RESUMEN
The 13 Hsp70 proteins in humans act on unique sets of substrates with diversity often being attributed to J-domain-containing protein (Hsp40 or JDP) cofactors. We were therefore surprised to find drastically different binding affinities for Hsp70-peptide substrates, leading us to probe substrate specificity among the 8 canonical Hsp70s from humans. We used peptide arrays to characterize Hsp70 binding and then mined these data using machine learning to develop an algorithm for isoform-specific prediction of Hsp70 binding sequences. The results of this algorithm revealed recognition patterns not predicted based on local sequence alignments. We then showed that none of the human isoforms can complement heat-shocked DnaK knockout Escherichia coli cells. However, chimeric Hsp70s consisting of the human nucleotide-binding domain and the substrate-binding domain of DnaK complement during heat shock, providing further evidence in vivo of the divergent function of the Hsp70 substrate-binding domains. We also demonstrated that the differences in heat shock complementation among the chimeras are not due to loss of DnaJ binding. Although we do not exclude JDPs as additional specificity factors, our data demonstrate substrate specificity among the Hsp70s, which has important implications for inhibitor development in cancer and neurodegeneration.
Asunto(s)
Proteínas de Escherichia coli , Proteínas de Choque Térmico , Humanos , Proteínas de Choque Térmico/metabolismo , Proteínas de Escherichia coli/química , Sitios de Unión , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Péptidos/metabolismo , Unión ProteicaRESUMEN
We recently reported on small-molecule inhibitors of the GroES/GroEL chaperone system as potential antibiotics against Escherichia coli and the ESKAPE pathogens but were unable to establish GroES/GroEL as the cellular target, leading to cell death. In this study, using two of our most potent bis-sulfonamido-2-phenylbenzoxazoles (PBZs), we established the binding site of the PBZ molecules using cryo-EM and found that GroEL was the cellular target responsible for the mode of action. Cryo-EM revealed that PBZ1587 binds at the GroEL ring-ring interface (RRI). A cellular reporter assay confirmed that PBZ1587 engaged GroEL in cells, but cellular rescue experiments showed potential off-target effects. This prompted us to explore a closely related analogue, PBZ1038, which is also bound to the RRI. Biochemical characterization showed potent inhibition of Gram-negative chaperonins but much lower potency of chaperonin from a Gram-positive organism, Enterococcus faecium. A cellular reporter assay showed that PBZ1038 also engaged GroEL in cells and that the cytotoxic phenotype could be rescued by a chromosomal copy of E. faecium GroEL/GroES or by expressing a recalcitrant RRI mutant. These data argue that PBZ1038's antimicrobial action is exerted through inhibition of GroES/GroEL, validating this chaperone system as an antibiotic target.
Asunto(s)
Antibacterianos , Chaperonina 10 , Escherichia coli , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Chaperonina 10/metabolismo , Chaperonina 10/antagonistas & inhibidores , Chaperonina 10/química , Escherichia coli/efectos de los fármacos , Chaperonina 60/metabolismo , Chaperonina 60/antagonistas & inhibidores , Chaperonina 60/química , Benzoxazoles/química , Benzoxazoles/farmacología , Benzoxazoles/síntesis química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/químicaRESUMEN
GroES/GroEL is the only bacterial chaperone essential under all conditions, making it a potential antibiotic target. Rationally targeting ESKAPE GroES/GroEL as an antibiotic strategy necessitates studying their structure and function. Herein, we outline the structural similarities between Escherichia coli and ESKAPE GroES/GroEL and identify significant differences in intra- and inter-ring cooperativity, required in the refolding cycle of client polypeptides. Previously, we observed that one-half of ESKAPE GroES/GroEL family members could not support cell viability when each was individually expressed in GroES/GroEL-deficient E. coli cells. Cell viability was found to be dependent on the allosteric compatibility between ESKAPE and E. coli subunits within mixed (E. coli and ESKAPE) tetradecameric GroEL complexes. Interestingly, differences in allostery did not necessarily result in differences in refolding rate for a given homotetradecameric chaperonin. Characterization of ESKAPE GroEL allostery, ATPase, and refolding rates in this study will serve to inform future studies focused on inhibitor design and mechanism of action studies.
Asunto(s)
Sitio Alostérico , Proteínas de Escherichia coli/química , Proteínas de Choque Térmico/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Chaperonina 10/química , Chaperonina 10/genética , Chaperonina 10/metabolismo , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismoRESUMEN
NRF2 is a basic leucine zipper (bZip) transcription factor that is the master regulator of redox homeostasis. Under basal conditions, the cellular level of NRF2 is low due to a posttranslational regulation by the ubiquitin proteasome system (UPS). But, when an organism is challenged with oxidative or xenobiotic stress, the NRF2 pathway is activated by inhibition of the E3 ubiquitin ligase complex that normally marks NRF2 for destruction. For several decades, researchers have searched for molecules that can intentionally activate NRF2, as this was shown to be a means to prevent certain diseases, at least in animal models. In the present era, there are many compounds known to activate the NRF2 pathway including natural products and synthetic compounds, covalent and non-covalent compounds, and others. However, it was also revealed that like many protective pathways, the NRF2 pathway has a dark side. Just as NRF2 can protect normal cells from damage, it can protect malignant cells from damage. As cells transform, they are exposed to many stressors and aberrant upregulation of NRF2 can facilitate transformation and it can help cancer cells to grow, to spread, and to resist treatment. For this reason, researchers are also interested in the discovery and development of NRF2 inhibitors. In the present review, we will begin with a general discussion of NRF2 structure and function, we will discuss the latest in NRF2 non-covalent activators, and we will discuss the current state of NRF2 inhibitors.
Asunto(s)
Terapia Molecular Dirigida/métodos , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Neoplasias , Animales , HumanosRESUMEN
While many studies have established the importance of protein homeostasis in tumor progression, little effort has been made to examine the therapeutic potential of targeting the HSP60 chaperonin system. In healthy cells, HSP60 is localized to the mitochondrial matrix; however, emerging evidence indicates HSP60 can be over-expressed and mis-localized to the cytosol of cancer cells, which is hypothesized to promote tumor cell survival and proliferation. This opens a potential avenue to selectively target the aberrant HSP60 in the cytosol as a chemotherapeutic strategy. In the present work, we examined a series of bis-aryl-α,ß-unsaturated ketone (ABK) HSP60 inhibitors for their ability to selectively target cancerous vs non-cancerous colon and intestine cells. We found that lead analogs inhibited migration and clonogenicity of cancer cells, with cytotoxicity correlating with the level of aberrant HSP60 in the cytosol.
RESUMEN
OBJECTIVE: This study exploits the intersection between molecular-targeted therapies and immune-checkpoint inhibition to define new means to treat pancreatic cancer. DESIGN: Patient-derived cell lines and xenograft models were used to define the response to CDK4/6 and MEK inhibition in the tumour compartment. Impacts relative to immunotherapy were performed using subcutaneous and orthotopic syngeneic models. Single-cell RNA sequencing and multispectral imaging were employed to delineate effects on the immunological milieu in the tumour microenvironment. RESULTS: We found that combination treatment with MEK and CDK4/6 inhibitors was effective across a broad range of PDX models in delaying tumour progression. These effects were associated with stable cell-cycle arrest, as well as the induction of multiple genes associated with interferon response and antigen presentation in an RB-dependent fashion. Using single-cell sequencing and complementary approaches, we found that the combination of CDK4/6 and MEK inhibition had a significant impact on increasing T-cell infiltration and altering myeloid populations, while potently cooperating with immune checkpoint inhibitors. CONCLUSIONS: Together, these data indicate that there are canonical and non-canonical features of CDK4/6 and MEK inhibition that impact on the tumour and immune microenvironment. This combination-targeted treatment can promote robust tumour control in combination with immune checkpoint inhibitor therapy.
Asunto(s)
Carcinoma Ductal Pancreático/terapia , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Terapia Molecular Dirigida , Neoplasias Pancreáticas/terapia , Animales , Técnicas de Cultivo de Célula , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Modelos Animales de Enfermedad , Humanos , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Native mass spectrometry (MS) has become a versatile tool for characterizing high-mass complexes and measuring biomolecular interactions. Native MS usually requires the resolution of different charge states produced by electrospray ionization to measure the mass, which is difficult for highly heterogeneous samples that have overlapping and unresolvable charge states. Charge detection-mass spectrometry (CD-MS) seeks to address this challenge by simultaneously measuring the charge and m/z for isolated ions. However, CD-MS often shows uncertainty in the charge measurement that limits the resolution. To overcome this charge state uncertainty, we developed UniDecCD (UCD) software for computational deconvolution of CD-MS data, which significantly improves the resolution of CD-MS data. Here, we describe the UCD algorithm and demonstrate its ability to improve the CD-MS resolution of proteins, megadalton viral capsids, and heterogeneous nanodiscs made from natural lipid extracts. UCD provides a user-friendly interface that will increase the accessibility of CD-MS technology and provide a valuable new computational tool for CD-MS data analysis.
Asunto(s)
Proteínas , Espectrometría de Masa por Ionización de Electrospray , Algoritmos , Iones , Programas InformáticosRESUMEN
Over the past few decades, an increasing variety of molecular chaperones have been investigated for their role in tumorigenesis and as potential chemotherapeutic targets; however, the 60 kDa Heat Shock Protein (HSP60), along with its HSP10 co-chaperone, have received little attention in this regard. In the present study, we investigated two series of our previously developed inhibitors of the bacterial homolog of HSP60/10, called GroEL/ES, for their selective cytotoxicity to cancerous over non-cancerous colorectal cells. We further developed a third "hybrid" series of analogs to identify new candidates with superior properties than the two parent scaffolds. Using a series of well-established HSP60/10 biochemical screens and cell-viability assays, we identified 24 inhibitors (14%) that exhibited > 3-fold selectivity for targeting colorectal cancer over non-cancerous cells. Notably, cell viability EC50 results correlated with the relative expression of HSP60 in the mitochondria, suggesting a potential for this HSP60-targeting chemotherapeutic strategy as emerging evidence indicates that HSP60 is up-regulated in colorectal cancer tumors. Further examination of five lead candidates indicated their ability to inhibit the clonogenicity and migration of colorectal cancer cells. These promising results are the most thorough analysis and first reported instance of HSP60/10 inhibitors being able to selectively target colorectal cancer cells and highlight the potential of the HSP60/10 chaperonin system as a viable chemotherapeutic target.
Asunto(s)
Antineoplásicos/farmacología , Benzoxazoles/farmacología , Chaperonina 10/antagonistas & inhibidores , Chaperonina 60/antagonistas & inhibidores , Neoplasias Colorrectales/tratamiento farmacológico , Salicilanilidas/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Benzoxazoles/síntesis química , Benzoxazoles/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Salicilanilidas/síntesis química , Salicilanilidas/química , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
In two previous studies, we identified compound 1 as a moderate GroEL/ES inhibitor with weak to moderate antibacterial activity against Gram-positive and Gram-negative bacteria including Bacillus subtilis, methicillin-resistant Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, and SM101 Escherichia coli (which has a compromised lipopolysaccharide biosynthetic pathway making bacteria more permeable to drugs). Extending from those studies, we developed two series of analogs with key substructures resembling those of known antibacterials, nitroxoline (hydroxyquinoline moiety) and nifuroxazide/nitrofurantoin (bis-cyclic-N-acylhydrazone scaffolds). Through biochemical and cell-based assays, we identified potent GroEL/ES inhibitors that selectively blocked E. faecium, S. aureus, and E. coli proliferation with low cytotoxicity to human colon and intestine cells in vitro. Initially, only the hydroxyquinoline-bearing analogs were found to be potent inhibitors in our GroEL/ES-mediated substrate refolding assays; however, subsequent testing in the presence of an E. coli nitroreductase (NfsB) in situ indicated that metabolites of the nitrofuran-bearing analogs were potent GroEL/ES inhibitor pro-drugs. Consequently, this study has identified a new target of nitrofuran-containing drugs, and is the first reported instance of such a unique class of GroEL/ES chaperonin inhibitors. The intriguing results presented herein provide impetus for expanded studies to validate inhibitor mechanisms and optimize this antibacterial class using the respective GroEL/ES chaperonin systems and nitroreductases from E. coli and the ESKAPE bacteria.
Asunto(s)
Antibacterianos/farmacología , Chaperonina 60/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Nitrofuranos/farmacología , Profármacos/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Chaperonina 60/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Bacterias Gramnegativas/metabolismo , Bacterias Grampositivas/metabolismo , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Nitrofuranos/síntesis química , Nitrofuranos/química , Profármacos/síntesis química , Profármacos/química , Relación Estructura-ActividadRESUMEN
All living organisms contain a unique class of molecular chaperones called 60â¯kDa heat shock proteins (HSP60 - also known as GroEL in bacteria). While some organisms contain more than one HSP60 or GroEL isoform, at least one isoform has always proven to be essential. Because of this, we have been investigating targeting HSP60 and GroEL chaperonin systems as an antibiotic strategy. Our initial studies focused on applying this antibiotic strategy for treating African sleeping sickness (caused by Trypanosoma brucei parasites) and drug-resistant bacterial infections (in particular Methicillin-resistant Staphylococcus aureus - MRSA). Intriguingly, during our studies we found that three known antibiotics - suramin, closantel, and rafoxanide - were potent inhibitors of bacterial GroEL and human HSP60 chaperonin systems. These findings prompted us to explore what other approved drugs, natural products, and known bioactive molecules might also inhibit HSP60 and GroEL chaperonin systems. Initial high-throughput screening of 3680 approved drugs, natural products, and known bioactives identified 161 hit inhibitors of the Escherichia coli GroEL chaperonin system (4.3% hit rate). From a purchased subset of 60 hits, 29 compounds (48%) re-confirmed as selective GroEL inhibitors in our assays, all of which were nearly equipotent against human HSP60. These findings illuminate the notion that targeting chaperonin systems might be a more common occurrence than we previously appreciated. Future studies are needed to determine if the in vivo modes of action of these approved drugs, natural products, and known bioactive molecules are related to GroEL and HSP60 inhibition.
Asunto(s)
Productos Biológicos/química , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Rafoxanida/química , Salicilanilidas/química , Suramina/química , Productos Biológicos/metabolismo , Chaperonina 10/antagonistas & inhibidores , Chaperonina 60/antagonistas & inhibidores , Escherichia coli/metabolismo , Humanos , Concentración 50 Inhibidora , Pliegue de Proteína , Rafoxanida/metabolismo , Salicilanilidas/metabolismo , Suramina/metabolismoRESUMEN
Current treatments for Mycobacterium tuberculosis infections require long and complicated regimens that can lead to patient non-compliance, increasing incidences of antibiotic-resistant strains, and lack of efficacy against latent stages of disease. Thus, new therapeutics are needed to improve tuberculosis standard of care. One strategy is to target protein homeostasis pathways by inhibiting molecular chaperones such as GroEL/ES (HSP60/10) chaperonin systems. M. tuberculosis has two GroEL homologs: GroEL1 is not essential but is important for cytokine-dependent granuloma formation, while GroEL2 is essential for survival and likely functions as the canonical housekeeping chaperonin for folding proteins. Another strategy is to target the protein tyrosine phosphatase B (PtpB) virulence factor that M. tuberculosis secretes into host cells to help evade immune responses. In the present study, we have identified a series of GroEL/ES inhibitors that inhibit M. tuberculosis growth in liquid culture and biochemical function of PtpB in vitro. With further optimization, such dual-targeting GroEL/ES and PtpB inhibitors could be effective against all stages of tuberculosis - actively replicating bacteria, bacteria evading host cell immune responses, and granuloma formation in latent disease - which would be a significant advance to augment current therapeutics that primarily target actively replicating bacteria.
Asunto(s)
Chaperonina 60/uso terapéutico , Mycobacterium tuberculosis/patogenicidad , Tuberculosis/tratamiento farmacológico , Proteínas Bacterianas/metabolismo , Chaperonina 60/farmacología , Humanos , Modelos Moleculares , PolifarmacologíaRESUMEN
Glucose-regulated protein 78 (GRP78) is the ER resident 70â¯kDa heat shock protein 70 (HSP70) and has been hypothesized to be a therapeutic target for various forms of cancer due to its role in mitigating proteotoxic stress in the ER, its elevated expression in some cancers, and the correlation between high levels for GRP78 and a poor prognosis. Herein we report the development and use of a high throughput fluorescence polarization-based peptide binding assay as an initial step toward the discovery and development of GRP78 inhibitors. This assay was used in a pilot screen to discover the anti-infective agent, hexachlorophene, as an inhibitor of GRP78. Through biochemical characterization we show that hexachlorophene is a competitive inhibitor of the GRP78-peptide interaction. Biological investigations showed that this molecule induces the unfolded protein response, induces autophagy, and leads to apoptosis in a colon carcinoma cell model, which is known to be sensitive to GRP78 inhibition.
Asunto(s)
Proteínas HSP70 de Choque Térmico/efectos de los fármacos , Hexaclorofeno/uso terapéutico , Ensayos Analíticos de Alto Rendimiento/métodos , Chaperón BiP del Retículo Endoplásmico , Hexaclorofeno/farmacología , HumanosRESUMEN
A Na2HPO4-catalyzed four-component reaction between a ketone, malononitrile, S8 and formamide has been realized for the first time. This reaction provides a concise approach to thieno[2,3-d]pyrimidin-4-amines, previously requiring 5 steps. The utility of this reaction was validated by preparing a multi-targeted kinase inhibitor and an inhibitor of the NRF2 pathway with excellent atom- and step-economy.
RESUMEN
A pulldown using a biotinylated natural product of interest in the 17ß-hydroxywithanolide (17-BHW) class, physachenolide C (PCC), identified the bromodomain and extra-terminal domain (BET) family of proteins (BRD2, BRD3, and BRD4), readers of acetyl-lysine modifications and regulators of gene transcription, as potential cellular targets. BROMOscan bromodomain profiling and biochemical assays support PCC as a BET inhibitor with increased selectivity for bromodomain (BD)-1 of BRD3 and BRD4, and X-ray crystallography and NMR studies uncovered specific contacts that underlie the potency and selectivity of PCC toward BRD3-BD1 over BRD3-BD2. PCC also displays characteristics of a molecular glue, facilitating proteasome-mediated degradation of BRD3 and BRD4. Finally, PCC is more potent than other withanolide analogues and gold-standard pan-BET inhibitor (+)-JQ1 in cytotoxicity assays across five prostate cancer (PC) cell lines regardless of androgen receptor (AR)-signaling status.
Asunto(s)
Factores de Transcripción , Witanólidos , Masculino , Humanos , Proteínas Nucleares , Dominios Proteicos , Proteínas de Ciclo CelularRESUMEN
A recent study illustrated that a fluorescence polarization assay can be used to identify substrate-competitive Hsp70 inhibitors that can be isoform-selective. Herein, we use that assay in a moderate-throughput screen and report the discovery of a druglike amino-acid-based inhibitor with reasonable specificity for the endoplasmic reticular Hsp70, Grp78. Using traditional medicinal chemistry approaches, the potency and selectivity were further optimized through structure-activity relationship (SAR) studies in parallel assays for six of the human Hsp70 isoforms. The top compounds were all tested against a panel of cancer cell lines and disappointingly showed little effect. The top-performing compound, 8, was retested using a series of endoplasmic reticulum (ER) stress-inducing agents and found to synergize with these agents. Finally, 8 was tested in a spheroid tumor model and found to be more potent than in two-dimensional models. The optimized Grp78 inhibitors are the first reported isoform-selective small-molecule-competitive inhibitors of an Hsp70-substrate interaction.
Asunto(s)
Chaperón BiP del Retículo Endoplásmico , Proteínas de Choque Térmico , Humanos , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/farmacología , Chaperonas Moleculares/química , Proteínas HSP70 de Choque Térmico , Estrés del Retículo Endoplásmico , Isoformas de ProteínasRESUMEN
The identification of modulators for proteins without assayable biochemical activity remains a challenge in chemical biology. The presented approach adapts a high-throughput fluorescence binding assay and functional chromatography, two protein-resin technologies, enabling the discovery and isolation of fluorescent natural product probes that target proteins independently of biochemical function. The resulting probes also suggest targetable pockets for lead discovery. Using human survivin as a model, we demonstrate this method with the discovery of members of the prodiginine family as fluorescent probes to the cancer target survivin.
RESUMEN
Increased protein synthesis is a requirement for malignant growth, and as a result, translation has become a pharmaceutical target for cancer. The initiation of cap-dependent translation is enzymatically driven by the eukaryotic initiation factor (eIF)4A, an ATP-powered DEAD-box RNA-helicase that unwinds the messenger RNA secondary structure upstream of the start codon, enabling translation of downstream genes. A screen for inhibitors of eIF4A ATPase activity produced an intriguing hit that, surprisingly, was not ATP-competitive. A medicinal chemistry campaign produced the novel eIF4A inhibitor 28, which decreased BJAB Burkitt lymphoma cell viability. Biochemical and cellular studies, molecular docking, and functional assays uncovered that 28 is an RNA-competitive, ATP-uncompetitive inhibitor that engages a novel pocket in the RNA groove of eIF4A and inhibits unwinding activity by interfering with proper RNA binding and suppressing ATP hydrolysis. Inhibition of eIF4A through this unique mechanism may offer new strategies for targeting this promising intersection point of many oncogenic pathways.
Asunto(s)
Descubrimiento de Drogas , Factor 4F Eucariótico de Iniciación/antagonistas & inhibidores , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Linfoma de Burkitt/patología , Línea Celular Tumoral , Humanos , Conformación de Ácido Nucleico , ARN Mensajero/químicaRESUMEN
As the GroES/GroEL chaperonin system is the only bacterial chaperone that is essential under all conditions, we have been interested in the development of GroES/GroEL inhibitors as potential antibiotics. Using Escherichia coli GroES/GroEL as a surrogate, we have discovered several classes of GroES/GroEL inhibitors that show potent antibacterial activity against both Gram-positive and Gram-negative bacteria. However, it remains unknown if E. coli GroES/GroEL is functionally identical to other GroES/GroEL chaperonins and hence if our inhibitors will function against other chaperonins. Herein we report our initial efforts to characterize the GroES/GroEL chaperonins from clinically significant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). We used complementation experiments in GroES/GroEL-deficient and -null E. coli strains to report on exogenous ESKAPE chaperone function. In GroES/GroEL-deficient (but not knocked-out) E. coli, we found that only a subset of the ESKAPE GroES/GroEL chaperone systems could complement to produce a viable organism. Surprisingly, GroES/GroEL chaperone systems from two of the ESKAPE pathogens were found to complement in E. coli, but only in the strict absence of either E. coli GroEL (P. aeruginosa) or both E. coli GroES and GroEL (E. faecium). In addition, GroES/GroEL from S. aureus was unable to complement E. coli GroES/GroEL under all conditions. The resulting viable strains, in which E. coligroESL was replaced with ESKAPE groESL, demonstrated similar growth kinetics to wild-type E. coli, but displayed an elongated phenotype (potentially indicating compromised GroEL function) at some temperatures. These results suggest functional differences between GroES/GroEL chaperonins despite high conservation of amino acid identity.IMPORTANCE The GroES/GroEL chaperonin from E. coli has long served as the model system for other chaperonins. This assumption seemed valid because of the high conservation between the chaperonins. It was, therefore, shocking to discover ESKAPE pathogen GroES/GroEL formed mixed-complex chaperonins in the presence of E. coli GroES/GroEL, leading to loss of organism viability in some cases. Complete replacement of E. coligroESL with ESKAPE groESL restored organism viability, but produced an elongated phenotype, suggesting differences in chaperonin function, including client specificity and/or refolding cycle rates. These data offer important mechanistic insight into these remarkable machines, and the new strains developed allow for the synthesis of homogeneous chaperonins for biochemical studies and to further our efforts to develop chaperonin-targeted antibiotics.
Asunto(s)
Chaperonina 10/genética , Chaperonina 60/genética , Escherichia coli/genética , Bacterias Gramnegativas/genética , Bacterias Grampositivas/genética , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Antibacterianos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Chaperonina 10/química , Chaperonina 10/metabolismo , Chaperonina 60/química , Chaperonina 60/metabolismo , Enterobacter/efectos de los fármacos , Enterobacter/genética , Enterobacter/metabolismo , Enterococcus faecium/efectos de los fármacos , Enterococcus faecium/genética , Enterococcus faecium/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Técnicas de Sustitución del Gen , Técnicas de Inactivación de Genes , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/metabolismo , Bacterias Grampositivas/efectos de los fármacos , Bacterias Grampositivas/metabolismo , Cinética , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC), like many KRAS-driven tumors, preferentially loses CDKN2A that encodes an endogenous CDK4/6 inhibitor to bypass the RB-mediated cell cycle suppression. Analysis of a panel of patient-derived cell lines and matched xenografts indicated that many pancreatic cancers have intrinsic resistance to CDK4/6 inhibition that is not due to any established mechanism or published biomarker. Rather, there is a KRAS-dependent rapid adaptive response that leads to the upregulation of cyclin proteins, which participate in functional complexes to mediate resistance. In vivo, the degree of response is associated with the suppression of a gene expression signature that is strongly prognostic in pancreatic cancer. Resistance is associated with an adaptive gene expression signature that is common to multiple kinase inhibitors, but is attenuated with MTOR inhibitors. Combination treatment with MTOR and CDK4/6 inhibitors had potent activity across a large number of patient-derived models of PDAC underscoring the potential clinical efficacy.