Development of a combined chemical biology tool using synthetic probes and tandem mass spectrometry to elucidate heat shock protein 90 KDA's C-terminal- domain binding sites

Heat shock protein 90 kDa (Hsp90) has long been an appealing drug target for cancer treatment due to its pivotal role in conformational maturation and refolding of many proteins directly related to malignant progression. Previously various Hsp90 N-terminal domain (NTD) inhibitors have progressed to clinical trials in patients, unfortunately these studies were suspended due to high toxicity-related issues. On the other hand, the lack of a high-resolution crystal structure of the C-terminal domain (CTD) of human Hsp90 hampers the discovery of next generations of CTD inhibitors and presents challenges for further structure-based drug design. This study attempts to provide validated evidence for the Novobiocin binding site in yeast Hsp90 CTD by combinatorial use of photochemistry-based affinity protein labelling and analytical tools including NMR and mass spectrometry. This research on completion will shed light on the development of Hsp90 CTD inhibitors, which provide an alternative route to effective development of cancer drug candidates based on inhibition/interruption of Hsp90. The use of ligands conjugated to photoaffinity labelling (PAL) reagents for chemical modification of active site residues can circumvent limitations such as lack of complete crystal structure in CTD region, allowing detailed information on the sites of protein-ligand interactions to be acquired through high resolution MS and MS/MS experiments. Bifunctional PAL compounds incorporated a diazirine group and a novobiocin component, based on a previously reported glucosyl-novobiocin scaffold, which has been reported to demonstrate 200-fold increase in CTD inhibitory effects towards Hsp90 activities compared to that of novobiocin. The synthesis of the diazirine group we developed alleviated the high temperature and highpressure conditions reported in literature. Also, in the functionalisation of the affinity providing novobiocin was based on the Williamson ether mechanism and resulted in regioisomers. An isomer due to rearrangement from coumarin to flavone in novobiocin core was identified by advanced NMR, and this phenomenon has recently been reported by our group. Further detailed studies of photoactivation revealed the physiochemical properties of these probes, and such information was very critical for the ease of data analysis in the protein labelling stage. These novobiocin derivatives were activated under UV irradiation in the presence of Hsp90 proteins and adducts were detected using detailed MS and MS/MS analysis. The protein-PAL complexes were identified using MALDITOF MS and related techniques to confirm the labelling of yeast Hsp90. Complexes were further subjected to enzymatic digestion and result products were analysed by tandem MS (Orbitrap MS). Currently, the peptide mapping of chemically labelled proteins and localisation of chemically labelled amino acid are frequently carried out by manual analysis, which is time-consuming and requires relatively high level of modified peptide in the enzymatic digestion mixture. In this project, by using PeptideShaker with custom modification of the pre-identified mass difference, the modified peptide (at W585) was identified at relatively low protein labelling yield. W585 in yeast Hsp90 has been recently reported to be related to client remodelling coincident with stabilizing yHsp90 in an open conformation. The peptide region in the vicinity of this residue was further analysed and the binding pocket was identified by correlating the experimental results to ICM Pro based molecular docking, providing a direct visualisation of the yeast Hsp90 CTD binding site. Even these probes demonstrated low affinity towards human Hsp90, given the close homology between Hsp90 tertiary structures from yeast and human origins, the combination of both experimental and computational results obtained yeast Hsp90 labelling is expected to cast light on the understanding of Hsp90-novobiocin binding rationale, and contribute to future structure based drug design (SBDD) in the search for more potent Hsp90 C-terminal domain inhibitors

Dopamine Transporter (DAT), Nicotinic Acetylcholine Receptor (nAChR), and Metabotropic Glutamate Receptor 2 (mGlu2) Irreversible Probes For Identifying Anti-Psychostimulant Therapeutics

Numerous in vitro and in vivo studies implicate that certain ligands that interact with DAT, nAChRs, and mGlu2 have tremendous potential as anti-addiction therapeutics. However, understanding how these promising anti-addiction compounds interact with their major drug targets at the molecular level is limited because of the absence of human DAT, nAChRs, and mGlu2 x-ray crystal structures. This knowledge gap is important towards rationally designing new therapeutics for psychostimulant abuse and addiction. The objective of this research was to develop irreversible chemical probes based on promising anti-addiction lead compounds (i.e., pyrovalerone, bupropion, BINA, etc) to map their binding sites and poses within the DAT, select nAChR subtypes, or mGlu2. The central hypothesis was that these compounds could be rationally derivatized, without significant alteration in their pharmacological activity, with a photoreactive group capable of forming a covalent bond to their target protein and a tag for application of a Binding Ensemble Profiling with (f)Photoaffinity Labeling (BEProFL) experimental approach. BEProFL rationally couples photoaffinity labeling, chemical proteomics, and computational molecular modeling to allow structure-function studies of the target proteins. This central hypothesis was tested by pursuing three specific aims: 1.) Identification of non-tropane photoprobes based on pyrovalerone (PV) suitable for DAT structure-function studies, 2.) Identification of bupropion (BP)-based photoprobes suitable for DAT, and nAChR structure-function studies, and 3.) Identification of irreversible mGlu2 PAM ligands as chemical probes suitable for mGlu2 structure-function studies. In the first aim, PV, a non-tropane DAT inhibitor, was structurally modified to contain a photoreactive group (i.e., an aryl azide) and a tag (i.e., 125I). These photoprobes were then pharmacologically evaluated to identify suitable candidates for DAT structure-function studies. In the second aim, BP was structurally modified to contain an aryl azide and 125I. This probe successfully identified the exact location of the bupropion-binding site within the Torpedo nAChR. Under the third aim, biphenyl-carboxylic acid indanone- and pyridone-based mGlu2 PAMs were structurally modified to contain a photoreactive group (e.g., aryl azide, acetophenone) and a tag (e.g., terminal alkyne, aliphatic azide). These compounds, at present, are being subjected to mGlu2 pharmacological evaluation to identify suitable chemical probe candidates for mGlu2 structure-function studies

The development of carbohydrate tools to understand nutrient uptake and metabolism in mycobacteria

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB). TB is the leading cause of death worldwide from a single-infectious agent, with 1.5 million deaths in 2018. Mtb can persist in the human host in a latent state for decades, and has evolved specialised mechanisms for long term intracellular survival. However, the nutrients that are available to Mtb intracellularly during infection are not fully understood. Trehalose, a non-mammalian disaccharide, has been shown to play a key role in the survival and virulence of Mtb. It can be found in the Mtb cytoplasm, and within the cell envelope. Understanding of trehalose uptake and metabolism by mycobacteria may lead to development of new therapeutic pathways. In this Thesis we have employed a chemical biology approach to develop tools to understand the fate of trehalose in mycobacteria. A novel modular chemical probe was designed that incorporates trehalose as ‘bait’ for the enrichment and identification of the mycobacterial proteins that specifically interact with this Mtb disaccharide. A new synthetic methodology was developed and preliminary pull-down experiments using model proteins was conducted. Trehalose was also conjugated onto polymer-stabilised gold nanoparticles to exploit their colorimetric (red to blue) transitions upon aggregation as a detection tool. Quantum dot (QD) nanoparticles were also used as they can be a useful bioimaging tool due to their fluorescent properties. A series of gold and QD nanoparticles conjugated with either glucose or trehalose were synthesized and used to investigate specific binding to lectins and mycobacteria. The AuNPs gave colorimetric responses to lectins, but weaker responses to mycobacteria. Electron microscopy suggested some bacteria interactions. Conjugated QDs were found to bind to M. smegmatis non-specifically. (Mtb) is the causative agent of tuberculosis (TB) which remains one of the most difficult infectious diseases to control in the world. Mtb possesses a low number of carbohydrate transporters: four ATP-binding cassette (ABC) importers and one major facilitator superfamily permease, potentially reflecting the poor nutrient availability inside the host. This study aimed to elucidate the physiological function of three of these putative sugar transporters (UspABC, UgpABCE and SugI) and a sugar metabolism enzyme (NagA) in the model organism Mycobacterium smegmatis and the human pathogen Mtb. M. smegmatis was used as the host strain for heterologous overexpression and characterisation of Mtb_uspC and Mtb_uspABC. Furthermore, gene knockout mutants were generated in M. smegmatis for the uspC, uspABC and nagA genes and in Mtb for the nagA, sugI and ugpABCE genes. The mutant strains were subjected to phenotypic (via Biolog microarrays), transcriptomic (via RNA-sequencing) and proteomic (via mass spectrometry) analyses to elucidate global quantitative differences between each mutant and the wild type strains. Combined, the results indicated that UspABC is an importer of hexose-phosphates, SugI and NagA have an important role in amino sugar biosynthesis and UgpABCE is associated with import of a phosphate source and, separately, with fatty acid elongation. Taken together, the findings presented here provide novel insights into the physiological role of nutrient acquisition by Mtb. This knowledge of nutrient import has the potential to inform future TB diagnostics and treatment

Synthetic Studies Of Biologically Active Molecules: Irl 2500, Bruceantin, And Biliatresone

Part 1: Carbon monoxide (CO) is a poisonous gas that binds to hemoglobin, preventing oxygen transport to the tissues. CO poisoning causes ~50,000 emergency room visits per year. Treatments for CO poisoning are extremely limited. IRL 2500 is a small molecule allosteric effector of hemoglobin that has been shown to decrease the half-life of CO-bound hemoglobin (COHb). Herein we describe the synthesis of a novel, structurally constrained derivative of IRL 2500 and its effect on COHb.Part 2: Bruceantin is a natural product that has long been studied for its anticancer activity. Inspired by the structure of bruceantin, we proposed to develop a homologous Pauson Khand reaction utilizing a strained bicyclic compound to generate bridged cyclohexenones via a [3+2+1] cycloaddition as a possible method to generate the bruceantin ring system. The synthesis of suitable bicyclic substrates for this reaction has been achieved, and their viability in a rhodium- catalyzed cycloaddition has been evaluated, with nitrogen-containing bicyclic compounds having been shown to undergo ring opening reactions. Progress towards the synthesis of substrates with stabilizing groups will also be discussed. Part 3: Biliary atresia is a rare disease that is the most common indication for liver transplant among the pediatric population. Biliatresone was discovered to be an environmental toxin that recapitulates the biliary atresia phenotype. Analogs of biliatresone the could be used to identify the cellular target of the toxin have been designed and synthesized for use in a photoaffinity pulldown experiment. The best analog has been shown to cause the biliary atresia phenotype in zebrafish and has been used in experiments aimed at determining the molecular target of biliatresone. Future study and determination of the molecular target will aid in our understanding of biliary atresia

Small Molecules Target Identification Using Medicinal Chemistry and Forward Genetics

Small molecule drugs have had a profound impact on medicine, and by extension, human health and our understanding of basic biology. Understanding the genetic drivers of disease has led to the description of dozens of potentially tractable protein targets, especially in oncology, and helped facilitate 'target-based' screens to identify therapeutic small molecules. Alternatively, small molecule libraries can be applied to find phenotypes of interest, such as anti-cancer activity, but this creates the challenge of identifying the small molecule's cellular target. Knowledge of a small molecule's target allows for therapeutic development and application in an appropriate clinical setting. To date, there are few robust methods to facilitate the target identification process and this has limited the latter approach. Herein, I discuss the application of two methods to identify the cellular target and mechanism-of-action for several anti-cancer small molecules. The first method utilized medicinal chemistry and biochemistry to identify the target of a tetracyclic dicarboximide (MM0299) with anti-glioblastoma activity. I discovered that MM0299 binds to and inhibits lanosterol synthase (LSS), an enzyme involved in cholesterol biosynthesis. I further demonstrated that MM0299's mode-of-action in glioblastoma is distinct, with MM0299-mediated inhibition of LSS causing diversion of cholesterol biosynthesis and production of the toxic cholesterol metabolite, 24(S),25-epoxycholesterol. An alternative, yet complementary, approach that I utilized for small molecule target identification is forward genetics-based identification of compound-resistant alleles. Relying on random mutagenesis endowed by its intrinsic mismatch repair deficiency, our lab has used the colorectal cancer cell line, HCT116, as a forward genetics tool. Despite its utility, there are inherent challenges to identifying compound-resistant alleles using HCT116 and I have developed an inducible forward genetics platform to circumvent these challenges. I have used this system to identify the targets of several small-molecule anti-cancer toxins including orphan compounds from high throughput screens, and natural products

Development and Application of Photoresponsive Small Molecule Probes to Modulate and Characterize Neuroreceptors

The field of neuroscience is rapidly evolving and so the development of novel tools that support innovative research is urgently needed. Two tools that have proven to be particularly impactful to the field of neuroscience are activity-based protein profiling (ABPP) and optogenetics, both of which have seen widespread application but with relatively limited advancement of the underlying technology. Limitations of ABPP and optogenetics have become increasingly apparent and research output is stalling due to lack of technological advancement. For example, few systems exist for targeting low-abundance and unstable proteins via ABPP and application of ABPP and optogenetic systems thus far has largely demonstrated the technology’s utility across families of receptors rather than subtype specific targets.1-11 Direct modulation of subtype specific endogenous neuroreceptor activity is key to connecting molecular and systems neuroscience, so next generation molecular tools to address these limitations possess huge potential. Here, we have demonstrated (1) The development and application of photoactivatable forms of WAY-161503 and N-desmethylclozapine (NDMC), designed as a system that enables spectrally multiplexed, spatiotemporal controlled modulation of native human serotonin receptor 2C (5-HT2C) calcium (Ca2+) signaling and (2) The design and use of bioactive photoaffinity probes for human dopamine receptor D2 (DRD2), which demonstrate excellent activity in “workhorse” biochemical assays, receptor labeling, cell labeling, and chemoproteomics. The advancements of these technologies demonstrate an important step forward for optogenetics, where modulation of endogenous neuroreceptors has proven to be a challenge, and for ABPP, where underrepresentation of low-abundance and unstable proteins has limited the scope of advancements in the field of proteomics. Future work will continue to develop neuroreceptor targeted photoactive probes, with lysergic acid (LSA) derivatives providing an initial family of targets that are both synthetically interesting and biologically important to the fields of neuroscience, chemical biology, and medicinal chemistry

DESIGN AND SYNTHESIS OF NEW CLPP ACTIVATORS: TOWARDS THE DEVELOPMENT OF A COMPUTATIONALLY GUIDED APPROACH

With the rise of antibiotic-resistant bacteria, there is an urgent need to develop antibacterials with new mechanisms of action. Because of its essential role in bacterial survival and the possibility to induce unselective substrate degradation via activation by small molecules, ClpP is an interesting target. Given the small number of chemotypes known to target ClpP ant the limited understanding of protein-ligand interactions involved in the binding of ClpP activators, we sought to develop a computational approach to provide insight into specific ligand-ClpP interactions and address some of the structure optimization issues in the field

Mechanistic Investigations of Transcriptional Activator Function for the Design of Synthetic Replacements.

Transcriptional activators play a critical role in regulating gene expression, precisely controlling the transcriptional response of their cognate genes in a signal-responsive fashion. Transcriptional activation occurs when the activator localizes to a specific DNA sequence and facilitates the assembly of the transcriptional machinery (RNA polymerase II and associated transcription factors) at a gene. Malfunctioning transcriptional activators are associated with a variety of human diseases; greater than 50% of all cancers, for example, are associated with mutations in the transcriptional activator p53. Thus, the development of activator artificial transcriptional factors (ATFs) that functionally replicate their natural counterparts is emerging as a potential therapeutic strategy. One of the biggest hurdles to this goal is the lack of knowledge about the binding interactions utilized by natural activators to up-regulate gene expression. The major goal of this work is to delineate the features of activator binding interactions in order to develop useful activator ATFs. Natural transcriptional activators exhibit a multi-partner binding profile in vitro and there is also evidence of this in vivo, although the identities of the binding partners are unknown. To investigate the feasibility that a single binding interaction could lead to transcription function, peptide ligands for the postulated activator target Med15(Gal11) were identified through a binding screen. Satisfyingly, localizing these ligands to a promoter in S. cerevisiae results in transcription activation that is Med15(Gal11)-specific. Activator ATFs constructed with these ligands were not, however, as active as natural activators. Activator ATFs with enhanced function could be created by incorporating ligands that were able to interact with more than one partner. Ligands that interact with both Med15(Gal11) and the SAGA component Tra1 upregulated transcription to higher levels than those targeting Med15(Gal11) alone. In addition, the incorporation of a masking interaction into the activator ATFs led to a profoundly positive impact on function. Finally, towards identifying the functionally relevant binding partners of transcriptional activators, a nonsense suppression strategy was adapted for incorporation of photoactivatable, crosslinking amino acids into natural transcriptional activators in S. cerevisiae. Crosslinking experiments with Gal4 thus modified revealed at least three binding partners that will be the subject of further study.Ph.D.ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/61795/1/chinmay_1.pd

Design and Application of Probes to Understand Peroxisomal Localisation of BODIPYs

BODIPYs (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) constitute an important class of fluorophores. The excellent qualities of BODIPY comprise relatively high fluorescence quantum yields and molar absorption coefficients, narrow emission bandwidths, high elevated stability towards chemicals and light, and the extra feature of excitation/emission wavelengths in the visible region. Previous work in our group generated a small set of BODIPY fluorophores, including one, nitro-BODIPY, 1,3,5,7‐tetramethyl-8‐(4‐nitrophenyl)‐BODIPY, that is intrinsically non-fluorescent but upon incubation of cells with this compound, fluorescence is observed at the target site. The target site in plant cells was identified as peroxisomes, as verified by co-localization with an SKL-FP construct (peroxisome-specific carboxyl-terminal targeting sequence fluorescent proteins). It is key to future applications to be able to understand how this is occurring at a molecular level. These objectives are challenged by a lack of knowledge of this protein target and the physicochemical profile of nitro-BODIPY. This leads to the specific aims of this project which are: 1. To develop a ‘toolbox’ of analogues that enable multicolour visualisation. 2. To confirm the nature of the binding process between nitro-BODIPY and the target protein and to determine its reversibility. 3. To identify the target protein of nitro-BODIPY in plant peroxisomes. Therefore, a family of red-shifted nitro-BODIPY probes were synthesised. Most of them showed selectivity towards plant peroxisomes and their emission wavelength could reach to a maximum of around 700 nm. Water-soluble probes were prepared and proved to exhibit selective labelling towards plant peroxisomes as well. In addition, a group of dual-fluorophore probes (nitro-BODIPY probes with a second fluorophore including Rhodamine B, Cy5, dansyl and NBD) were developed to determine whether nitro-BODIPY only localizes at peroxisome or somewhere else. Although several dual-fluorophore probes showed selective labelling of the peroxisome, the reporter function of the second fluorophore in all probes was challenged by various degrees of FRET. Finally, a photoaffinity labelling probe (nitro-BODIPY core attatched with a benzophenone photoaffinity group) was prepared to determine the target proteins of nitro-BODIPY in plant peroxisomes. Several potential protein targets of nitro-BODIPY in plant peroxisomes were detected by this photoaffinity labelling probe via SDS-PAGE technique