Native Mass Spectrometry-Guided Screening Identifies Hit Fragments for HOP-HSP90 PPI Inhibition

Contemporary medicinal chemistry considers fragment‐based drug discovery (FBDD) and inhibition of protein‐protein interactions (PPI) as important means of expanding the volume of druggable chemical space. However, the ability to robustly identify valid fragments and PPI inhibitors is an enormous challenge, requiring the application of sensitive biophysical methodology. Accordingly, in this study, we exploited the speed and sensitivity of nanoelectrospray (nano‐ESI) native mass spectrometry to identify a small collection of fragments which bind to the TPR2AB domain of HOP. Follow‐up biophysical assessment of a small selection of binding fragments confirmed binding to the single TPR2A domain, and that this binding translated into PPI inhibitory activity between TPR2A and the HSP90 C‐terminal domain. An in‐silico assessment of binding fragments at the PPI interfacial region, provided valuable structural insight for future fragment elaboration strategies, including the identification of losartan as a weak, albeit dose‐dependent inhibitor of the target PPI

Mass spectrometry-based methods for characterizing transient protein–protein interactions

The dynamic associations of transient protein–protein interactions (PPIs) are critical mediators of myriad biochemical processes. These specific, low-affinity interactions are often mediated by conserved amino acid sequences or short linear motifs (SLiMs) that interact with corresponding binding domains. The short-lived and dynamic nature of these interactions make their biophysical characterization a significant challenge. This review focuses on the development and future directions of mass spectrometry (MS)-based techniques for elucidating and characterizing SLiM-mediated PPIs. This includes the application of protein footprinting techniques to infer the location of SLiM binding sites and the growing role of native MS for direct observation of protein–SLiM interactions, highlighting their potential for the assessment of small molecule modulation of transient PPIs and the identification of interfacial SLiMs.</p

Strategies and Functional Consequences of Inhibiting Protein-Protein Interactions.

Networks of protein-protein interactions (PPIs) are essential in all aspects of cellular biology. At the nodes of these networks are multi-protein complexes that are often composed of dynamic, exchangeable modules assembled around a central enzyme. In this thesis, I have used the molecular chaperone heat shock protein 70 (Hsp70) as a model to develop ways of creating inhibitors of PPIs that tune the assembly and function of multi-protein complexes. Hsp70 is an ATPase and master regulator of protein homeostasis that interacts with co-chaperones, including nucleotide exchange factors (NEFs) and J-proteins. There is interest in creating chemical inhibitors that selectively interrupt PPIs between Hsp70 and its co-chaperones, as these molecules would be powerful chemical probes for validating Hsp70 as a target in cancer and other diseases. In this dissertation, I first review how advances in chemical screening methodologies, structural and computational biology, and proteomics have paved the way for the discovery of potent PPI inhibitors, even for difficult targets such as Hsp70 complexes. In Chapter 2, I develop a new high throughput screening (HTS) method in which Hsp70 is combined with co-chaperones and the ATPase activity of the combination is measured. I use this method to identify new inhibitors of Hsp70, characterizing their binding sites and molecular mechanism by NMR, mutagenesis and biochemical approaches. Importantly, I found that this HTS method reveals inhibitors of multiple PPIs within the Hsp70 system, including the interactions with NEFs and J proteins. This approach allowed me to find that Hsp70-NEF complexes control the stability of inhibitor of apoptosis (IAP) protein family members. In Chapter 3, I characterize IAPs as new “clients” of the Hsp70 system and explore the physical interaction between these proteins. That work establishes IAPs as the first sensitive, selective biomarkers suitable for use in pre-clinical studies of Hsp70 inhibitors. Finally, I show how inhibiting the Hsp70-NEF interaction has effects throughout the broader PPI network in Chapter 4. Together, these findings not only have important implications for Hsp70 drug discovery, but they also illustrate, more broadly, how small molecules can be used to re-shape multi-protein complexes and propagate changes throughout PPI networks.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/135910/1/ceslaura_1.pd

Strategies for Modulating the Diverse Activities of Heat Shock Protein 70.

Heat shock protein 70 (Hsp70) is an essential regulator of protein homeostasis. Dysfunction of protein homeostasis is directly linked to many diseases, including cancer and neurodegeneration. Thus, an understanding of Hsp70’s roles in this process is expected to provide insights into the mechanisms of disease and, potentially, provide new opportunities for therapies. However, Hsp70 is also involved in essential cellular functions, so it is not clear how to safely target it. In this thesis, I first review how Hsp70 cooperates with co-chaperones to enable its many activities. Hsp70 binds to distinct co-chaperones to form complexes that have individual functions in protein folding, degradation and trafficking, suggesting that inhibition of the protein-protein interactions (PPIs) between Hsp70 and its co-chaperones might be one promising way to safely modulate this system. In Chapter 2, I performed a comprehensive, comparative study on how five TPR domain-containing co-chaperones bind to Hsp70 in vitro. These experiments highlighted the opportunities and challenges of targeting this PPI. In Chapter 3, I demonstrate how allosteric networks in Hsp70 can be manipulated, using both chemical and genetic approaches, in order to regulate binding to co-chaperones and tune chaperone activity in unexpected ways. Taking all this information together, I show in Chapter 4 that allosteric inhibitors of Hsp70 have surprisingly potent antibiotic activity in drug-resistant bacteria, which seem to rely on robust protein homeostasis. By better understanding allostery and PPIs in the Hsp70 network, I made new insights into Hsp70 biology and also discovered new lead compounds for therapeutic development.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/116624/1/vaa_1.pd

Advanced Technologies for Protein Complex Production and Characterization

This edited volume discusses the identification, discovery, characterization, structure determination and modeling of multicomponent macromolecular complexes, and as such, it fully complements the first volume (ISBN 978-3-319-27214-6), which targeted methods of recombinant production of protein complexes. This book is divided in 8 sections offering a selection of technologies widely used in the characterization of protein and protein-nucleic acid complexes for different purposes and at different scales. From native electrophoresis methods, that are accessible to any reasonably well-equipped laboratory, to the sophisticated setup required for structure determination by cryo-electron microscopy or X-ray crystallography, this book contains a wide variety of clearly explained analytic and preparative approaches, connected with the production techniques developed on the previous volume. The readers will find an integral connection between this book and the first volume, which ensures a comprehensive and updated discussion of the main topics of the discipline. Taken together, these volumes constitute a cohesive and authoritative source of the research on multicomponent macromolecular complexes. In here, we focus on characterization of protein complexes in the broadest sense, which is not typically covered in other sources. Moreover, all chapters are carefully written by world renowned scientists and active researchers, making this volume ideal, not only as a reference source, but also as a companion book for the daily laboratory work. This book is aimed for a wide range of scientists, from science students to experienced researchers, working on protein and protein-nucleic acid complexes, who need a thorough understanding of protein production and complex characterization

Protein-ligand interactions in native mass spectrometry: Applications in medicinal and environmental chemistry

Protein-ligand interactions play a crucial role in various biological processes of living organisms. Thus, characterising protein-ligand interactions is important for understanding the mechanism of ligands in signalling pathways as well as developing novel drugs. This thesis describes the application of native mass spectrometry (MS) to studying protein-ligand interactions in medicinal chemistry and environmental chemistry, and the use of additives to stabilise the proteinligand complexes in the gas phase for improved native MS measurements. Chapter 2 describes the synthesis and biological evaluation of peptides targeting heat shock protein 27 (Hsp27). Hsp27’s main function is to bind misfolded proteins and prevent them from aggregating. However, the aberrant activity of Hsp27 has been implicated in numerous diseases such as cancers and neurodegenerative diseases. An analogue of peptides derived from the N-terminal domain of Hps27 was identified to bind to Hsp27 using native MS for the first time. To date, these are also the smallest peptides reported to bind to Hsp27 and affect citrate synthase aggregation. This native MS approach may be useful for identifying other promising compounds targeting Hsp27. The aim of the research detailed in Chapter 3 is to identify per and polyfluoroalkyl substances (PFASs) that can bind to the major whey protein β-lactoglobulin (BLG) in bovine milk. PFASs are a topic of global health concern owing to their persistence in the environment, bioaccumulation and toxic effects in humans and animals. Native MS was used to investigate the binding affinities and the binding site of PFASs to BLG. Six out of 19 of the PFASs investigated can bind strongly to two isoforms of BLG at the same binding site as endogenous ligands. These results demonstrate a potential route for the bioaccumulation of PFASs in cow milk and an approach to remove PFASs from BLG. Chapter 4 aims to investigate the effect of the Hofmeister series of anions on protein-ligand interactions in native MS. For β-lactoglobulin and stearic acid system, the addition of citrate and tartrate to the ESI solutions resulted in significant stabilisation of the complex such that the binding affinities increased by a respective 42% and 40% compared to using ammonium acetate alone. For lysozyme binding to tri-N-acetylchitotriose, bromide and thiocyanate dramatically decreased the dissociation constants especially at lower charge states. Therefore, some specific anions can be added to the solution to stabilise protein-ligand complexes in the gas phase and improve their dissociation constants

Structure-based Search for novel c-di-AMP Synthase Inhibiting Fragments

One of the major threats for the global health system is the increasing number of antibiotic resistant bacterial strains. The misuse of antibiotics during the Covid pandemic aggravates these problems. Therefore, the development of new antibiotics is mandatory to fight the rising threat of multi-resistant bacteria. The essential second messenger c-di-AMP was discovered in 2008 and is mainly found in gram positive bacteria. It has been identified in several human pathogens like Listeria monocytogenes, Staphylococcus aureus or Enterococcus faecalis. It is a key player in the regulation of several pathways like DNA integrity scanning, cell wall metabolism or osmolyte homeostasis. Five different protein classes are able to synthesize c-di-AMP They all have the diadenylate cyclase domain (DAC) in common and need to dimerize to produce c-di-AMP in a metal-ion dependent manner from two ATP molecules. The protein class CdaA consists of three N-terminal transmembrane helices followed by the DAC domain and is often the sole diadenylate cyclase in several human pathogenic bacteria like Staphylococcus aureus, Streptococcus pneumoniae or Enterococcus faecium. The essentiality of c-di-AMP renders CdaA as potential target for the development novel antibiotic substances. The major goal of this thesis was the establishment of a crystallization system of CdaA suitable for a fragment screening campaign in order to identify starting points for the development of inhibitors of CdaA. Therefore, the first part of this work is focused on the crystallization of CdaA from the soil bacterium Bacillus subtilis and the human pathogens Listeria monocytogenes, Staphylococcus aureus, Streptococcus pneumoniae and Enterococcus faecium. To obtain constructs well suited for crystallography, the CdaAs of these organisms were N- and C-terminal truncated to solely consist of the DAC domain. The purity and homogeneity of every purified CdaA were verified via SDS-PAGE and DLS. To prove the enzymatic functionality of the purified proteins, the coralyne activity assay was applied with varying divalent cations. All purified CdaAs exhibit activity in the presence of manganese ions while their activity in presence of magnesium- or cobalt-ions differs. To explore these variations, crystallization trials of the different CdaAs in presence of their favorable metal-ion and ATP were carried out. As a result, the crystal structure of Enterococcus faecium CdaA in complex with c-di-AMP and manganese as well as the crystal structure of Streptococcus pneumoniae CdaA in complex with c-di-AMP and magnesium were solved at a resolution of 2.1 Å (EfCdaA) and 2.2 Å (SpCdaA). Unfortunately, both models represent a post catalytic state where the metal ion is not coordinated in a catalytically active way, giving no further insights into the structural basis for the differing metal ion specificity. Nevertheless, if both obtained structures are compared to the Listeria monocytogenes CdaA structure in complex with c-di-AMPa conserved interaction pattern with c-di-AMP could be observed, rendering the interacting amino acids as possible targets for inhibitor design. In order to identify potential fragments which can reduce the activity of CdaA, the CdaAs from all previously mentioned organisms were applied to crystallization trials in their APO state. For the CdaA of Enterococcus faecium and Bacillus subtilis these trials were successful. However, the crystallization system for Enterococcus faecium CdaA does not matches the requirements for a crystallographic fragment screening campaign as the crystals diffracted just to 2.4 Å and required several months to grow. In contrast, the Bacillus subtilis APO crystals diffracted up to 1.6 Å and were highly reproduceable. Further optimization of the crystallization condition led to a crystallization system which is greatly suitable for a fragment screening campaign. Through the following fragment campaign using the F2X-Entry screen, 32 unique fragments were identified to interact with BsCdaA. Mapping these fragments on a Consurf model of CdaA reduced the number to six different fragments which bound in the highly conserved active/dimerization site. These fragments were also biochemically characterized using the coralyne and malachite green assay. As none of the fragments reduced the cyclase activity of BsCdaA, an alternative approach was carried out to identify an inhibitory compound. Here, the structure of BsCdaA in complex with AMP, which were determined in this work served as starting point for the identification of an inhibitory compound. By a computational defragmentation approach employing SeeSar, adenine was identified as fragment of AMP which is largely responsible for the biding capability of AMP. Combing the methods of computer aided drug design and structural information gained by X-ray crystallography, the Janus kinase inhibitor Ruxolitinib was identified as potential inhibitory compound for CdaA. The conserved binding mode of Ruxolitinib towards CdaA was proven as Ruxolitinib exhibits the same interaction pattern for CdaA from Bacillus subtilis and Listeria monocytogenes as it can be concluded by the X-ray structures obtained in this work as Ruxolitinib binds in the active site of the protein, it most likely acts as competitive inhibitor for CdaA. Moreover, the IC50 value of Ruxolitinib was determined as 2.7 µM for BsCdaA. Determining the cyclase activity of CdaA from Listeria monocytogenes, Staphylococcus aureus, Streptococcus pneumoniae or Enterococcus faecium in presence of Ruxolitinib also show a reduced activity, underlining the conservation of the protein-ligand interaction. Besides the in vitro studies, also in vivo experiments utilizing different Bacillus subtilis strains were carried out. The results from these experiments suggest a high specificity of Ruxolitinib towards Bacillus subtilis CdaA in vivo.2023-03-3

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Development and prospective application of chemoinformatic tools to explore new ligand chemistry and protein biology

Drug discovery and design is a tedious and expensive process whose small chances of success necessitates the development of novel chemoinformatic approaches and concepts. Their common goal is the efficient and robust identification of promising chemical matter and the reliable prediction of its properties. Computer-aided drug discovery and design (CADDD) and its multifarious installments throughout the different phases of the drug discovery pipeline contribute significantly to the expansion of the hits, the understanding of their structure-activity relationship and their rational diversification. They alleviate the development’s costs and its time-demand thus support the search for the needle in the haystack – a potent hit. The HTS-driven brute-force nature of current and of the decades’ past discovery and design strategies compelled researchers to develop ideas and algorithms in order to interfere with the pipeline and prevent its frequent failures. In the introduction, I describe the drug discovery and design pipeline and point out interfaces where CADDD contributes to its success. In Part 1 of this thesis, I present a novel methodology that supports the early-stage hit discovery processes through a fragment-based reduced graph similarity approach (RedFrag). It is a chimeric algorithm that combines fingerprint-based similarity calculation with scaffold-hopping-enabling graph isomorphism. We thoroughly investigated its performance retro- and prospectively. It uses a new type of reduced graph that does not suffer from information loss during its construction and bypasses the necessity of feature definitions. Built upon chemical epitopes resulting from molecule fragmentation, the reduced graph embodies physico-chemical and 2D-structural properties of a molecule. Reduced graphs are compared with a continuous-similarity-distance-driven maximal common subgraph algorithm, which calculates similarity at the fragmental and topological levels. The second chapter, Part 2, is dedicated to PrenDB: A digital compendium of the reaction space of prenyltransferases of the dimethylallyltryptophan synthase (DMATS) superfamily. Their catalytical transformations represent a major skeletal diversification step in the biosynthesis of secondary metabolites including the indole alkaloids. DMATS enzymes thus contribute significantly to the biological and pharmacological diversity of small molecule metabolites. The attachment of the prenyl donor to lead- or drug-like molecules renders the prenyltransferases useful in the access of chemical space that is difficult to reach by conventional synthesis. In PrenDB, we collected the substrates, enzymes and products. We then used a newly developed algorithm based on molecular fragmentation to automatically extract reactive chemical epitopes. The analysis of the collected data sheds light on the thus far explored substrate space of DMATS enzymes. We supplemented the browsable database with algorithmic prediction routines in order to assess the prenylability of novel compounds and did so for a set of 38 molecules. In a case study, Part 3, we investigated the regioselectivity of five prenyltransferases in the presence of unnatural prenyl donors. Detailed biochemical investigations revealed the acceptance of these dimethylallyl pyrophosphate (DMAPP) analogs by all tested enzymes with different relative activities and regioselectivities. In order to understand the activity profiles and their differences on a molecular level we investigated the interaction within the enzyme-prenyl donor-substrate system with molecular dynamics. Our experiments show that the reactivity of a prenyl donor strongly correlates with the distance of its electrophilic, reactive atom and the nucleophilic center of the substrate molecule. It renders the first step towards a better mechanistic understanding of the reactivity of prenyltransferases and expands significantly the potential usage and rational design of tryptophan prenylating enzymes as biocatalysts for Friedel–Crafts alkylation. Lastly, in Part 4, we present the synergistic potential of combined ligand- and structure-based drug discovery methodologies applied to the β2-adrenergic receptor (β2AR). The β2AR is a G protein-coupled receptor (GPCR) and a well-explored target. By the joint application of fingerprint-based similarity, substructure-based searches and docking we discovered 13 ligands – ten of which were novel – of this particular GPCR. Of note, two of the molecules used as starting points for the similarity and substructure searches distinguish themselves from other β2AR antagonists by their unique scaffold. Thus, the usage of a multistep hierarchical or parallel screening approach enabled us to use these unique structural features and discover novel chemical matter beyond the bounds of the ligand space known so far and emphasize the intrinsic complementarity of ligand- and structure-based approaches. The molecules described in this work allow us to explore the ligand space around the previously reported molecules in greater detail, leading to insights into their structure-activity relationship. In addition, we also characterized our hits with experimental binding and selectivity data and discussed it based on their putative binding modes derived by docking