the nanoparticle is a key design principle for acoustically responsive fusogenic nanoparticles. We have optimized a cationic LNP (designated LNPLH) with two different phase transitions near physiological conditions for delivering mRNA. A bicistronic mRNA encoding a single domain intracellular antibody fragment and green fluorescent protein (GFP) was introduced into a range of human cancer cell types using LNPLH, and the protein expression was measured via fluorescence corresponding to the GFP expression. The LNPLH/mRNA complex demonstrated low toxicity and high delivery, which was significantly enhanced when the transfection occurred in the presence of acoustic shock waves. The results suggest that the thermodynamic state of LNPs provides an important criterion for stimulus responsive fusogenic nanoparticles to deliver macrodrugs to the inside of cells.
Assuntos
Lipídeos/química , Nanopartículas/química , RNA Mensageiro/química , Transfecção/métodos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Ondas de Choque de Alta Energia , Humanos , Microscopia de Fluorescência , Nanopartículas/toxicidade , RNA Mensageiro/metabolismoRESUMO
The RAS gene family is frequently mutated in human cancers, and the quest for compounds that bind to mutant RAS remains a major goal, as it also does for inhibitors of protein-protein interactions. We have refined crystallization conditions for KRAS169Q61H-yielding crystals suitable for soaking with compounds and exploited this to assess new RAS-binding compounds selected by screening a protein-protein interaction-focused compound library using surface plasmon resonance. Two compounds, referred to as PPIN-1 and PPIN-2, with related structures from 30 initial RAS binders showed binding to a pocket where compounds had been previously developed, including RAS effector protein-protein interaction inhibitors selected using an intracellular antibody fragment (called Abd compounds). Unlike the Abd series of RAS binders, PPIN-1 and PPIN-2 compounds were not competed by the inhibitory anti-RAS intracellular antibody fragment and did not show any RAS-effector inhibition properties. By fusing the common, anchoring part from the two new compounds with the inhibitory substituents of the Abd series, we have created a set of compounds that inhibit RAS-effector interactions with increased potency. These fused compounds add to the growing catalog of RAS protein-protein inhibitors and show that building a chemical series by crossing over two chemical series is a strategy to create RAS-binding small molecules.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Proteína Oncogênica p21(ras)/antagonistas & inibidores , Cristalografia por Raios X , Desenvolvimento de Medicamentos , Estrutura Molecular , Proteína Oncogênica p21(ras)/metabolismo , Ligação Proteica , Ressonância de Plasmônio de SuperfícieRESUMO
Targeting specific protein-protein interactions (PPIs) is an attractive concept for drug development, but hard to implement since intracellular antibodies do not penetrate cells and most small-molecule drugs are considered unsuitable for PPI inhibition. A potential solution to these problems is to select intracellular antibody fragments to block PPIs, use these antibody fragments for target validation in disease models and finally derive small molecules overlapping the antibody-binding site. Here, we explore this strategy using an anti-mutant RAS antibody fragment as a competitor in a small-molecule library screen for identifying RAS-binding compounds. The initial hits are optimized by structure-based design, resulting in potent RAS-binding compounds that interact with RAS inside the cells, prevent RAS-effector interactions and inhibit endogenous RAS-dependent signalling. Our results may aid RAS-dependent cancer drug development and demonstrate a general concept for developing small compounds to replace intracellular antibody fragments, enabling rational drug development to target validated PPIs.
Assuntos
Sítios de Ligação de Anticorpos , Fragmentos de Imunoglobulinas/química , Transdução de Sinais , Anticorpos/química , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Cristalografia por Raios X , Células HEK293 , Humanos , Mutação , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Bibliotecas de Moléculas Pequenas , Ressonância de Plasmônio de Superfície , Proteínas ras/químicaRESUMO
The RAS family of proteins is amongst the most highly mutated in human cancers and has so far eluded drug therapy. Currently, much effort is being made to discover mutant RAS inhibitors and in vitro screening for RAS-binding drugs must be followed by cell-based assays. Here, we have developed a robust set of bioluminescence resonance energy transfer (BRET)-based RAS biosensors that enable monitoring of RAS-effector interaction inhibition in living cells. These include KRAS, HRAS and NRAS and a variety of different mutations that mirror those found in human cancers with the major RAS effectors such as CRAF, PI3K and RALGDS. We highlighted the utility of these RAS biosensors by showing a RAS-binding compound is a potent pan-RAS-effector interactions inhibitor in cells. The RAS biosensors represent a useful tool to investigate and characterize the potency of anti-RAS inhibitors in cells and more generally any RAS protein-protein interaction (PPI) in cells.
Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Técnicas Biossensoriais/métodos , Mutação , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Transferência de Energia , Células HEK293 , Humanos , Ligação Proteica , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de SinaisRESUMO
Lens epithelium-derived growth factor (LEDGF)/p75 is the dominant binding partner of HIV-1 integrase in human cells. The crystal structure of the HIV integrase-binding domain (IBD) of LEDGF has been determined in the absence of ligand. IBD was overexpressed in Escherichia coli, purified and crystallized by sitting-drop vapour diffusion. X-ray diffraction data were collected at Diamond Light Source to a resolution of 2.05â Å. The crystals belonged to space group P21, with eight polypeptide chains in the asymmetric unit arranged as an unusual octamer composed of four domain-swapped IBD dimers. IBD exists as a mixture of monomers and dimers in concentrated solutions, but the dimers are unlikely to be biologically relevant.