Chemical Proteomic Profiling of Small Molecule-Protein Interactions

Wang, Yujia The Scripps Research Institute ProQuest Dissertati 2021 해외박사(DDOD)

A central goal in chemical biology and drug discovery is to develop small molecule probes to illuminate the functions of proteins in human diseases. The proportion of human proteome that can be targeted by chemical probes remains yet limited, underscoring the need of versatile strategies to expedite ligand discovery. We previously reported a chemical proteomic platform that utilized fully functionalized (photoreactive, clickable) fragments (FFFs) to broadly identify proteins that can interact with fragment compounds within native biological environments. Despite proven valuable in assessing the overall proteomic interaction potential of individual fragments, decerning selective small molecule-protein interactions from the non-specific background mainly driven by hydrophobic interaction remains a challenge. Careful manual review of complicated probe-vs-probe enrichment data is required for identifying authentic recognition events. In this thesis, we describe a general strategy to address this bottleneck in the form of next-generation sets of FFF probes consisting of physicochemically matched fragment pairs differing only in absolute stereochemistry. In Chapter 1, using a set of eight pairs of enantiomeric FFF probes—or “enantioprobes”—we expeditiously identified > 170 of stereochemistry-dependent small molecule-protein interactions for a diverse range of proteins including those lack chemical probes and traditionally considered as ‘undruggable’. Intriguingly, we showed that the interactions occur at functionally relevant sites on several proteins. In Chapter 2, to expand the scope and throughput of the pairwise chemical proteomic experiments, we developed a multiplexed quantitative proteomic approach capable of analyzing up to 10 separate population of cells treated under various conditions. We applied this strategy to explore the ligandable proteome in an AML cancer cell line using a set of structurally more complex enantiomeric FFF probes (elaborated enantioprobes). We showed that by elaborating simple fragment-like enantioprobes into drug-like small molecules with higher molecular weight, the stereoselective binding and chemotype selective enrichment of enantioprobes can be improved. Taken together, our findings demonstrate that incorporating chirality into FFF libraries provides a robust and streamlined method to map small molecule-protein interactions in cells. These stereoselective interactions can then form the basis for more advanced chemical probes targeting a broad range of proteins for basic and translational research.

Adventures in Total Synthesis

Stratton, Thomas P The Scripps Research Institute ProQuest Dissertati 2021 해외박사(DDOD)

Alkaloid total synthesis has challenged the field of chemical research for well over a century. The array of reactivity puzzles that present themselves to the chemical researcher pursuing such an endeavor pushes the limits of known reactivity and can also inspire the discovery of previously unknown transformations. In addition to its unmatched importance as an academic pursuit, alkaloid total synthesis has enabled access to biologically-important chemical matter, which in turn has allowed for further studies surrounding their mechanisms of action, compound stability and reactivity, and potentially can open the door to unnatural analog synthesis. This dissertation details the pursuit and successful total synthesis of two classic unanswered challenges in natural products synthesis: herqulines B and C; and tagetitoxin. During the course of our synthesis campaign towards the herqulines, several generations of synthesis planning were explored prior to the discovery that a simple and direct approach, one predicated on four consecutive reductions of an advanced biaryl macrocyclic intermediate, was key to unlocking the instability puzzle that was presented. Tagetitoxin, an enigmatic phytotoxin that possesses unusual bioactivity and has been the subject of several structural revisions, has recently been synthesized by our laboratory. Several unusual transformations were employed towards tagetitoxin including rearrangements, bromocyclization, and the first use of chiral PSI reagents in total synthesis. Both enantiomers were divergently synthesized, and with the aid of a bioassay, total synthesis has enabled confirmation of tagetitoxin’s structure and assignment of the natural isolate’s (+)-configuration.

Pd-Catalyzed Functionalization of Distal C–H Bonds

Tanaka, Keita The Scripps Research Institute ProQuest Dissertati 2020 해외박사(DDOD)

Direct conversion of targeted C–H bonds into desired functional groups is the most ideal transformation in the organic synthesis, simplifying the preparation of complex molecules. Countless research efforts by a number of researchers improved the scope of substrates and transformations, however there is a lot of room for improvement in terms of the site-selectivity. One of the powerful strategies to achieve the desired selectivity is to use directing groups. The major problem of this strategy is that as the distance between the targeted C–H bond and the directing group gets large, both reactivity and selectivity drop significantly. Since Pd catalysis can enable a wide range of C–C and C–heteroatom bond formation reactions, the development of Pd-catalyzed remote C–H functionalization is the major focus of this thesis.The work in this dissertation is divided into two parts. Part I describes the development of a bifunctional template for the selective remote C–H functionalization of aromatic nitrogen heterocycles. We have achieved meta-C–H activation of arenes by developing a template where the directing group and substrate are connected by the linker. However, this method was not applicable to aromatic nitrogen heterocycles, which have no functional group handles to install the templates. Here, a bifunctional template was developed, which could be installed on the substrates by means of coordination, and it allowed to achieve selective functionalization of distal C–H bonds in various aromatic nitrogen heterocycles. Part II describes the development of a new auxiliary for the C(sp3)–H functionalization of alcohols. Pd-catalyzed C(sp3)–H activation of alcohols typically shows β-selectivity due to the required distance between the chelating atom in the attached directing group and the targeted C–H bonds. Herein we report the design of a hemilabile directing group which exploits the chelation of a readily removable benzyl ether moiety to direct γ- or δ-C–H carbonylation and olefination of alcohols.

Application of Oxygenase Enzymes for Chemoenzymatic Synthesis of Natural Products & Exploration of Iron(II) α-Ketoglutarate Sequence Space for Non-canonical Amino Acid Native Substrate Promiscuity

King-Smith, Emma The Scripps Research Institute ProQuest Dissertati 2022 해외박사(DDOD)

Enzymatic transformations are powerful tools for synthetic chemistry, characterized by their unrivaled selectivity and nontoxic properties. Whilst their utility in the synthesis of natural products and active pharmaceutical ingredients has been modestly explored, their adoption has been limited to late-stage process chemistry and specialized academic labs. To facilitate the democratization of chemoenzymatic synthesis, we developed a paradigm which blends the unparalleled selectivity of enzymatic oxidations with the flexibility of state-of-the-art synthetic paradigms. These chemoenzymatic syntheses do not require mutagenesis campaigns and are broadly applicable to a breadth of targets. First, we will delve into the synthesis of tambromycin, a non-ribosomal peptide natural product with promising cytotoxicity in human cancerous B– and T– cells. We show how an iron(II) α-ketoglutarate (Fe(II)/αKG) L-lysine dioxygenase, KDO1, can be effectively used to deliver the first synthesis of tambromycin. Next we look towards the development of a P450 monooxygenase to facilitate a modular total synthesis to eight meroterpenoids, compounds which have members known to potently inhibit acyl- CoA:cholesterol acyltransferase and acetycholinesterase activity. We then repurpose enzymes from the platensimycin biosynthetic pathway and our aforementioned P450 to develop rapid divergent syntheses of nine diterpenoids. Finally, we develop a methodology to expand the enzyme toolbox for synthetic chemists via rational exploration of the Fe(II)/αKG protein family, PF10014. Utilizing both machine learning and genomic enzymology techniques, we identified three enzymes with native promiscuity for readily available non-canonical amino acids, with minimal screening (less than 1% of sequence space).

Diagnostics for Onchocerciasis and High-Throughput Screens for Modulators of the Oncoprotein MYC and the Immune Regulators PLD3 and PLD4

Shirey, Ryan J The Scripps Research Institute ProQuest Dissertati 2022 해외박사(DDOD)

Point-of-care diagnostic for onchocerciasis. The parasitic disease onchocerciasis is the second leading cause of preventable blindness. Efforts to combat this disease are limited by the lack of a robust, point-of-care diagnostic for active Onchocerca infection. To address this need, we have developed a lateral flow immunoassay (LFIA) that detects the disease biomarker N-acetyl-tyramine-O-glucuronide (NATOG) in urine. This LFIA is portable, inexpensive, and simple to use. This assay was validated with patient samples where it accurately identified 85% of analyzed patient samples (N = 27) relative to diagnosis by mass spectroscopy.Optimization of MYC Inhibitor probes. Dysregulation of MYC occurs in over half of all human tumors and is recognized as a general hallmark of cancer. However, chemical probes of MYC function are limited. KJ-Pyr-9 is a potent and specific MYC inhibitor active in vivo but possesses pharmacokinetic liabilities. We have utilized field-effect transistors, surface plasmon resonance, and a microtumor formation assay to guide optimization of this scaffold. This effort has produced a lead with increased stability and solubility culminating in a 40-fold increase in exposure (AUC) in vivo. Additionally, PROTACs, crosslinkers, and biotinylated derivatives were explored.High-throughput screen for MYC inhibitors. We report the discovery of a fluorescent small molecule probe that exhibits an emission increase in the presence of MYC. Hydrogen–deuterium exchange mass spectrometry analysis, rationalized by induced-fit docking, and verified by point mutation, revealed it binds to the “coiled-coil” region of the leucine zipper domain. Application of this probe in a high-throughput screening assay, has yielded molecules with greater drug-like properties than KJ-Pyr-9.High-throughput screen for modulators of PLD3 and PLD4. PLD3 and PLD4 are endosomal exonucleases that regulate the innate immune response, and their polymorphisms are correlated with inflammatory diseases. Chemical probes are lacking to explore the disease pathways and assess therapeutic potential of these proteins. To address this, we have performed a high-throughput screen to identify modulators of PLD3 and PLD4. This work confirms the viability of modulating these enzymes selectively despite their homology and the potential to discover immunosuppressant and immunostimulatory compounds.

Gene Networks Governing in vivo and ex vivo Function of Adult Skeletogenic Stem/Progenitor Cells

Booker, Cori Nicole The Scripps Research Institute ProQuest Dissertati 2021 해외박사(DDOD)

Disuse osteoporosis (DO) is a form of bone loss that occurs in response to prolonged mechanical unloading of the skeleton and results in a significantly increased risk of fracture. Skeletal pathology in DO results from imbalances in bone formation by osteoblasts vs. bone resorption by osteoclasts. It also induces increases in marrow adipose tissue (MAT) volume. However, whether DO also impairs the function of skeletal stem cells (SSCs), which serve as precursors of bone and fat tissue in adult bone marrow, has not been fully elucidated. To address this question, we performed RNA sequencing-based (RNA-seq) transcriptional profiling on CD31–CD45–Ter119–LepR+ SSCs isolated prospectively from the bone marrow of mice subjected to 8 or 14 weeks of hindlimb unloading (HU) and 8 wk of HU followed by an 8 wk recovery period (HUR) compared to sex- and age-matched ambulatory controls (AMB). Transcriptional profiling results were correlated with physiological changes in HU compared to AMB. Gene ontology and KEGG analyses based on differentially expressed genes indicated that 8 wks of HU induced a quiescent-like state coupled with downregulation of metabolic, bone-anabolic, and neuroregulatory pathways in SSCs compared to AMB, which was even more pronounced in 14 wk HU. These changes mirrored HU-induced increases in MAT volume, leptin expression, and density of TH+ nerve fibers in the niche. Psuedo-time course analysis based on K-means clustering of the RNA-seq data indicated that HUR reactivated growth and metabolic pathways in SSCs, however bone anabolic pathways remained repressed, consistent with permanent alterations to trabecular bone microarchitecture. Together these data indicate that SSC gene expression is sensitive to HU and that equivalent periods of HUR are not sufficient to revert the SSC transcriptome to an ambulatory state. When RNA-seq studies were repeated in CD11b–CD34–CD45–Sca1+ mesenchymal stem cells (MSCs) enriched from the bone marrow of 8 or 14 wk HU and AMB mice via immunodepletion, only minor differences in gene expression were detected. This indicated that cultured primary cells retain little to no memory of their HU status. MSCs are commonly used in in vitro studies as surrogates for SSCs to infer information about their function. However, few, if any, studies have directly compared the relatedness of these populations. Therefore, we also conducted a meta-analysis comparing the transcriptomes of SSCs, immunodepleted MSCs (IdMSCs) isolated from AMB control mice, and that from IdMSCs that were also subjected to a 7 day period of culture expansion (ExMSCs). This analysis revealed a unique complement of cell surface proteins, ECM structural components, and paracrine factors expressed by SSCs vs IdMSCs, which reflect culture adaptation of the latter. This analysis also showed that culture expansion upregulated key genes that encode clinically relevant paracrine factors in ExMSCs. Together, these data demonstrate that SSCs and MSCs are functionally distinct, and that culture adaptation endows MSCs with unique functional attributes that are widely exploited for therapeutic purposes. Overall, discoveries from HU experiments and the SSC vs MSC meta-analysis provide a roadmap for downstream basic and translational experimentation.

1,2,3-Triazines: Synthesis, Methodology, and Applications

Quinones, Ryan E The Scripps Research Institute ProQuest Dissertati 2022 해외박사(DDOD)

Systematic studies on the reactivity of the 1,2,3-triazine heterocycle are described.A comprehensive study of the reaction scope of methyl 1,2,3-triazine-5-carboxylate (3a) with alkyl and aryl amidines is disclosed, reacting at room temperature at remarkable rates (< 5 min, 0.1 M in CH3CN) nearly 10000-fold faster than that of unsubstituted 1,2,3-triazine and providing the product pyrimidines in high yields. C4 Methyl substitution of the 1,2,3-triazine (3b) had little effect on the rate of the reaction, whereas C4/C6 dimethyl substitution (3c) slowed the room-temperature reaction (< 24 h, 0.25 M) but displayed an unaltered scope, providing the product pyrimidines in similarly high yields. Measured second-order rate constants of the reaction of 3a–c, the corresponding nitriles 3e and 3f, and 1,2,3-triazine itself (3d) with benzamidine and substituted derivatives quantitated the remarkable reactivity of 3a and 3e, verified the inverse electron demand nature of the reaction (Hammett ρ = −1.50 for substituted amidines, ρ = +7.9 for 5-substituted 1,2,3-triazine), and provided a quantitative measure of the impact of 4-methyl and 4,6-dimethyl substitution on the reactivity of the methyl 1,2,3-triazine-5-carboxylate and 5-cyano-1,2,3-triazine core heterocycles.Simple and direct nucleophilic addition of secondary amines, including imidazole, to 1,2,3-triazine (3d) under mild reaction conditions (THF, 25–65 °C, 12–48 h), requiring no additives, cleanly provides β-aminoenals 9 in good yields (21 examples, 31–79%). The reaction proceeds by amine nucleophilic addition to C4 of the 1,2,3-triazine, in situ loss of N2, and subsequent imine hydrolysis to provide 9.

Advancing the Tiancimycins as Payloads for Antibody-Drug Conjugates

Adhikari, Ajeeth The Scripps Research Institute ProQuest Dissertati 2021 해외박사(DDOD)

Enediyne natural products are excellent candidates for evaluation as novel payloads for antibody-drug conjugates (ADCs). Presented below is the discovery and advancement of a novel enediyne natural product family, the tiancimycins (TNMs), as ADC payloads. The TNMs were discovered by mining actinobacterial genomes for conserved biosynthetic genes that encode the machinery necessary to produce enediyne natural products. The TNMs are potent cytotoxins whose structures can be readily diversified through genetic manipulation of the producing microorganism, Streptomyces sp. CB03234. We demonstrate the value of accessing the TNMs by microbial fermentation through utilization of S. sp. CB03234 as a biotechnology platform to formulate novel conjugation strategies. This is highlighted by complementary biocatalytic and semisynthetic approaches to functionalize two distinct sites on the TNM scaffold to install linkers for conjugation to engineered antibodies. Furthermore, we describe a conjugation strategy to prepare site-specific TNM-based ADCs and perform their preliminary biological evaluation against selected breast cancer cell lines, revealing significant influences from the site of linker attachment. These findings provide insights to be considered when advancing the TNMs as ADC payloads and motivate their further development.

Selection and Characterization of RNA Aptamers to Doxycycline

Tickner, Zachary John The Scripps Research Institute ProQuest Dissertati 2021 해외박사(DDOD)

Aptamers, named for the Latin aptus (fitting) and the Greek meros (region), are short biopolymers which bind specific targets with high affinity and are useful for a variety of applications in the chemical and biological sciences. RNA aptamers were the first to be isolated and are currently used as sensors, allosteric regulators, and targeting motifs in both laboratory and therapeutic settings, while naturally-selected RNA aptamers play important roles in multiple domains of life. Here we present selection and characterization of high-affinity RNA aptamers to doxycycline (dox), a structural isomer of the small-molecule antibiotic tetracycline (tet). Chapter 1 presents an introduction to the directed Darwinian evolution of small-molecule RNA aptamers, beginning with three seminal 1990 papers which laid the foundation for the field. In the subsequent 30 years aptamers have been used in a variety of applications while many new selection strategies and analytical methods have been developed. This chapter also discusses the chemical, structural, and binding properties of small molecule RNA, DNA, and xeno nucleic acid (XNA) aptamers. An overview of the in-vitro selection process is provided and alternative methods briefly mentioned. Applications of small molecule RNA aptamers are presented with an emphasis on those relevant to novel dox aptamers.Chapters 2 and 3 describe the isolation and characterization of novel dox aptamers. Chapter 2 covers how dox aptamers were enriched from tet aptamer-templated libraries over four rounds of in-vitro Darwinian selection using affinity column SELEX. Library design, with an emphasis on selective randomization and the role of constant regions during selections, is discussed along with methods for preparing affinity resins and the selection process itself. Chapter 3 describes experiments exploring the binding and structural properties of dox aptamers, including rearrangement of the tet aptamer structure used as a selection library template. These novel dox aptamers bind with high affinity and specificity and are immediately useful as fluorescent biosensors.Chapter 4 discusses strategies for improving the folding and binding properties of dox aptamers through further structural studies, techniques such as mutagenesis and truncation, and new selections. One potential use for dox aptamers is in gene expression control systems known as riboswitches: efforts to develop dox-dependent, self-cleaving riboswitches known as aptazymes are described. Next, lessons learned from dox aptamer selections are discussed: structural rearrangements from the library template used in dox aptamer selections may provide insight on future selection library designs, and dox aptamer enrichment required several changes to selection methods and analytical methods. Finally, preliminary efforts toward selecting RNA aptamers to the drug cabotegravir for use in a novel HIV prophylaxis system are described.

Small Molecule Tools to Modulate RNA Repeat Expansion Biology

Angelbello, Alicia J The Scripps Research Institute ProQuest Dissertati 2021 해외박사(DDOD)

Since the dissemination of the human genome sequence, the important roles of RNA, specifically non-coding RNA, in disease and healthy biology have been revealed. Indeed, RNA can cause disease by a variety of mechanisms, including oncogenic non-coding RNAs overexpressed in cancers or RNA repeat expansions that cause neuromuscular disorders. Common ways to target disease-causing RNAs include antisense oligonucleotides (ASOs) that recognize RNA sequences and small molecules that bind to 3D folds in an RNA structure. In many cases, small molecules targeting RNA structure can be advantageous over ASOs due to increased selectivity and cell permeability. This thesis details an expansion of small molecule approaches to target RNA repeat expansions, specifically the r(CUG) repeat that causes myotonic dystrophy type 1 (DM1). These small molecule approaches include the identification of novel scaffolds and lead optimization of compounds that bind r(CUG)exp and improve DM1 defects in cells and in vivo. Another approach to more potently target r(CUG)exp is through direct cleavage by using RNA-binding compounds attached to bleomycin A5. This small molecule cleaver allowed for selective cleavage of r(CUG)exp in patient-derived myotubes and in a mouse model of DM1 with transcriptome-wide improvement of DM1-associated defects. To improve selectivity of small molecule cleavers targeting r(CUG)exp, a bleomycin derivative without the disaccharide (deglycobleomycin) was used to ablate DNA damage observed at high concentrations of bleomycin-conjugated compounds while maintaining cellular activity. To expand the scope of targeted cleavage by bleomycin-small molecule conjugates, the optimal RNA substrates for cleavage by bleomycin A5 were defined in base paired RNAs. Bleomycin A5 preferred to cleave AU pairs and purine rich sequences. The final approach to more potently target r(CUG)exp is through an RNA-templated tetrazine ligation in which an r(CUG)exp binding compound appended with tetrazine and cyclopropene reactive handles reacted in situ and formed potent oligomers upon binding to the RNA. In cells, the compound improved DM1 defects at picomolar concentrations. The RNA-templated reaction was adapted to a fluorogenic variant to image r(CUG)exp in living cells and muscle fibers. Together, this work provides multiple approaches to potently and selectively target RNA repeats.