Inhibition of Ral GTPases Using a Stapled Peptide Approach

Aberrant Ras signalling drives numerous cancers and drugs to inhibit this are urgently required. This compelling clinical need, combined with recent innovations in drug discovery including the advent of biologic therapeutic agents, has propelled Ras back to the forefront of targeting efforts. Activated Ras has proved extremely difficult to target directly and the focus has moved to the main downstream Ras-signalling pathways. In particular, the Ras-Raf and Ras-PI3K pathways have provided conspicuous enzyme therapeutic targets, which were more accessible to conventional drug-discovery strategies. The Ras-RalGEF-Ral pathway is a more difficult challenge for traditional medicinal development and there have therefore been few inhibitors reported that disrupt this axis. We have used our structure of a Ral-effector complex as a basis for the design and characterization of α-helical stapled peptides that bind selectively to active, GTP-bound Ral proteins and that compete with downstream effector proteins. The peptides have been thoroughly characterized biophysically. Crucially, the lead peptide enters cells and is biologically active, inhibiting isoform-specific RalB-driven cellular processes. This therefore provides a starting point for therapeutic inhibition of the Ras-RalGEF-Ral pathway.This work was supported by a Cambridge Cancer Centre Pump Priming award to CA, DO and HRM, a BBSRC Studentship to NSC, and a National Institutes for Health grant (CA71443) and the Welch Foundation (grant number I-1414) to MAW.This is the final version of the article. It first appeared from the American Society for Biochemistry and Molecular Biology via https://doi.org/10.1074/jbc.M116.72024

Inhibition of Ral GTPases Using a Stapled Peptide Approach.

Aberrant Ras signaling drives numerous cancers, and drugs to inhibit this are urgently required. This compelling clinical need combined with recent innovations in drug discovery including the advent of biologic therapeutic agents, has propelled Ras back to the forefront of targeting efforts. Activated Ras has proved extremely difficult to target directly, and the focus has moved to the main downstream Ras-signaling pathways. In particular, the Ras-Raf and Ras-PI3K pathways have provided conspicuous enzyme therapeutic targets that were more accessible to conventional drug-discovery strategies. The Ras-RalGEF-Ral pathway is a more difficult challenge for traditional medicinal development, and there have, therefore, been few inhibitors reported that disrupt this axis. We have used our structure of a Ral-effector complex as a basis for the design and characterization of α-helical-stapled peptides that bind selectively to active, GTP-bound Ral proteins and that compete with downstream effector proteins. The peptides have been thoroughly characterized biophysically. Crucially, the lead peptide enters cells and is biologically active, inhibiting isoform-specific RalB-driven cellular processes. This, therefore, provides a starting point for therapeutic inhibition of the Ras-RalGEF-Ral pathway.This work was supported by a Cambridge Cancer Centre Pump Priming award to CA, DO and HRM, a BBSRC Studentship to NSC, and a National Institutes for Health grant (CA71443) and the Welch Foundation (grant number I-1414) to MAW.This is the final version of the article. It first appeared from the American Society for Biochemistry and Molecular Biology via https://doi.org/10.1074/jbc.M116.72024

Regulation of Hematopoiesis and Methionine Homeostasis by mTORC1 Inhibitor NPRL2

Nitrogen permease regulator-like 2 (NPRL2) is a component of a conserved complex that inhibits mTORC1 (mammalian Target Of Rapamycin Complex 1) in response to amino acid insufficiency. Here, we show that NPRL2 is required for mouse viability and that its absence significantly compromises fetal liver hematopoiesis in developing embryos. Moreover, NPRL2 KO embryos have significantly reduced methionine levels and exhibit phenotypes reminiscent of cobalamin (vitamin B12) deficiency. Consistent with this idea, NPRL2 KO liver and mouse embryonic fibroblasts (MEFs) show defective processing of the cobalamin-transport protein transcobalamin 2, along with impaired lysosomal acidification and lysosomal gene expression. NPRL2 KO MEFs exhibit a significant defect in the cobalamin-dependent synthesis of methionine from homocysteine, which can be rescued by supplementation with cyanocobalamin. Taken together, these findings demonstrate a role for NPRL2 and mTORC1 in the regulation of lysosomal-dependent cobalamin processing, methionine synthesis, and maintenance of cellular re-methylation potential, which are important during hematopoiesis

Exploiting albumin as a mucosal vaccine chaperone for robust generation of lung-resident memory T cells

Tissue-resident memory T cells (TRMs) can profoundly enhance mucosal immunity, but parameters governing TRM induction by vaccination remain poorly understood. Here, we describe an approach exploiting natural albumin transport across the airway epithelium to enhance mucosal TRM generation by vaccination. Pulmonary immunization with albumin-binding amphiphile conjugates of peptide antigens and CpG adjuvant (amph-vaccines) increased vaccine accumulation in the lung and mediastinal lymph nodes (MLNs). Amph-vaccines prolonged antigen presentation in MLNs over 2 weeks, leading to 25-fold increased lung-resident T cell responses over traditional immunization and enhanced protection from viral or tumor challenge. Mimicking such prolonged exposure through repeated administration of soluble vaccine revealed that persistence of both antigen and adjuvant was critical for optimal TRM induction, mediated through T cell priming in MLNs after prime, and directly in the lung tissue after boost. Thus, vaccine persistence strongly promotes TRM induction, and amph-conjugates may provide a practical approach to achieve such kinetics in mucosal vaccines

Integration of data-intensive, machine learning and robotic experimental approaches for accelerated discovery of catalysts in renewable energy-related reactions

Technological advancements in recent decades have greatly transformed the field of material chemistry. Juxtaposing the accentuating energy demand with the pollution associated, urgent measures are required to ensure energy maximization, while reducing the extended experimental time cycle involved in energy production. In lieu of this, the prominence of catalysts in chemical reactions, particularly energy related reactions cannot be undermined, and thus it is critical to discover and design catalyst, towards the optimization of chemical processes and generation of sustainable energy. Most recently, artificial intelligence (AI) has been incorporated into several fields, particularly in advancing catalytic processes. The integration of intensive data set, machine learning models and robotics, provides a very powerful tool in modifying material synthesis and optimization by generating multifarious dataset amenable with machine learning techniques. The employment of robots automates the process of dataset and machine learning models integration in screening intermetallic surfaces of catalyst, with extreme accuracy and swiftness comparable to a number of human researchers. Although, the utilization of robots in catalyst discovery is still in its infancy, in this review we summarize current sway of artificial intelligence in catalyst discovery, briefly describe the application of databases, machine learning models and robots in this field, with emphasis on the consolidation of these monomeric units into a tripartite flow process. We point out current trends of machine learning and hybrid models of first principle calculations (DFT) for generating dataset, which is integrable into autonomous flow process of catalyst discovery. Also, we discuss catalyst discovery for renewable energy related reactions using this tripartite flow process with predetermined descriptors

Machine learning and robot-assisted synthesis of diverse gold nanorods via seedless approach

The challenge of data-driven synthesis of advanced nanomaterials can be minimized by using machine learning algorithms to optimize synthesis parameters and expedite the innovation process. In this study, a high-throughput robotic platform was employed to synthesize over 1356 gold nanorods with varying aspect ratios via a seedless approach. The developed models guided us in synthesizing gold nanorods with customized morphology, resulting in highly repeatable morphological yield with quantifiable structure-modulating precursor adjustments. The study provides insight into the dynamic relationships between key structure-modulating precursors and the structural morphology of gold nanorods based on the expected aspect ratio. The high-throughput robotic platform-fabricated gold nanorods demonstrated precise aspect ratio control when spectrophotometrically investigated and further validated with the transmission electron microscopy characterization. These findings demonstrate the potential of high-throughput robot-assisted synthesis and machine learning in the synthesis optimization of gold nanorods and aided in the development of models that can aid such synthesis of as-desired gold nanorods

Murine CD8 T‐cell functional avidity is stable in vivo but not in vitro: Independence from homologous prime/boost time interval and antigen density

It is known that for achieving high affinity antibody responses, vaccines must be optimized for antigen dose/density, and the prime/boost interval should be at least 4 weeks. Similar knowledge is lacking for generating high avidity T-cell responses. The functional avidity (FA) of T cells, describing responsiveness to peptide, is associated with the quality of effector function and the protective capacity in vivo. Despite its importance, the FA is rarely determined in T-cell vaccination studies. We addressed the question whether different time intervals for short-term homologous vaccinations impact the FA of CD8 T-cell responses. Four-week instead of 2-week intervals between priming and boosting with potent subunit vaccines in C57BL/6 mice did not improve FA. Equally, similar FA was observed after vaccination with virus-like particles displaying low versus high antigen densities. Interestingly, FA was stable in vivo but not in vitro, depending on the antigen dose and the time interval since T-cell activation, as observed in murine monoclonal T cells. Our findings suggest dynamic in vivo modulation for equal FA. We conclude that low antigen density vaccines or a minimal 4-week prime/boost interval are not crucial for the T-cell's FA, in contrast to antibody responses

Exocyst protein subnetworks integrate Hippo and mTOR signaling to promote virus detection and cancer.

The exocyst is an evolutionarily conserved protein complex that regulates vesicular trafficking and scaffolds signal transduction. Key upstream components of the exocyst include monomeric RAL GTPases, which help mount cell-autonomous responses to trophic and immunogenic signals. Here, we present a quantitative proteomics-based characterization of dynamic and signal-dependent exocyst protein interactomes. Under viral infection, an Exo84 exocyst subcomplex assembles the immune kinase Protein Kinase R (PKR) together with the Hippo kinase Macrophage Stimulating 1 (MST1). PKR phosphorylates MST1 to activate Hippo signaling and inactivate Yes Associated Protein 1 (YAP1). By contrast, a Sec5 exocyst subcomplex recruits another immune kinase, TANK binding kinase 1 (TBK1), which interacted with and activated mammalian target of rapamycin (mTOR). RALB was necessary and sufficient for induction of Hippo and mTOR signaling through parallel exocyst subcomplex engagement, supporting the cellular response to virus infection and oncogenic signaling. This study highlights RALB-exocyst signaling subcomplexes as mechanisms for the integrated engagement of Hippo and mTOR signaling in cells challenged by viral pathogens or oncogenic signaling