Author ORCID Identifier

https://orcid.org/0000-0003-2696-5989

Defense Date

2021

Document Type

Thesis

Degree Name

Master of Science

Department

Pharmaceutical Sciences

First Advisor

Dr. Yana Cen

Second Advisor

Dr. Jiong Li

Third Advisor

Dr. Karolina Aberg

Abstract

Fig.1. SAM analog for the profiling of MTase activity. A. Chemical structure of probe 1; B. General scheme of the labeling and capture strategy.

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Methylation is a fundamental mechanism used in the biological system to modify the structure and function of biomolecules such as proteins, DNA, RNA, and metabolites.1 Methyl groups are installed by a large and diverse class of S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTases), which transfer the sulfonium methyl group of SAM to either carbon, nitrogen, oxygen, or other heteroatoms on biomolecules.2 Dysregulated MTase activity contributes to numerous diseases, including cancer, metabolic disorders, neurodegenerative diseases.3 Presently, there is intense interest in pursuing MTases as therapeutic targets for the treatment of the aforementioned diseases.

The human MTase family has more than 200 members.4 However, a large fraction of this family remains poorly characterized in terms of their endogenous substrates and biological functions. The need for innovative chemical probes to evaluate MTase function in the native biological matrix becomes apparent.5–8 In this current project, we are developing an activity-based protein profiling (ABPP) probe to directly investigate MTase activity in a complex biological sample. This probe (probe 1, Fig. 1A) is a structural analog of SAM with several major components: 1) a methylated nitrogen “warhead” mimics the positively charged sulfur in SAM;9 2) a photocrosslinkable azido group for the covalent modification of the target enzyme;8 3) a biotin tag for the subsequent affinity capture by streptavidin beads. The general scheme of the labeling and enrichment strategy is shown in Fig.1B. This strategy will selectively enrich for the active MTase content in a complex cellular context. Side-by-side comparison of functional MTase profiles under different physiological and pathological conditions, combined with proteomics analysis, should unwind the intricate interaction loops between human MTases and various cellular pathways, as well as empower the better manipulation of these enzymes for therapeutic purposes.

Our original synthetic plan started with commercially available 8-bromoinsoine and acetyl protection of the 2', 3', and 5'-hydroxyl groups of the ribose ring (Fig. 2A).6 It was expected that after the installation of the azido group and the coupling with the biotin tag, the 5’-O-acetyl group could be selectively deprotected to enable the subsequent “warhead” installation. However, the selective deprotection using a published protocol was proven to be unsuccessful.6 Instead of the mono-deprotected probe 2, the mono-protected probe 3 was obtained. In the revised synthetic plan, the 2' and 3'-OHs were protected as an acetonide.10The 5'-OH can then be selectively protected by the acetyl group. The azido group was readily installed at 8-

Fig.2. Synthetic plans for probe 1. A. Original plan involves the tri-O-acetylation of the hydroxyl groups on the ribose ring. However, the selective deprotection of 5'-OH was challenging; B. Revised plan features the selective protection/deprotection of 5’-OH.

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position with NaN3. The biotin tag was tethered to the C6-position of the adenine ring through a 1,6-hexadiamine linker.9,11 The 5'-OH can then be selectively deprotected to afford the photocrosslink-able adenosine derivative probe 4 (Fig. 2B). The later stage of the synthesis focuses on the installation of the “warhead”. The 5'-OH was activated as a mesylate, which can then be replaced by a methylamino group.6 The subsequent reductive amination with tert-butyl (S)-2-[N-(tert-butoxycarbonyl) amino]-4-oxobutanoate allowed the “warhead” to be fully incorporated into the structure. Finally, the global deprotection should provide the desired SAM analog, probe 1. We are still in the process of finalizing the last step of the synthesis.

Fig.3. Photoaffinity labeling and affinity capture of recombinant proteins using synthetic probe. A. Schematic representation of the labeling and enrichment protocols; B. Western blots showing the labeling of EHMT1 (left) and ADA (right) with probe 3. The biotinylated proteins were detected with anti-biotin antibody.

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In parallel to the synthetic effort, we also established and optimized the photoaffinity labeling and affinity enrichment assays. Recombinantly expressed and purified human euchromatin histone lysine methyltransferase 1 (EHMT1) was used for the initial assay development. Several parameters such as irradiation time, probe dosage, and elution conditions were fine-tuned to ensure accurate profiling of active enzymes. The protein was incubated with probe 3, followed by UV irradiation to trigger the covalent conjugation (Fig. 3A).8,12 The unbound free probes were then filtered off. Subsequently, streptavidin beads were introduced to the sample to capture the biotinylated protein. Ultimately, EHMT1 was eluted off the beads and analyzed by western blot using anti-biotin antibody. EHMT1 was only strongly labeled by probe 3 at high micromolar concentrations (Fig. 3B, left). We reasoned that probe 3 is an adenosine analog rather than a SAM analog. It may demonstrate selectivity towards adenosine-binding proteins. Indeed, probe 3 labeled adenosine deaminase (ADA) in a concentration-dependent manner (Fig. 3B, right). Even at the lowest probe concentration, ADA can still be labeled and enriched. The labeling can be competed off using a known ADA inhibitor, suggesting the on-target effect of the probe.

The labeling strategy was also applied to cell lysates. It has the advantage of enriching the active enzymes independently of protein abundance, allowing the capture of dynamic enzyme activity changes in response to environmental or cellular stimuli.

In the current study, the facile synthesis of SAM analogs was developed. Photoaffinity labeling and affinity enrichment protocols were developed. The probes were able to label recombinant proteins, and demonstrated target selectivity. The synthesis of probe 1 will be completed. These innovative chemical probes will be used to profile MTase activity in their native matrix to better understand their roles in different cellular events.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

7-6-2021

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