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Target occupancy is often insufficient to elicit biological activity, particularly for RNA, compounded by the longstanding challenges surrounding the molecular recognition of RNA structures by small molecules. Here we studied molecular recognition patterns between a natural-product-inspired small-molecule collection and three-dimensionally folded RNA structures. Mapping these interaction landscapes across the human transcriptome defined structure-activity relationships. Although RNA-binding compounds that bind to functional sites were expected to elicit a biological response, most identified interactions were predicted to be biologically inert as they bind elsewhere. We reasoned that, for such cases, an alternative strategy to modulate RNA biology is to cleave the target through a ribonuclease-targeting chimera, where an RNA-binding molecule is appended to a heterocycle that binds to and locally activates RNase L 1 . Overlay of the substrate specificity for RNase L with the binding landscape of small molecules revealed many favourable candidate binders that might be bioactive when converted into degraders. We provide a proof of concept, designing selective degraders for the precursor to the disease-associated microRNA-155 (pre-miR-155), JUN mRNA and MYC mRNA. Thus, small-molecule RNA-targeted degradation can be leveraged to convert strong, yet inactive, binding interactions into potent and specific modulators of RNA function., (© 2023. The Author(s).)

The KRAS oncogene product is considered a major target in anticancer drug discovery. However, direct interference with KRAS signalling has not yet led to clinically useful drugs. Correct localization and signalling by farnesylated KRAS is regulated by the prenyl-binding protein PDEδ, which sustains the spatial organization of KRAS by facilitating its diffusion in the cytoplasm. Here we report that interfering with binding of mammalian PDEδ to KRAS by means of small molecules provides a novel opportunity to suppress oncogenic RAS signalling by altering its localization to endomembranes. Biochemical screening and subsequent structure-based hit optimization yielded inhibitors of the KRAS-PDEδ interaction that selectively bind to the prenyl-binding pocket of PDEδ with nanomolar affinity, inhibit oncogenic RAS signalling and suppress in vitro and in vivo proliferation of human pancreatic ductal adenocarcinoma cells that are dependent on oncogenic KRAS. Our findings may inspire novel drug discovery efforts aimed at the development of drugs targeting oncogenic RAS.

Biological membranes define the boundaries of the cellular compartments in higher eukaryotes and are active in many processes such as signal transduction and vesicular transport. Although post-translational lipid modification of numerous proteins in signal transduction is crucial for biological function, analysis of protein-protein interactions has mainly focused on recombinant proteins in solution under defined in vitro conditions. Here we present a new strategy for the synthesis of such lipid-modified proteins. It involves the bacterial expression of a carboxy-terminally truncated non-lipidated protein, the chemical synthesis of differently lipidated peptides representing the C terminus of the proteins, and their covalent coupling. Our technique is demonstrated using Ras constructs, which exhibit properties very similar to fully processed Ras, but can be produced in high yields and are open for selective modifications. These constructs are operative in biophysical and cellular assay systems, showing specific recognition of effectors by Ras lipoproteins inserted into the membrane surface of biosensors and transforming activity of oncogenic variants after microinjection into cultured cells.

Allogeneic reactions are the major limitation to organ transplantation. These are manifested as rejection of the grafted tissue, and also, in the case of bone marrow transplantation (BMT), graft-versus-host disease (GVHD). Recent methods of avoiding GVHD, by depleting T cells from donor marrow, have led to an increased incidence of marrow graft rejection. Current recipient conditioning protocols involving drugs or irradiation cannot safely be increased, so alternatives must be found. Monoclonal antibodies can be used to control immune responses in vivo, and would be useful in this context if we could define and deplete the cells responsible for marrow rejection. We show here that elimination of residual L3T4+ and Lyt-2+ cells from mice receiving fully mismatched bone marrow abrogates rejection and promotes tolerance to donor-type skin grafts, even in sub-lethally irradiated recipients.

One therapeutic use for monoclonal antibody technology is the elimination of categories of unwanted cells by virtue of their distinct cell surface antigens. The efficiency of cell destruction by complement lysis or opsonization depends on a number of factors such as antibody specificity and isotype as well as certain properties of the target antigen. In some instances cells can escape destruction by redistributing and eventually losing the antigen-antibody complexes from their surface. This process, known as antigenic modulation, generally depends on bivalent antibody binding. Starting from the observation that rabbit antisera can be made more effective at killing tumour cells if they are first rendered univalent by limited proteolysis, we have now prepared a number of monovalent rat monoclonal antibodies to human cell-surface antigens. We find that these antibodies are no longer able to bring about modulation of their target antigens and have an enhanced facility for lysis with human complement. These special properties should greatly increase the therapeutic potential of monoclonal antibodies.

H-2 restriction is an established characteristic of T-cell behaviour and, in effect, it means that mouse T cells are activated against foreign antigens only if those antigens are presented in a membrane association with molecules of the mouse major histocompatibility complex, H-2. Whether T-cell inactivation or tolerance is also H-2-restricted is a question which has been tested directly and indirectly several times in the past. In each case the answer was 'No' but in each case the answer was inconclusive. Doubts arose because of the observation that activation of T cells, in vivo, is an H-2-restricted event which appears unrestricted because of antigen processing by the host. If antigen processing is involved in the induction of tolerance, then tolerance might also be an H-2-restricted process disguised to appear unrestricted. We report here a study designed to minimize antigen processing in which we find that T-cell tolerance induction to 'self' minor histocompatibility (H) antigens is indeed H-2-restricted.

A major aim in immunology has been to understand how the immune system evokes characteristic responses to infection, foreign tissue grafts and tumours. The current view of immunoregulation is based mainly on studies of lymphocyte subsets, either in vitro or by adoptive transfer to irradiated recipients. Many reagents are available for defining T-cell subsets, but only recently have there been helper T-cell-specific antibodies against the mouse equivalent of the Leu3/T4 (man) and W3/25 (rat) antigens. It is clear that monoclonal antibodies will eventually replace antilymphocyte globulin for immunosuppression in organ grafting, but although there has been some clinical success, most monoclonal reagents cause only transient reductions in their target cells in vivo. This uncertainty in the potency of monoclonal antibodies has led some workers to consider them as targeting agents for such highly cytotoxic drugs as ricin A (ref. 21). We show here that unmodified monoclonal antibodies can be extremely effective at depleting cells in vivo and can be used for the selective manipulation of different aspects of the immune response.

A human IgGI antibody has been reshaped for serotherapy in humans by introducing the six hypervariable regions from the heavy- and light-chain variable domains of a rat antibody directed against human lymphocytes. The reshaped human antibody is as effective as the rat antibody in complement and is more effective in cell-mediated lysis of human lymphocytes.

A major goal in immunology has been to find a means of selectively abolishing an individual's potential to mount an immune response to certain antigens, while preserving responsiveness to others. The facility to induce such specific immunological unresponsiveness in an adult would have major implications for tissue-grafting, the control of allergy and for treatment of autoimmune disease. Classical work has shown that immunosuppressive regimes, such as irradiation, anti-lymphocyte globulin or thoracic duct drainage, may facilitate tolerance induction. We describe here a technique by which the immune system of mice can be manipulated to be tolerant to certain protein antigens by administering these during a brief pulse of treatment with a monoclonal antibody directed to the L3T4 molecule on helper T lymphocytes. This technique has the potential to form the basis of a novel generalized means of tolerance induction.