Rule of five in 2015 and beyond: Target and ligand structural limitations, ligand chemistry structure and drug discovery project decisions

Identifying drug targets within cellular contexts is critical to advancing both target-based and phenotypic drug discovery, particularly for small molecule degraders, such as molecular glues (MG). In most common cases of MG, target identification involves two key mechanism of actions (MoA): determining specific protein target(s) that are engaged by the E3 ligase when a drug binds the E3 ligase, and, conversely, identifying the E3 ligase (adaptor) that is recruited to form the ternary complex when the drug directly targets the protein itself. Limited proteolysis combined with mass spectrometry (LiP-MS) has emerged as a robust, label-free method for deconvoluting small molecule and peptide targets directly within cell lysates, avoiding the need for compound modification or cell line manipulation. This approach leverages a non-specific protease under controlled conditions to detect conformational changes or steric hindrance induced by drug-protein interactions or protein-protein interactions. Using quantitative MS, LiP monitors over 250, 000 peptides, representing more than 8, 000 proteins in a mammalian cell line, allowing for proteome-wide profiling of drug-target interactions. A comprehensive concentration-response study spanning seven concentrations, combined with machine learning-based LiP-scoring, provides a precise ranking of targets and predicts binding sites with peptide-level resolution. Beyond global target identification, we also established a high-throughput HR-LiP workflow that offers detailed peptide-level insights into small molecule-protein interactions directly in cell lysates from cell lines overexpressing target proteins or complexes. This workflow bypasses the need for protein purification, mitigating risks of protein truncation or misfolding. To evaluate LiP-MS’ performance on molecular glue MoAs, we performed global target ID experiments using two well-characterized models: the cyclin K degraders CR8 and SR-4835 and the GSPT1 degraders MRT-2359 and Eragidomide. While the cyclin K degraders bind directly to CDK12, inducing its interaction with DDB1 and leading to the degradation of cyclin K within the CDK12 complex, the GSPT1 compounds engage CRBN, which in turn recruits and degrades GSPT1 as a neosubstrate. Applying HR-LiP, we mapped the binding site of the BRD4 inhibitor JQ1 on full-length BRD4, a large protein challenging to study via standard methods, using a mammalian transient overexpression system. Similarly, we located binding sites for EGFR inhibitors gefitinib and afatinib on the membrane-bound EGFR protein. Additionally, we pinpointed the SMER28 binding site on the hexameric ATPase VCP, an autophagy enhancer, demonstrating the method's applicability to large protein complexes. In conclusion, LiP-MS provides a versatile toolbox for identifying drug targets and mapping binding sites within complex cellular environments. Citation Format: Martin Soste, Polina Shichkova, Roland Bruderer, Matevz Stefancic, Daniel Redfern, Francesca Cavallo, Kuhulika Bhalla, Helen Burston, Prasamit S. Baruah, Roland Hjerpe, Stuart Thomson, Allan Jordan, Yuehan Feng. Drug target ID and binding site mapping in complex cellular environments using LiP-MS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4499.

Periodically, advances in understanding factors that influence success in drug discovery become formulated as rules or guidelines; these become broadly accepted for a time, until new knowledge or a sufficient number of exceptions are identified to highlight the missed opportunities that result from overly rigid adherence. We are coming out of one such cycle now, as it becomes recognized that the concept of ‘druglikeness’, which has been highly effective in focusing discovery efforts on compounds with potential for achieving good ‘pharmaceutical properties’ of absorption, distribution, metabolism and excretion (ADME), has at the same time impeded exploration of new pharmacologically useful chemotypes that do not resemble typical known drugs. At the same time, attention is turning increasingly to new classes of drug targets that are less amenable to inhibition by conventional drug-like molecules, such as protein–protein interaction (PPI) interfaces, intrinsically disordered proteins, DNA-binding, carbohydratebinding and some phosphopeptide-binding proteins. This trend has led to growing interest in identifying new chemotypes and drug modalities that possess both an ability to bind to poorly druggable sites and a high potential for good ADME properties. The features that make a target undruggable by conventional drug-like compounds generally include some combination of the following: a binding site, such as many PPI interface sites, that due to its topology (cavities are too small are too large) cannot generate a large area of intimate contact with a drug-like small ligand; a binding site that is exclusively hydrophobic, and thus lacks the ability to engage in charged or polar interactions with the ligand, as are typically required for high affinity and selectivity; a binding site that has evolved to interact with a highly polar or charged ligand, such as a sugar, a nucleic acid polymer, or a phosphopeptide, and thus is too polar for high complementarity with an amphiphilic drug-like ligand; a site that is intrinsically disordered, such that there is potentially a large entropic cost associated with organizing the binding site around a ligand. Several promising approaches have been developed to address highly challenging drug targets such as those described above. These approaches exploit compound chemotypes that differ from typical drugs, and thus require us to move beyond conventional views of druglikeness when evaluating prospects for good ADME properties.

Natural products are of significant interest to us in many ways. The air we breathe, the water we drink, the foods we eat are all natural products. Many drug products and toxins are also “natural”. Natural product sciences now face challenges at many fronts. At a global and economical level, biodiversity is dimin-ishing everyday as the rain forest gives away to farmland and the coral reef is destroyed by pollution. As a result, many potentially valuable natural products are lost forever before we even know their very existence. An immediate implica-tion beyond the direct loss is that we have less natural products to “copy” from. We pharmacolo-gists may not have the power to change the world, but we can certainly contribute. At a social level, natural products are generally perceived as safe and largely devoid of side effects. This belief has been taken advantage of to promote “natural health products”. Beneficial effects of some are substantiated, but the only benefit of great many is only psychological. Some even created much damage to the believers. Aristolochic acid nephropathy is but only one example. Pharmacologists can, and are obliged to educate the public. At a scientific level, pharmacologists who are interested in natural products also face many challenges. First, rapid advances in related scientific disciplines (e.g. molecular biology, immunology) made us look like dino-saurs. We need to learn new concepts and technologies, and more importantly, to incorporate what we learn in our re-search. Second, modern chemistry and material sciences have blurred the boundary between natural products and synthetic materials. A great many of substances are neither natural nor purely man-made. We need to venture out of our comfort zones to embrace such great opportunities. Third, the use of natural products by human beings to combat diseases and promote health has never been with sin-gle known chemical identity until very recently. Many substances/recipes are known to have solid impact on human health; some have been validated by good clinical practice. Acceptance of application for herbal medications with multi-ple ingredients by the US FDA signals to the world a change of attitude towards acknowledging “good clinical observation”. This change probably also reflects a much more fundamental issue: the human body is a very complex organism. Very few diseases could be traced to a single cause. Multiple causes and pathological changes un-der most diseases call for intervention at multiple sites. One commonly encountered phenomenon in this area is the loss of biological activity in search of the “active compound”. Some, and probably most, are due to technical reasons, but we need to consider the alternative: 1 + 1 may be much more than 2. It is a general policy that studies accepted by this journal must be of known chemical entity or entities. This policy is needed to ensure the scientific quality but creates a set of problems. Frankly, we do not have a clear strategy to deal with this dilemma. Last but not least, we also face challenges at a practical level. First, many natural products with biological activity have poor solubility in water. As a result, research on these natural products is often difficult. Second, natural products are often limited in supply. Semi or total synthesis is a discipline that attempts to address this issue. Third, many natu-ral compounds with biological activity have complex chemical structures. Considering the almost infinite combination of chemical modifications, investigating the structure-activity relationship is tremendously time- and resource-consuming. Great challenges are often synonymous to great opportunities. The creation of this journal is a testimony to our collective effort in rising up to the challenges and turn opportunities to advances. We welcome submission of schol-arly manuscripts that may advance the pharmacology of natural sciences from every possible angle.

It was a great privilege for us to participate in editing this special issue of The Chemical Record on “Carbohydrate Chemistry”. Carbohydrates are biomolecules helping to sustain all life forms. The discovery and development of carbohydrate-based drugs, vaccines, adjuvants and drug delivery systems have emerged as current topics of great interest, founded on advances in carbohydrate chemistry made over the last century or more by many scientists, who have followed the great pioneering work of Emil Fischer. The increasing recognition of the role of glycans in biomedicine is leading to an increasing number of biological and clinical scientists entering the fields of glycobiology and glycomedicine. Progress in biology and medicine are dependent on access to carbohydrate probes to address fundamental questions or for the development of ligands for carbohydrate binding receptors to inspire the next generation of therapeutics or diagnostics. Increasingly, synthetic vaccines are being prepared to combat infectious disease that are based on microbial carbohydrates or glycoconjugate antigens. Often, the tools needed or structurally well-defined compounds can only be prepared by synthesis. New and improved synthetic methods and strategies, including new ligand design and synthesis will help advance these fields. A collection of 14 personal accounts and 8 record reviews from scientists at various stages of their scientific career is presented herein. They cover a range of topics and highlight major areas of interest and challenges faced therein. A number of these articles address recent advances in synthetic methodology for stereoselective glycosylation. These include self-promoting glycosylation, development of glycosyl sulfonate donors, stereoselective sialylation and mannosylation, as well as the use of unsaturated monosaccharides in glycosylation reactions. Updates to synthetic strategies to give structurally well-defined and homogeneous glycans is addressed. Strategies discussed include chemoenzymatic as well as solid phase and automated synthetic approaches, with the latter aimed making oligosaccharide synthesis available for non-specialists. The synthesis of a number of biologically important target oligosaccharides and their derivatives are highlighted. These include articles on the synthesis of mycobacterial phenolic glycolipids and saponins. Such target syntheses provide structurally well-defined materials for biological studies. Many oligosaccharide target compounds, especially those from bacterial origin, contain rare sugars with access to large quantities often problematic. A review on the state of the art of synthesis of rare l-hexoses is included, which are needed for microbial oligosaccharide synthesis and other applications. Building links between synthesis and immunology is highlighted in a review where cancer vaccine development based on mucin O-glycosylation is discussed. Glycosylation structure and presentation is shown to have an enormous influence on the immune response mediated by antibodies. Such insights are enable by achievements in oligosaccharide and glycopeptide synthesis. Chemical synthesis of polysaccharide fragments and their mimetics also features. There are two reviews on heparin/heparan sulfate (HS), which are sulphated polysaccharides of considerable biological and pharmacological importance, with the synthesis of well-defined native HS oligosaccharides remaining challenging. Recent progress on improving heparin-derived pentasaccharide anticoagulant fondaparinux, as well as other methodological and technical improvements for substances of this type are presented. Progress in generating mimetics of these polysaccharides with useful properties is appraised and applications of various assay and molecular modelling tools to gain understanding of interaction of such compounds their target proteins are highlighted. Other polysaccharides, such as the β-glucans also have well documented medicinal uses. Approaches taken to synthesise well defined β-glucan fragments, important to gain understanding of their structure bioactivity relationships is reviewed. Also included is a discussion of chemistry used to conjugate β-glucan oligosaccharides to proteins. There is also a cohort of articles discussing the applications of monosaccharides, including glycals or unsaturated monosaccharides as valuable chiral pool precursors to natural products and biologically or pharmacologically active substances. The synthesis and structural identification of naturally occurring amipurimycin and miharamycin A/B is reviewed. Also discussed are cyclisations and other strategies for synthesis of piperidine iminosugars, some of which have found clinical application and their derivatives; this includes applications of asymmetric synthesis of building blocks for producing polyhydroxylated compounds. Approaches to saccharide natural products and their analogues are discussed such as polyhydroxypyrrolidines (N-heterocycles), conduramines and aminocyclitols (carbacycles) among many others targets. Reagents to give rise to glycomimetics containing sulphur and selenium are discussed. There is an extensive review on synthesis of other well-known type of glycomimetics, the C-glycosides. Therein is a focus on C-glycosidic analogues of natural products or drug molecules, including synthesis of C-flavonoids. The successes of carbohydrates and natural products in drug discovery is inspiring synthesis of ‘natural product like’ compounds with carbohydrates being incorporated into new molecular scaffolding. An account of research on pharmacological chaperones for β-galactosidases demonstrates how glycomimetics contribute to generating new pharmaceutically active molecules or medicines. Synthesis of antibody-drug conjugates, where cytotoxic agents need to be conjugated to monoclonal antibodies (mABs) has emerged as a strategy in biopharmaceutical development. One challenge has been site specific conjugation. Chemoenzymatic methods have recently been developed to remove heterogeneous glycan fragments from the mABs and then incorporate homogeneous azide-containing replacements for subsequent bioorthogonal functionalisation. This is leading to structurally homogeneous conjugates which may have improved pharmacological properties, and which may have benefits in manufacturing, such as better control in batch-to-batch consistency. Synthesis of fluorophore containing glycoconjugates with application in imaging is also highlighted; specifically, methods for synthesis of BODIPY-carbohydrate hybrids are detailed. This includes mention of applications of such conjugates ranging from study of glycan uptake into cells, activity based profiling of carbohydrate binding protein targets and photodynamic therapy. The carbohydrate synthesis field needs to continue to make progress in methods to produce ever more complex target compounds, such as in improving protecting group chemistry, glycosylation reactions as well as strategies for synthesis of glycomimetics and target oriented synthesis of naturally occurring carbohydrate derivatives. The development of chemistries for the preparation of glycoconjugates in the absence of protecting groups is also very important. Although this research field is growing well, grand challenges still remain. Much of the science presented in this issue is relevant to biomedical and pharmaceutical sciences. Yet carbohydrate chemistry has and will continue to have a key role in sustainability sciences, given carbohydrates are renewable. Progress in chemistry of carbohydrates and attaining greater understanding of their reactivity will no doubt be critical for future developments. We are very optimistic that the field will continue to develop in an exciting way. We greatly thank all contributing authors who have made this issue possible. Thanks also go to Dr. Dinesh Talwar of Wiley-VCH for his work to put the issue together and for his patience with authors, extending the deadline for submission of articles, which was a great help during the pandemic.

Heterologous biosynthesis as a platform for producing new generation natural products

Multidrug resistance occurs when a tumor develops resistance to multiple chemotherapeutic drugs, which may include antitumor drugs with different chemical structures and mechanisms. Multidrug resistance limits the treatment effects of antitumor drugs, and is the main cause of chemotherapy failure. Multidrug resistance is caused by numerous factors including changes in ATP-binding cassette transporters, target proteins, detoxification, deoxyribonucleic acid repair, drug metabolic enzymes, and signal pathways of apoptosis. Clinical research indicates that natural products have great potential to treat tumors and reverse multidrug resistance. Natural products, which often have multiple targets, could play an important role in tumor treatment, have beneficial effects on tumor inhibition, improve symptoms, reduce radiotherapy and chemotherapy side effects, enhance immunity, and prolong survival. Because natural products often have few adverse reactions and less drug resistance, the antitumor activities of natural products have attracted extensive research. We aimed to review the basic research and clinical application of natural products in the reversal of multidrug resistance.

Natural products have been attracting much interest around the world for their diverse applications, especially in drug and food industries. Plants have been a major source of many different natural products. However, plants are affected by weather and environmental conditions and their successful extraction is rather limited. Chemical synthesis is inefficient due to the complexity of their chemical structures involving enantioselectivity and regioselectivity. For these reasons, an alternative means of overproducing valuable natural products using microorganisms has emerged. In recent years, various metabolic engineering strategies have been developed for the production of natural products by microorganisms. Here, the strategies taken to produce natural products are reviewed. For convenience, natural products are classified into four main categories: terpenoids, phenylpropanoids, polyketides, and alkaloids. For each product category, the strategies for establishing and rewiring the metabolic network for heterologous natural product biosynthesis, systems approaches undertaken to optimize production hosts, and the strategies for fermentation optimization are reviewed. Taken together, metabolic engineering has enabled microorganisms to serve as a prominent platform for natural compounds production. This article examines both the conventional and novel strategies of metabolic engineering, providing general strategies for complex natural compound production through the development of robust microbial‐cell factories.

Advancements in next-generation sequencing (NGS) technologies are generating a vast amount of data. This exacerbates the current challenge of translating NGS data into actionable clinical interpretations. We have comprehensively combined germline and somatic nonsynonymous single-nucleotide variations (nsSNVs) that affect drug binding sites in order to investigate their prevalence. The integrated data thus generated in conjunction with exome or whole-genome sequencing can be used to identify patients who may not respond to a specific drug because of alterations in drug binding efficacy due to nsSNVs in the target protein’s gene. To identify the nsSNVs that may affect drug binding, protein–drug complex structures were retrieved from Protein Data Bank (PDB) followed by identification of amino acids in the protein–drug binding sites using an occluded surface method. Then, the germline and somatic mutations were mapped to these amino acids to identify which of these alter protein–drug binding sites. Using this method we identified 12 993 amino acid–drug binding sites across 253 unique proteins bound to 235 unique drugs. The integration of amino acid–drug binding sites data with both germline and somatic nsSNVs data sets revealed 3133 nsSNVs affecting amino acid–drug binding sites. In addition, a comprehensive drug target discovery was conducted based on protein structure similarity and conservation of amino acid–drug binding sites. Using this method, 81 paralogs were identified that could serve as alternative drug targets. In addition, non-human mammalian proteins bound to drugs were used to identify 142 homologs in humans that can potentially bind to drugs. In the current protein–drug pairs that contain somatic mutations within their binding site, we identified 85 proteins with significant differential gene expression changes associated with specific cancer types. Information on protein–drug binding predicted drug target proteins and prevalence of both somatic and germline nsSNVs that disrupt these binding sites can provide valuable knowledge for personalized medicine treatment. A web portal is available where nsSNVs from individual patient can be checked by scanning against DrugVar to determine whether any of the SNVs affect the binding of any drug in the database.

A DNA-encoded library for the identification of natural product binders that modulate poly (ADP-ribose) polymerase 1, a validated anti-cancer target

Proteins interact with small molecules through specific molecular recognition, which is central to essential biological functions in living systems. Therefore, understanding such interactions is crucial for basic sciences and drug discovery. Here, we present Structure template-based ab initio ligand design solution (Stalis), a knowledge-based approach that uses structure templates from the Protein Data Bank libraries of whole ligands and their fragments and generates a set of molecules (virtual ligands) whose structures represent the pocket shape and chemical features of a given target binding site. Our benchmark performance evaluation shows that ligand structure-based virtual screening using virtual ligands from Stalis outperforms a receptor structure-based virtual screening using AutoDock Vina, demonstrating reliable overall screening performance applicable to computational high-throughput screening. However, virtual ligands from Stalis are worse in recognizing active compounds at the small fraction of a rank-ordered list of screened library compounds than crystal ligands, due to the low resolution of the virtual ligand structures. In conclusion, Stalis can facilitate drug discovery research by designing virtual ligands that can be used for fast ligand structure-based virtual screening. Moreover, Stalis provides actual three-dimensional ligand structures that likely bind to a target protein, enabling to gain structural insight into potential ligands. Stalis can be an efficient computational platform for high-throughput ligand design for fundamental biological study and drug discovery research at the proteomic level. © 2019 Wiley Periodicals, Inc.

Abnormal protein aggregation has been linked to many neurodegenerative diseases, including Parkinson's disease (PD). The main pathological hallmark of PD is the formation of Lewy bodies and Lewy neurites, both containing the pre-synaptic protein α-synuclein (α-syn). Native α-syn, under normal conditions, exists in a soluble unfolded state but undergoes misfolding and aggregation into toxic aggregates under pathological conditions. Toxic α-syn species can cause oxidative stress, membrane penetration, synaptic and mitochondrial dysfunction, leading to neuronal death and eventually neurodegeneration. Currently, early diagnosis and treatments targeting PD pathogenesis are urgently needed. Given its critical role in PD, α-syn is an attractive target for the development of both diagnostic tools and effective therapeutics. This thesis consists of a series of published and unpublished papers. In Chapter 1, which was published as a review, the progress towards discovering imaging probes and aggregation inhibitors for α-syn was summarized. Since a key property of such required therapeutic agents is specific binding to the target protein, relevant strategies and techniques in the discovery of α-syn-targeted drugs are discussed. As my PhD project aimed to screen small molecules capable of binding to α-syn specifically and then discover new α-syn aggregation inhibitors from the screened structures, relevant techniques were discussed at the end of Chapter 1. Mass spectrometry was chosen to discover specific α-syn binding molecules as this technique allows rapid detection of direct interactions between molecules and proteins. The materials and methods that were used in the included publications, were summarized in detail in Chapter 2. To provide sufficient protein for our study, the in-house α-syn having equally good quality as the commercial protein, was successfully generated in Chapter 3. Also, high yield of pure protein can be acquired from medium scale of bacteria culture, saving plenty of time and money for preparing proteins for large-scale screening. The protein expression and purification was a part of the supplementary data in the publication included in Chapter 4, where an automated screening system based on the connection of a mass spectrometer and the auto-sampler from a high performance liquid chromatograph was successfully established. This system allows computer-controlled sample loading and data acquisition with high stability and reproducibility. We first discovered a new inhibitor by screening over 4,300 pure molecules. The new compound, 3-[(3-methoxyphenyl)carbamoyl]-7-[(E)-2-phenylethenyl]-4,7- dihydropyrazolo [1,5-a]pyrimidine-5-carboxylic acid, not only significantly inhibited the misfolding and aggregation of α-syn, protected neuroblastoma cells from α-syn toxicity, but also has a more specific binding site compared with positive controls. The capability of the MS-based screening was further extended to the discovery of active components from natural products (manuscript in submission). A total of 29 marine fractions from our collaborators, were tested by MS and a new cholesterol derivative with significant inhibition of α-syn aggregation, was discovered and isolated from the active fraction. This MS-guided isolation of active components from natural products can also be applied to investigating traditional Chinese medicines with known therapeutic effects. Post-translational modifications (PTMs) of α-syn, especially enzymatic glycosylation with N-acetylglucosamine (GlcNAc) onto the proteins hydroxylated amino acid residues, have been reported to affect the pathogenic self-assembly of α-syn. As such, manipulation of the proteins' O-GlcNAcylation statuses has been proposed to offer a therapeutic route toward addressing PD. In Chapter 5, small peptides with different sequences and modification sites were synthesized by our collaborators. In the thioflavin-T assay, which is a golden standard for measuring α-syn aggregation, two peptides with O-GlcNAcylation at the serine site exhibited significant inhibition. Therefore, small glycopeptides that couple the protective effects of O-GlcNAc with the selectivity of recognition sequences may prove useful tools to modulate α-syn aggregation (manuscript under review). Other sources of compounds including new analogs of anle138b, which is a well-studied α-syn aggregation modulator, were evaluated. Two derivatives of anle138b exerted promising effects on the aggregation of α-syn. Interestingly, these synthesized compounds and peptides did not form protein-ligand complexes in the mass spectra, indicating that these molecules, unlike the compounds we discovered in Chapter 4, may interact with α-syn aggregates instead of α-syn monomers. In the last chapter, general conclusions of the thesis were made and future directions were also discussed.

Natural products have proven to be valuable sources for the identification of new drug candidates, and also as tools for chemical biology and medicinal chemistry research [1]. In fact, if we trace back human history, many natural products have also long been used to treat various human disorders and distinguished by their drug-like properties [2]. To date, tens of thousands of bioactive compounds have been isolated from plants, microbes, marine invertebrates, and other sources [3]. Consequently, these chemical structures have been employed by chemists as references to scan the diversity space for drug discovery efforts [4]. It is estimated that 50–70% of launched drugs in the marketplace are either natural products themselves or natural product-derived molecules [5]. So, what features of natural products make them effective drug candidates? Natural products play important roles in biomedical research and drug discovery largely attributable to their structural complexity and diversity, which Nature has engineered to facilitate optimal functions of living systems. Structural complexity and diversity enable natural products to modulate biological targets of human diseases. The drug-likeness of natural products often possesses common factors including molecular complexity, ability to bind to proteins, structural rigidity, and three-dimensionality [6]. Comparedwith synthetic molecules, the chemical structures of natural products are more constrained, which allows the accumulation of reliable structure–activity relationship (SAR) data for studying protein target–chemical ligand interactions [7]. Recently, much attention has been devoted to the natural products which are an obvious violation of ‘‘Rule-of-Five’’ [8], but still possess reasonable biopharmaceutical and pharmacokinetic properties, for example, ciclosporin A. Thus, exploitation of such types of natural products could not only significantly broaden the chemical space beyond the ‘‘Rule-of-Five’’ domain for the purpose of drug discovery, but also give us the opportunity to appreciate and investigate how Nature selects and optimizes natural products which are able to bind to and disrupt the function of biological targets through evolution. We should also recognize that natural products were optimized in living systems presumably not for the same purpose as is desired to serve in a biomedical research

Better than one: A novel class of PROTAC that recruits target protein for ubiquitination and subsequent degradation by the proteasome has been successfully synthesized and tested for its activity to degrade a target protein. Detailed facts of importance to specialist readers are published as "Supporting Information". Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

This thesis is separated in three main parts including a general introduction, three chapters based on research projects and the last chapters with the experimental procedures and analytical data. The focus of this work is based on the isolation of natural products and their application in chemistry and biology. Chapter 1: The first chapter consists in a general introduction about natural products, where their importance in drug discovery and in the interaction between living organisms is highlighted. Several examples of natural products from different origins are described. Chapter 2: The study of a system living in obligatory symbiosis is addressed in this chapter. Psychotria kirkii is a plant having the particularity to be in symbiosis with a bacterial strain named Candidatus Burkholderia kirkii and the presence of this microorganism was found to be essential for the plant’s survival. Interestingly, the bacterial genomic sequencing and analysis revealed the presence of a putative C7N aminocyclitol. This project led to the isolation, total synthesis and biological evaluation of kirkamide, a new C7N aminocyclitol from Candidatus Burkholderia kirkii. In the course of the chemical composition analysis of Psychotria kirkii, another new natural product (streptol glucoside) was isolated and its structure was elucidated. The last part of this chapter focuses on detection and quantification methods for the analysis of these natural products. Chapter 3: After completing a genome-driven bioassay-guided fractionation, a natural product possessing an unusual N-nitrosohydroxylamine functional group was isolated. The formation of the N-N bond in biological system has yet to be elucidated. Chapter 3 addresses the question regarding the biosynthetic analysis of the N-nitrosohydroxylamine compound. Furthermore, the mechanism of the N-N bond formation was investigated in detail using model compounds. Chapter 4: The potential of secondary metabolites has been recognized for the discovery of biological active compounds. The strength of natural products resides in the diversity and complexity of their chemical structures. In this chapter the isolation, structure elucidation and biological evaluation of new natural products from cyanobacteria are described. Three new cyanobactins were isolated from the cyanobacteria Microcystis aeruginosa EAWAG 251. The stereoassignement of these natural products, named balgacyclamides A–C, is presented with their biological activity investigations against the parasite Plasmodium falciparum. The last part of the chapter describes a bioassay-guided fractionation leading to the discovery of two new glycolipopeptides from cyanobacteria. The efforts achieved towards the structure elucidation using HRMS tandem mass spectrometry, NMR and amino acid analysis are presented.

Family 18 chitinases play key roles in the life cycles of a variety of organisms ranging from bacteria to man. Very recently it has been shown that one of the mammalian chitinases is highly overexpressed in the asthmatic lung and contributes to the pathogenic process through recruitment of inflammatory cells. Although several potent natural product chitinase inhibitors have been identified, their chemotherapeutic potential or their use as cell biological tools is limited due to their size, complex chemistry, and limited availability. We describe a virtual screening-based approach to identification of a novel, purine-based, chitinase inhibitor. This inhibitor acts in the low micromolar (Ki=2.8+/-0.2 microM) range in a competitive mode. Dissection of the binding mode by x-ray crystallography reveals that the compound, which consists of two linked caffeine moieties, binds in the active site through extensive and not previously observed stacking interactions with conserved, solvent exposed tryptophans. Such exposed aromatics are also present in the structures of many other carbohydrate processing enzymes. The compound exhibits favorable chemical properties and is likely to be useful as a general scaffold for development of pan-family 18 chitinase inhibitors.