RESUMO
Membrane anchoring of farnesylated KRAS is critical for activation of RAF kinases, yet our understanding of how these proteins interact on the membrane is limited to isolated domains. The RAS-binding domain (RBD) and cysteine-rich domain (CRD) of RAF engage KRAS and the plasma membrane, unleashing the kinase domain from autoinhibition. Due to experimental challenges, structural insight into this tripartite KRAS:RBD-CRD:membrane complex has relied on molecular dynamics simulations. Here, we report NMR studies of the KRAS:CRAF RBD-CRD complex. We found that the nucleotide-dependent KRAS-RBD interaction results in transient electrostatic interactions between KRAS and CRD, and we mapped the membrane interfaces of the CRD, RBD-CRD, and the KRAS:RBD-CRD complex. RBD-CRD exhibits dynamic interactions with the membrane through the canonical CRD lipid-binding site (CRD ß7-8), as well as an alternative interface comprising ß6 and the C terminus of CRD and ß2 of RBD. Upon complex formation with KRAS, two distinct states were observed by NMR: State A was stabilized by membrane association of CRD ß7-8 and KRAS α4-α5 while state B involved the C terminus of CRD, ß3-5 of RBD, and part of KRAS α5. Notably, α4-α5, which has been proposed to mediate KRAS dimerization, is accessible only in state B. A cancer-associated mutation on the state B membrane interface of CRAF RBD (E125K) stabilized state B and enhanced kinase activity and cellular MAPK signaling. These studies revealed a dynamic picture of the assembly of the KRAS-CRAF complex via multivalent and dynamic interactions between KRAS, CRAF RBD-CRD, and the membrane.
Assuntos
Membrana Celular/metabolismo , Cisteína/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Sítios de Ligação , Cisteína/química , Humanos , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Mutations in RAS oncogenes occur in ~ 30% of human cancers, with KRAS being the most frequently altered isoform. RAS proteins comprise a conserved GTPase domain and a C-terminal lipid-modified tail that is unique to each isoform. The GTPase domain is a 'switch' that regulates multiple signaling cascades that drive cell growth and proliferation when activated by binding GTP, and the signal is terminated by GTP hydrolysis. Oncogenic RAS mutations disrupt the GTPase cycle, leading to accumulation of the activated GTP-bound state and promoting proliferation. RAS is a key target in oncology, however it lacks classic druggable pockets and has been extremely challenging to target. RAS signaling has thus been targeted indirectly, by harnessing key downstream effectors as well as upstream regulators, or disrupting the proper membrane localization required for signaling, by inhibiting either lipid modification or 'carrier' proteins. As a small (20 kDa) protein with multiple conformers in dynamic equilibrium, RAS is an excellent candidate for NMR-driven characterization and screening for direct inhibitors. Several molecules have been discovered that bind RAS and stabilize shallow pockets through conformational selection, and recent compounds have achieved substantial improvements in affinity. NMR-derived insight into targeting the RAS-membrane interface has revealed a new strategy to enhance the potency of small molecules, while another approach has been development of peptidyl inhibitors that bind through large interfaces rather than deep pockets. Remarkable progress has been made with mutation-specific covalent inhibitors that target the thiol of a G12C mutant, and these are now in clinical trials. Here we review the history of RAS inhibitor development and highlight the utility of NMR and integrated biophysical approaches in RAS drug discovery.
Assuntos
Descoberta de Drogas/métodos , Proteínas de Membrana/antagonistas & inibidores , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Mutação , Prenilação/efeitos dos fármacos , Ligação Proteica , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Bibliotecas de Moléculas Pequenas/químicaRESUMO
KRAS homo-dimerization has been implicated in the activation of RAF kinases, however, the mechanism and structural basis remain elusive. We developed a system to study KRAS dimerization on nanodiscs using paramagnetic relaxation enhancement (PRE) NMR spectroscopy, and determined distinct structures of membrane-anchored KRAS dimers in the active GTP- and inactive GDP-loaded states. Both dimerize through an α4-α5 interface, but the relative orientation of the protomers and their contacts differ substantially. Dimerization of KRAS-GTP, stabilized by electrostatic interactions between R135 and E168, favors an orientation on the membrane that promotes accessibility of the effector-binding site. Remarkably, "cross"-dimerization between GTP- and GDP-bound KRAS molecules is unfavorable. These models provide a platform to elucidate the structural basis of RAF activation by RAS and to develop inhibitors that can disrupt the KRAS dimerization. The methodology is applicable to many other farnesylated small GTPases.
Assuntos
Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Sítios de Ligação , Dimerização , Humanos , Espectroscopia de Ressonância Magnética/métodos , Simulação de Dinâmica Molecular , Proteínas Proto-Oncogênicas p21(ras)/químicaRESUMO
Small GTPases (sGTPases) are critical switch-like regulators that mediate several important cellular functions and are often mutated in human cancers. They are activated by guanine nucleotide exchange factors (GEFs), which specifically catalyze the exchange of GTP for GDP. GEFs coordinate signaling networks in normal cells, and are frequently deregulated in cancers. sGTPase signaling pathways are complex and interconnected; however, most GEF assays do not reveal such complexity. In this Communication, we describe the development of a unique real-time NMR-based multiplexed GEF assay that employs distinct isotopic labeling schemes for each sGTPase protein to enable simultaneous observation of six proteins of interest. We monitor nucleotide exchange of KRas, Rheb, RalB, RhoA, Cdc42 and Rac1 in a single system, and assayed the activities of GEFs in lysates of cultured human cells and 3D organoids derived from pancreatic cancer patients. We observed potent activation of RhoA by lysates of HEK293a cells transfected with GEF-H1, along with weak stimulation of Rac1, which we showed is indirect. Our functional analyses of pancreatic cancer-derived organoids revealed higher GEF activity for RhoA than other sGTPases, in line with RNA-seq data indicating high expression of RhoA-specific GEFs.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Bioensaio , Fatores de Troca do Nucleotídeo Guanina/classificação , Células HEK293 , Humanos , Espectroscopia de Ressonância Magnética , Neoplasias Pancreáticas/patologia , Proteína rhoA de Ligação ao GTP/químicaRESUMO
RAS-like protein expressed in many tissues 1 (RIT1) is a disease-associated RAS subfamily small guanosine triphosphatase (GTPase). Recent studies revealed that germ-line and somatic RIT1 mutations can cause Noonan syndrome (NS), and drive proliferation of lung adenocarcinomas, respectively, akin to RAS mutations in these diseases. However, the locations of these RIT1 mutations differ significantly from those found in RAS, and do not affect the three mutational "hot spots" of RAS. Moreover, few studies have characterized the GTPase cycle of RIT1 and its disease-associated mutants. Here we developed a real-time NMR-based GTPase assay for RIT1 and investigated the effect of disease-associated mutations on GTPase cycle. RIT1 exhibits an intrinsic GTP hydrolysis rate similar to that of H-RAS, but its intrinsic nucleotide exchange rate is â¼4-fold faster, likely as a result of divergent residues near the nucleotide binding site. All of the disease-associated mutations investigated increased the GTP-loaded, activated state of RIT1 in vitro, but they could be classified into two groups with different intrinsic GTPase properties. The S35T, A57G, and Y89H mutants exhibited more rapid nucleotide exchange, whereas F82V and T83P impaired GTP hydrolysis. A RAS-binding domain pulldown assay indicated that RIT1 A57G and Y89H were highly activated in HEK293T cells, whereas T83P and F82V exhibited more modest activation. All five mutations are associated with NS, whereas two (A57G and F82V) have also been identified in urinary tract cancers and myeloid malignancies. Characterization of the effects on the GTPase cycle of RIT1 disease-associated mutations should enable better understanding of their role in disease processes.
Assuntos
Adenocarcinoma , Neoplasias Pulmonares , Mutação de Sentido Incorreto , Proteínas de Neoplasias , Síndrome de Noonan , Neoplasias Urológicas , Proteínas ras , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma de Pulmão , Substituição de Aminoácidos , Linhagem Celular , Guanosina Trifosfato/química , Humanos , Hidrólise , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Síndrome de Noonan/genética , Síndrome de Noonan/metabolismo , Domínios Proteicos , Neoplasias Urológicas/genética , Neoplasias Urológicas/metabolismo , Proteínas ras/química , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
OBJECTIVES: To investigate the radiosensitizing effects of cepharanthine (CEP) in the human cervical adenocarcinoma HeLa cell line and to examine the underlying mechanisms. MATERIALS/METHODS: Survival of HeLa cells after treatment with or without ionizing radiation (IR) and CEP administration was investigated. MTT assays and apoptosis analysis were used to assess cytotoxicity. Nude mouse xenografts were established to evaluate the antitumor effects of CEP and IR in vivo. Expression of signal transducer and activator of transcription 3 (STAT3) and its downstream signaling molecules as well as cyclooxygenase-2 (COX-2) were examined by Western blot analysis. RESULTS: Clonogenic assays showed that treatment with CEP and IR resulted in significant radiosensitization. Cepharanthine and IR treatment achieved maximum cytotoxic effects on HeLa cells with regard to apoptosis induction. Cepharanthine efficiently decreased IR-induced STAT3 and COX-2 activation. The STAT3 target genes, including the antiapoptotic Bcl-2 and the cell cycle regulator c-Myc, were decreased concomitantly. In vivo administration of CEP (20 mg/kg every 2 days) combined with radiation in HeLa xenografts enhanced tumor growth delay and apoptosis (indicated by activated caspase-3 Western blot analysis), with reduced expression of STAT3, Bcl-2, c-Myc, and COX-2. CONCLUSIONS: Cepharanthine was shown to induce radiation sensitization in HeLa cells in vitro and in vivo. The inhibitory effects of CEP on STAT3 signaling pathway and COX-2 help us to better understand the radiosensitization of CEP.
Assuntos
Benzilisoquinolinas/farmacologia , Inibidores de Ciclo-Oxigenase 2/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Células HeLa , Humanos , Fator de Transcrição STAT3/fisiologia , Transdução de Sinais/efeitos dos fármacosRESUMO
T cell receptor (TCR) repertoires are critical for antiviral immunity. Determining the TCR repertoire composition, diversity, and dynamics and how they change during viral infection can inform the molecular specificity of host responses to viruses such as SARS-CoV-2. To determine signatures associated with COVID-19 disease severity, here we perform a large-scale analysis of over 4.7 billion sequences across 2130 TCR repertoires from COVID-19 patients and healthy donors. TCR repertoire analyses from these data identify and characterize convergent COVID-19-associated CDR3 gene usages, specificity groups, and sequence patterns. Here we show that T cell clonal expansion is associated with the upregulation of T cell effector function, TCR signaling, NF-kB signaling, and interferon-gamma signaling pathways. We also demonstrate that machine learning approaches accurately predict COVID-19 infection based on TCR sequence features, with certain high-power models reaching near-perfect AUROC scores. These analyses provide a systems immunology view of T cell adaptive immune responses to COVID-19.
Assuntos
COVID-19 , Humanos , SARS-CoV-2 , Linfócitos T , Receptores de Antígenos de Linfócitos T/genética , Aprendizado de MáquinaRESUMO
The SARS-CoV-2 variant, Omicron (B.1.1.529), rapidly swept the world since its emergence. Compared with previous variants, Omicron has a high number of mutations, especially those in its spike glycoprotein that drastically dampen or abolish the efficacy of currently available vaccines and therapeutic antibodies. Several major sublineages of Omicron evolved, including BA.1, BA.1.1, BA.2, BA.2.12.1, BA.3, BA.4/5, and BA.2.75, which rapidly changing the global and regional landscape of the pandemic. Although vaccines are available, therapeutic antibodies remain critical for infected and especially hospitalized patients. To address this, we have designed and generated a panel of human/humanized therapeutic bispecific antibodies against Omicron and its sub-lineage variants, with activity spectrum against other lineages. Among these, the top clone CoV2-0213 has broadly potent activities against multiple SARS-CoV-2 ancestral and Omicron lineages, including BA.1, BA.1.1, BA.2, BA.2.12.1, BA.3, BA.4/5, and BA.2.75. We have solved the cryo-EM structure of the lead bi-specific antibody CoV-0213 and its major Fab arm MB.02. Three-dimensional structural analysis shows distinct epitope of antibody - spike receptor binding domain (RBD) interactions and reveals that both Fab fragments of CoV2-0213 can simultaneously target one single spike RBD or two adjacent ones in the same spike trimer, further corroborating its mechanism of action. CoV2-0213 represents a unique and potent broad-spectrum SARS-CoV-2 neutralizing bispecific antibody (nbsAb) against the currently circulating major Omicron variants (BA.1, BA.1.1, BA.2, BA.2.12.1, BA.2.75, BA.3, and BA.4/5). CoV2-0213 is primarily human and ready for translational testing as a countermeasure against the ever-evolving pathogen.
RESUMO
As a clinical vaccine, lipid nanoparticle (LNP) mRNA has demonstrated potent and broad antibody responses, leading to speculation about its potential for antibody discovery. Here, we developed RAMIHM, a highly efficient strategy for developing fully human monoclonal antibodies that employs rapid mRNA immunization of humanized mice followed by single B cell sequencing (scBCR-seq). We immunized humanized transgenic mice with RAMIHM and generated 15 top-ranked clones from peripheral blood, plasma B, and memory B cell populations, demonstrating a high rate of antigen-specificity (93.3%). Two Omicron-specific neutralizing antibodies with high potency and one broad-spectrum neutralizing antibody were discovered. Furthermore, we extended the application of RAMIHM to cancer immunotherapy targets, including a single transmembrane protein CD22 and a multi-transmembrane G protein-coupled receptor target, GPRC5D, which is difficult for traditional protein immunization methods. RAMIHM-scBCR-seq is a broadly applicable platform for the rapid and efficient development of fully human monoclonal antibodies against an assortment of targets.
Assuntos
Anticorpos Monoclonais , Imunização , Camundongos , Humanos , Animais , Anticorpos Monoclonais/genética , RNA Mensageiro/genética , Vacinação , Anticorpos Neutralizantes/genética , Camundongos TransgênicosRESUMO
PURPOSE: Neoadjuvant therapy modality can increase the operability rate and mitigate pathological risks in locally advanced cervical cancer, but treatment response varies widely. It remains unclear whether genetic alterations correlate with the response to neoadjuvant therapy and disease-free survival (DFS) in locally advanced cervical cancer. MATERIALS AND METHODS: A total of 62 locally advanced cervical cancer (stage IB-IIA) patients who received neoadjuvant chemoradiation plus radical hysterectomy were retrospectively analyzed. Patients' tumor biopsy samples were comprehensively profiled using targeted next generation sequencing. Pathologic response to neoadjuvant treatment and DFS were evaluated against the association with genomic traits. RESULTS: Genetic alterations of PIK3CA were most frequent (37%), comparable to that of Caucasian populations from The Cancer Genome Atlas. The mutation frequency of genes including TERT, POLD1, NOS2, and FGFR3 was significantly higher in Chinese patients whereas RPTOR, EGFR, and TP53 were underrepresented in comparison to Caucasians. Germline mutations were identified in 21% (13/62) of the cohort and more than half (57%) had mutations in DNA damage repair genes, including BRCA1/2, TP53 and PALB2. Importantly, high tumor mutation burden, TP53 polymorphism (rs1042522), and KEAP1 mutations were found to be associated with poor pathologic response to neoadjuvant chemoradiation treatment. KEAP1 mutations, PIK3CA-SOX2 co-amplification, TERC copy number gain, and TYMS polymorphism correlated with an increased risk of disease relapse. CONCLUSION: We report the genomic profile of locally advanced cervical cancer patients and the distinction between Asian and Caucasian cohorts. Our findings highlight genomic traits associated with unfavorable neoadjuvant chemoradiation response and a higher risk of early disease recurrence.
Assuntos
Terapia Neoadjuvante , Neoplasias do Colo do Útero , Classe I de Fosfatidilinositol 3-Quinases , Receptores ErbB , Feminino , Genômica , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch , Fator 2 Relacionado a NF-E2 , Recidiva Local de Neoplasia/patologia , Estadiamento de Neoplasias , Estudos Retrospectivos , Resultado do Tratamento , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/terapiaRESUMO
Although COVID-19 vaccines have been developed, multiple pathogenic coronavirus species exist, urging on development of multispecies coronavirus vaccines. Here we develop prototype lipid nanoparticle (LNP)-mRNA vaccine candidates against SARS-CoV-2 Delta, SARS-CoV, and MERS-CoV, and we test how multiplexing LNP-mRNAs can induce effective immune responses in animal models. Triplex and duplex LNP-mRNA vaccinations induce antigen-specific antibody responses against SARS-CoV-2, SARS-CoV, and MERS-CoV. Single-cell RNA sequencing profiles the global systemic immune repertoires and respective transcriptome signatures of vaccinated animals, revealing a systemic increase in activated B cells and differential gene expression across major adaptive immune cells. Sequential vaccination shows potent antibody responses against all three species, significantly stronger than simultaneous vaccination in mixture. These data demonstrate the feasibility, antibody responses, and single-cell immune profiles of multispecies coronavirus vaccination. The direct comparison between simultaneous and sequential vaccination offers insights into optimization of vaccination schedules to provide broad and potent antibody immunity against three major pathogenic coronavirus species.
Assuntos
COVID-19 , Coronavírus da Síndrome Respiratória do Oriente Médio , Vacinas Virais , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Humanos , Lipossomos , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Nanopartículas , RNA Mensageiro/genética , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genética , Vacinação , Vacinas Sintéticas , Vacinas de mRNARESUMO
Lipid nanoparticle (LNP)-mRNA vaccines offer protection against COVID-19; however, multiple variant lineages caused widespread breakthrough infections. Here, we generate LNP-mRNAs specifically encoding wild-type (WT), B.1.351, and B.1.617 SARS-CoV-2 spikes, and systematically study their immune responses. All three LNP-mRNAs induced potent antibody and T cell responses in animal models; however, differences in neutralization activity have been observed between variants. All three vaccines offer potent protection against in vivo challenges of authentic viruses of WA-1, Beta, and Delta variants. Single-cell transcriptomics of WT- and variant-specific LNP-mRNA-vaccinated animals reveal a systematic landscape of immune cell populations and global gene expression. Variant-specific vaccination induces a systemic increase of reactive CD8 T cells and altered gene expression programs in B and T lymphocytes. BCR-seq and TCR-seq unveil repertoire diversity and clonal expansions in vaccinated animals. These data provide assessment of efficacy and direct systems immune profiling of variant-specific LNP-mRNA vaccination in vivo.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Anticorpos Neutralizantes , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Humanos , Imunidade , Lipossomos , Nanopartículas , RNA Mensageiro/genética , VacinaçãoRESUMO
INTRODUCTION: Mutations in BRAF occur in 2% to 4% of patients with lung adenocarcinoma. Combination dabrafenib and trametinib, or single-agent vemurafenib is approved only for patients with cancers driven by the V600E BRAF mutation. Targeted therapy is not currently available for patients harboring non-V600 BRAF mutations. METHODS: A lung adenocarcinoma patient-derived xenograft model (PHLC12) with wild-type and nonamplified EGFR was tested for response to EGFR tyrosine kinase inhibitors (TKIs). A cell line derived from this model (X12CL) was also used to evaluate drug sensitivity and to identify potential drivers by small interfering RNA knockdown. Kinase assays were used to test direct targeting of the candidate driver by the EGFR TKIs. Structural modeling including, molecular dynamics simulations, and binding assays were conducted to explore the mechanism of off-target inhibition by EGFR TKIs on the model 12 driver. RESULTS: Both patient-derived xenograft PHLC12 and the X12CL cell line were sensitive to multiple EGFR TKIs. The BRAFG469V mutation was found to be the only known oncogenic mutation in this model. Small interfering RNA knockdown of BRAF, but not the EGFR, killed X12CL, confirming BRAFG469V as the oncogenic driver. Kinase activity of the BRAF protein isolated from X12CL was inhibited by treatment with the EGFR TKIs gefitinib and osimertinib, and expression of BRAFG469V in non-EGFR-expressing NR6 cells promoted growth in low serum condition, which was also sensitive to EGFR TKIs. Structural modeling, molecular dynamic simulations, and in vitro binding assays support BRAFG469V being a direct target of the TKIs. CONCLUSIONS: Clinically approved EGFR TKIs can be repurposed to treat patients with non-small cell lung cancer harboring the BRAFG469V mutation.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/genéticaRESUMO
The Omicron variant of SARS-CoV-2 recently swept the globe and showed high level of immune evasion. Here, we generate an Omicron-specific lipid nanoparticle (LNP) mRNA vaccine candidate, and test its activity in animals, both alone and as a heterologous booster to WT mRNA vaccine. Our Omicron-specific LNP-mRNA vaccine elicits strong antibody response in vaccination-naïve mice. Mice that received two-dose WT LNP-mRNA show a > 40-fold reduction in neutralization potency against Omicron than WT two weeks post boost, which further reduce to background level after 3 months. The WT or Omicron LNP-mRNA booster increases the waning antibody response of WT LNP-mRNA vaccinated mice against Omicron by 40 fold at two weeks post injection. Interestingly, the heterologous Omicron booster elicits neutralizing titers 10-20 fold higher than the homologous WT booster against Omicron variant, with comparable titers against Delta variant. All three types of vaccination, including Omicron alone, WT booster and Omicron booster, elicit broad binding antibody responses against SARS-CoV-2 WA-1, Beta, Delta variants and SARS-CoV. These data provide direct assessments of an Omicron-specific mRNA vaccination in vivo, both alone and as a heterologous booster to WT mRNA vaccine.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Lipossomos , Camundongos , Nanopartículas , RNA Mensageiro/genética , SARS-CoV-2/genética , Vacinação , Vacinas Sintéticas , Vacinas de mRNARESUMO
The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has high transmissibility and recently swept the globe. Due to the extensive number of mutations, this variant has high level of immune evasion, which drastically reduced the efficacy of existing antibodies and vaccines. Thus, it is important to test an Omicron-specific vaccine, evaluate its immune response against Omicron and other variants, and compare its immunogenicity as boosters with existing vaccine designed against the reference wildtype virus (WT). Here, we generated an Omicron-specific lipid nanoparticle (LNP) mRNA vaccine candidate, and tested its activity in animals, both alone and as a heterologous booster to existing WT mRNA vaccine. Our Omicron-specific LNP-mRNA vaccine elicited strong and specific antibody response in vaccination-naive mice. Mice that received two-dose WT LNP-mRNA, the one mimicking the commonly used Pfizer/Moderna mRNA vaccine, showed a >40-fold reduction in neutralization potency against Omicron variant than that against WT two weeks post second dose, which further reduced to background level >3 months post second dose. As a booster shot for two-dose WT mRNA vaccinated mice, a single dose of either a homologous booster with WT LNP-mRNA or a heterologous booster with Omicron LNP-mRNA restored the waning antibody response against Omicron, with over 40-fold increase at two weeks post injection as compared to right before booster. Interestingly, the heterologous Omicron LNP-mRNA booster elicited neutralizing titers 10-20 fold higher than the homologous WT booster against the Omicron variant, with comparable titers against the Delta variant. All three types of vaccination, including Omicron mRNA alone, WT mRNA homologous booster, and Omicron heterologous booster, elicited broad binding antibody responses against SARS-CoV-2 WA-1, Beta, and Delta variants, as well as other Betacoronavirus species such as SARS-CoV, but not Middle East respiratory syndrome coronavirus (MERS-CoV). These data provided direct proof-of-concept assessments of an Omicron-specific mRNA vaccination in vivo, both alone and as a heterologous booster to the existing widely-used WT mRNA vaccine form.
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COVID-19 pathogen SARS-CoV-2 has infected hundreds of millions and caused over 5 million deaths to date. Although multiple vaccines are available, breakthrough infections occur especially by emerging variants. Effective therapeutic options such as monoclonal antibodies (mAbs) are still critical. Here, we report the development, cryo-EM structures, and functional analyses of mAbs that potently neutralize SARS-CoV-2 variants of concern. By high-throughput single cell sequencing of B cells from spike receptor binding domain (RBD) immunized animals, we identify two highly potent SARS-CoV-2 neutralizing mAb clones that have single-digit nanomolar affinity and low-picomolar avidity, and generate a bispecific antibody. Lead antibodies show strong inhibitory activity against historical SARS-CoV-2 and several emerging variants of concern. We solve several cryo-EM structures at ~3 Å resolution of these neutralizing antibodies in complex with prefusion spike trimer ectodomain, and reveal distinct epitopes, binding patterns, and conformations. The lead clones also show potent efficacy in vivo against authentic SARS-CoV-2 in both prophylactic and therapeutic settings. We also generate and characterize a humanized antibody to facilitate translation and drug development. The humanized clone also has strong potency against both the original virus and the B.1.617.2 Delta variant. These mAbs expand the repertoire of therapeutics against SARS-CoV-2 and emerging variants.
Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19 , SARS-CoV-2 , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Glicoproteína da Espícula de Coronavírus , Proteínas do Envelope ViralRESUMO
Pretreatment neutrophil-to-lymphocyte ratio (NLR) has been reported to be associated with the prognosis of inoperable gastric cancer patients with systemic therapy. However, no consensus on the association has been reached. In this study, we mainly evaluated whether pretreatment NLR predicted the benefit of inoperable gastric cancer patients with systemic therapy, including chemotherapy, targeted therapy and immunotherapy. PubMed, Embase and Cochrane Library databases were systematically searched from inception up to September 16th, 2020. A total of 36 studies including 8614 patients were involved in the meta-analysis. Pooled data revealed that high pretreatment NLR was significantly associated with poor outcomes of OS (HR = 1.78, 95% CI = [1.59, 1.99]) and PFS (HR = 1.63, 95% CI = [1.39, 1.91]) in gastric cancer. Subgroup analyses stratified by country, study type, case load, analysis of HR, cutoff of pretreatment NLR, or treatment types arrived at the same conclusion. Pooled data based on different effect models and sensitivity analyses did not change the conclusion. Overall, high pretreatment NLR predicts the poor prognosis of inoperable gastric cancer patients with systemic therapy. Measurement of pretreatment NLR will assist clinicians with patient counseling and clinical treatment guiding accordingly.
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Contagem de Leucócitos , Contagem de Linfócitos , Linfócitos , Neutrófilos , Neoplasias Gástricas/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Biomarcadores , Terapia Combinada , Feminino , Humanos , Imunoterapia , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Neoplasias Gástricas/tratamento farmacológico , Resultado do TratamentoRESUMO
COVID-19 pathogen SARS-CoV-2 has infected hundreds of millions and caused over 5 million deaths to date. Although multiple vaccines are available, breakthrough infections occur especially by emerging variants. Effective therapeutic options such as monoclonal antibodies (mAbs) are still critical. Here, we report the development, cryo-EM structures, and functional analyses of mAbs that potently neutralize SARS-CoV-2 variants of concern. By high-throughput single cell sequencing of B cells from spike receptor binding domain (RBD) immunized animals, we identified two highly potent SARS-CoV-2 neutralizing mAb clones that have single-digit nanomolar affinity and low-picomolar avidity, and generated a bispecific antibody. Lead antibodies showed strong inhibitory activity against historical SARS-CoV-2 and several emerging variants of concern. We solved several cryo-EM structures at â¼3 Å resolution of these neutralizing antibodies in complex with prefusion spike trimer ectodomain, and revealed distinct epitopes, binding patterns, and conformations. The lead clones also showed potent efficacy in vivo against authentic SARS-CoV-2 in both prophylactic and therapeutic settings. We also generated and characterized a humanized antibody to facilitate translation and drug development. The humanized clone also has strong potency against both the original virus and the B.1.617.2 Delta variant. These mAbs expand the repertoire of therapeutics against SARS-CoV-2 and emerging variants.
RESUMO
KRAS is frequently mutated in several of the most lethal types of cancer; however, the KRAS protein has proven a challenging drug target. K-RAS4B must be localized to the plasma membrane by prenylation to activate oncogenic signaling, thus we endeavored to target the protein-membrane interface with small-molecule compounds. While all reported lead compounds have low affinity for KRAS in solution, the potency of Cmpd2 was strongly enhanced when prenylated K-RAS4B is associated with a lipid bilayer. We have elucidated a unique mechanism of action of Cmpd2, which simultaneously engages a shallow pocket on KRAS and associates with the lipid bilayer, thereby stabilizing KRAS in an orientation in which the membrane occludes its effector-binding site, reducing RAF binding and impairing activation of RAF. Furthermore, enrichment of Cmpd2 on the bilayer enhances potency by promoting interaction with KRAS. This insight reveals a novel approach to developing inhibitors of membrane-associated proteins.