In Fragment-Based Lead Discovery (FBLD) highly sensitive biochemical and biophysical screening technologies are used to detect the low-affinity binding of low-molecular-weight compounds (the so-called fragments) to biological targets that are involved in pathophysiological processes. Knowledge of the molecular interactions between fragment hit(s) and the target protein allows the rational generation of high-quality leads for drug development. Thus, high-resolution (e.g. X-ray crystallography) and relatively high-throughput structure determination technologies are key to this approach but they can become a limiting factor for both technical and economical reasons. As a matter of fact, when the experimental characterization of binding mode fails, the success rate of FBLD approaches drastically drops. To overcome current limitations in FLBD, here we propose the development of a computational framework based on advanced-sampling molecular dynamics simulations to map the binding of fragments to protein surfaces on the proteome scale---thus generating the Fragmentome Altas. For each individual target this will allow to: a) systematically identify fragments binding to protein surfaces and cryptic pockets; b) reconstruct the mechanism of binding with atomistic spatiotemporal resolution; c) characterize the molecular determinants of affinity and kinetics in fragment-protein complexes. This insight is fundamental for optimizing and evolving fragments to lead compounds with desired thermodynamics and kinetics features. To date, neither experimental nor computational approaches can provide such information at affordable costs while maintaining the high throughput needed for screening campaigns. The successful implementation of this ambitious project lies in the unique combination of expertise of its participants, and it will allow a novel state-of-the-art for modern drug discovery to be established. The Fragentome Atlas will be a freely accessible on-line server.

This project aims to optimize and validate a promising therapeutic tool for combined cancer therapy, 2shRNA, in an ex vivo model system. Combined therapies are of great significance nowadays, due to their key role in fighting, for instances, resistance processes during cancer treatment. Nonetheless, the drug combinations approved to date face several problems, such as cooperative toxicity effects, lack of efficiency and poor bioavailability. We have designed and synthesized 2shRNA, a new bifunctional RNA tool that can simultaneously attack two therapeutic targets involved in drug resistance pathways, and that can additionally bind other molecules such as peptide carriers or fluorophores, to improve delivery and efficacy. The 2shRNA nanostructure displayed low toxicity and excellent anti-proliferative properties in resistant HER2+ breast cancer cell lines. The present proposal is aimed at optimizing and validating this novel and promising RNA tool by combining state-of-the-art bioinformatics design and cycles of chemical refinement with biological evaluation in PDx-derived primary cell cultures and biodistribution studies in PDx mouse models. The proposed strategy presents a novel therapeutic approach with great potential to circumvent drug resistance in breast cancer, which is a major health challenge for our society. The ability of our biostable RNA tool to administer two drugs in a single dose could improve the quality of life of the patients, as fewer doses might be needed to stall disease progression.

Mutational processes that generate structural variants (SV) in human and other genomes are understudied and merit attention, and similarly so for the functional impact of the SVs on gene function and regulation. SVs may explain some of the missing heritability in population studies, they may provide candidate pathogenic variants in pedigree studies of hereditary diseases, as well as constitute yet-undiscovered driver events that were anticipated from cancer genomics. Reasons why the SV mutational processes and SV functional impact are understudied are both of a technical and a conceptual nature; both aspects will be addressed in the STRUCTOMATIC project. We will study structural variation in human somatic cells by combining diverse computational and experimental approaches, drawing on a genomic resource of hundreds of tumors and healthy tissues sequenced using long-read WGS that we will generate. We will further perform mutation accumulation and directed evolution experiments using cell line models of chromosomal instability, generating further genomic data that will support observational analyses of tumor genomes. The project aims are: thoroughly cataloguing the diversity of SVs in multiple somatic cell types including those not detectable by short-read WGS, elucidating the underlying mutational mechanisms that generate SVs, their heterogeneity across the human chromosomes as well as their variation between individuals, and developing rigorous statistical methodologies for identifying positive and negative selection on SVs in human somatic cells. Characterizing the landscape of somatic SVs is crucial for a more complete understanding of the genetic basis of carcinogenesis and of the variable cancer risk across tissues and individuals, and may also provide evidence for hypothesized roles of somatic genetic variation in aging-related pathologies more generally.

Myeloid cells, such as dendritic cells (DCs) and neutrophils, reside in various organs to respond to insults and are key to control immunity and inflammation. However, their aberrant activities in the elderly can cause immunosenecence and inflammaging. Different tissues comprise highly distinct milieus that impose context-dependent biochemical challenges on their resident cells, which further change when tissues age. Yet, it is not known how DCs and neutrophils survive in their different homing organs and maintain their functionality. I hypothesize that DCs and neutrophils have to adjust their metabolism to the distinct environments of their tissues of residence. This is important, because metabolic changes upon stimulation were shown to affect the functions of those cells in vitro. Given their power to orchestrate immune responses, it is now vital to reveal the precise metabolic adaptions of DCs and neutrophils to their distinct homing organs and to determine how that affects their activities. This pioneering knowledge will expose organ-dependent metabolic vulnerabilities of DCs and neutrophils to uphold their homeostatic and immune functions, which I envisage to cause their dysfunction in aging. I will uncover the metabolic adaptions of DCs and neutrophils to >10 healthy and aged tissues using independent innovative approaches and adapted cutting-edge techniques. I will reveal the relevance of such tissue-dependent metabolic adjustments for their presence and functions in organs, and dissect the underlying molecular mechanisms. I will expose the tissue-specificity of DC and neutrophil dysfunction in aging and the role of their metabolism. The discovery of tissue-dependent metabolic adaptions by DCs and neutrophils that impact their functions will transform translational immunology research to integrate the tissue-context and reveal novel organ-specific therapeutic strategies to combat immune dysfunction in aging and beyond.