Induced modification and degradation of intracellular proteins in lysosomes

Abstract

Compounds including small molecules and methods for the targeted, induced methylation of specific targeted intracellular proteins of interest (POIs), for inducing intracellular lysosomal degradation and other protein manipulation downstream of protein methylation.

Description

Reference No.: 2024-9AV-1, UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025INDUCED MODIFICATION AND DEGRADATION OF INTRACELLULAR PROTEINS IN LYSOSOMESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 681,323 filed August 9, 2024, the specification of which is incorporated herein in its entirety by reference.FIELD OF THE INVENTION

[0002] Compounds and methods for the targeted, induced methylation of specific targeted intracellular proteins of interest (POIs), for inducing intracellular lysosomal degradation and other protein manipulation downstream of protein methylation.BACKGROUND OF THE INVENTION

[0003] In all cells, proteins can be chemically modified to redirect features like function, location, and turnover. Dysregulation of protein modifications is linked to several diseases. Arginine methylation is a very common modification in human cells and affects protein interactions, signaling, degradation, and more. Prior tools have been developed to induce protein modifications like acetylation and phosphorylation. When applied, these modification inducers can effectively reshape cell biology and function. Despite the prevalence and biological significance of arginine methylation, there has previously been no tool available to chemically regulate the methylation of arginine residues.

[0004] Previously, proximity-based inducers have been used to induce degradation of disease-causing proteins through the proteasome via PRoteasomal TArgeting Chimeras (PROTACs) via proximity-based addition of ubiquitin tags onto proteins. While this system has proven to be efficacious, it is limited therapeutically by improper target ubiquitination, unfavorable ternary complex formation, and acquired mutation. The other site of protein degradation in the cell, the lysosome, could resolve such issues but has previously remained intractable due to the lack of a modification like ubiquitin.

[0005] Previous efforts to induce targeted degradation of intracellular proteins have focused on the use of the ubiquitin-proteasomal pathway (UPP). The UPP, however, is relatively limited in its potential application. Previous efforts have been unable to access the lysosome for targeted degradation of intracellular proteins.

[0006] Previously existing tools to selectively regulate protein methylation are limited inReference No.: 2024-9AV-1, UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025 selectivity. Each methyltransferase has upwards of thousands of methylated substrates. The most selective tools are small molecule inhibitors against specific PRMTs, but these small molecules will affect the methylation status of many protein substrates in the cell. The present invention enables a much higher level of substrate selectivity in the regulation of protein methylation. Additionally, the ability of protein methylation to induce lysosomal degradation of certain proteins broadens the range of proteins that are tractable to targeted protein degradation therapeutic strategies. When challenges arise in proteasomal targeting strategies, such as unfavorable ubiquitination or POI mutation, the present invention may be used to mediate the degradation of the offending protein via the lysosome. The present invention thus offers a valuable, alternative degradation route.

[0007] The present invention provides for the ability to exploit intracellular methylation to either target proteins to the lysosome for degradation, or to cause other protein manipulation downstream of protein methylation (for example, preventing the target POI from entering the nucleus).BRIEF SUMMARY OF THE INVENTION

[0008] It is an objective of the present invention to provide compositions and methods that allow for the targeted, induced methylation of specific targeted intracellular proteins of interest (POIs), for inducing intracellular lysosomal degradation and other protein manipulation downstream of protein methylation, as specified in the independent claims. Embodiments of the invention are given in the dependent claims. Embodiments of the present invention can be freely combined with each other if they are not mutually exclusive.

[0009] The present invention describes the chemically induced methylation of intracellular proteins. Arginine methylation regulates cell fate, and with the present invention, it is possible to precisely tune protein function and lifespan. In some embodiments of the present invention, a heterobifunctional small molecule binds to both an intracellular protein target (i.e., a protein of interest, or POI) and a methyltransferase enzyme. Induced proximity between these proteins drives the methylation of the POI. The resulting outcome of induced methylation then depends on the protein being targeted.

[0010] Current systems exist to regulate methylation by altering the function of methyltransferases. However, these approaches are broad and affect global methylation patterns, with only nine methyltransferases and thousands of methylated proteins in the cell. In contrast,Reference No.: 2024-9AV-1 , UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025 the present invention enables the selective methylation of a specific protein target without off-target effects on methylation. The present invention may be firstly used to regulate protein function. For instance, existing work shows that methylation can affect whether a protein is able to enter the nucleus and regulate gene expression. By inducing methylation, it is possible to precisely tune one specific feature of a protein without inhibiting total protein function. Methylation can also signal protein degradation in both degradation sites of the cell: the proteasome and lysosome. The lysosome has been largely unused in targeted protein degradation due to the lack of a protein modification that mirrors ubiquitination for the proteasome. By inducing protein methylation chemically, it is possible to target a specific protein to be degraded through the lysosome in a manner that is independent of ubiquitin or the proteasome. Given the host of challenges with proteasomal targeted degradation, including effective target ubiquitination, sufficient proteasomal activity, and acquired mutation, opening a new route for targeted protein degradation will close a significant technical gap. One of the methyltransferases highlighted in this design is protein methyltransferase 1 (PRMT1) because it is expressed in every tissue, can be bound with small molecules, and performs 85% of cellular methylation. PRMT1 is often overexpressed in cancer, making it a suitable candidate to induce the targeted methylation of disease-causing proteins.

[0011] The invention is modeled after a heterobifunctional small molecule that binds to a methyltransferase enzyme and a protein of interest (POI). In some embodiments, the methyltransferase used is protein methyltransferase 1 (PRMT1), as chosen in these embodiments for its biological relevance. The present invention may take at least two forms, where the small molecule-mediated interaction between a methyltransferase (e.g., PRMT1) and POI is either direct or indirect. In the direct form, the small molecule binds directly to both the methyltransferase and the POI, forming a clinically translatable system. In the indirect form, the methyltransferase and the POI are attached to opposing fusion proteins that bind the small molecule, which provides clinical applicability for rapid screening of POI compatibility with the methyltransferase (e.g., PRMT1) inducer system. In cases of direct binding, the heterobifunctional small molecule is composed of a methyltransferase (e.g., PRMT1) ligand and a POI-binding ligand that is bridged by a linker such as polyethylene glycol (PEG). In some embodiments, the methyltransferase-binding ligand reacts via a hyperreactive cysteine residue (C101) that is largely unique to the methyltransferase (e.g., PRMT1) without affecting the catalytic activity of the methyltransferase. The POI-binding ligand is variable to the POI being targeted, where the structure mimics existing or predicted inhibitors. Small molecules of theReference No.: 2024-9AV-1 , UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025 present invention may be synthesized using organic synthetic techniques. In cases of indirect proximity, commercially available small molecules may be used to induce proximity between fusion proteins, which are tethered to the methyltransferase and POI. For example, human cells in culture express HaloTag-PRMTl and SNAP-tag-POI fusion proteins following transfection. A commercially available small molecule, HaXS8, dimerizes the HaloTag and SNAP -tag fusion proteins, which indirectly brings PRMT1 and POI into nanometer proximity.

[0012] The present invention provides for the ability to use a small molecule capable of exploiting intracellular methylation to either target proteins to the lysosome for degradation or to cause other protein manipulation downstream of protein methylation (for example, preventing the target POI from entering the nucleus). In some embodiments, the present invention utilizes a novel small molecule to bring a target POI into proximity with a methyltransferase (e.g., a methylase), allowing methylation of the target POI, including POIs that are not endogenously methylated under normal physiological conditions. In some embodiments, the present invention comprises a '‘double-sided” small molecule, wherein one “side” contains a POI binding moiety while the other “side” contains a methyltransferase binding moiety. In some embodiments, the methylated POI is then trafficked through cellular processes (e.g., microautophagy) to the lysosome for subsequent lysosomal degradation.

[0013] In some embodiments, the present invention features a compound effective for targeted intracellular methylation of a protein of interest (POI). The compound may comprise a methyltransferase-binding moiety configured to bind a methyltransferase protein, a target protein-binding moiety configured to bind the POI, and a linker moiety connecting the methyltransferase-binding moiety and the target protein-binding moiety. In some embodiments, the methyltransferase-binding moiety binds directly to the methyltransferase protein. In other embodiments, the methyltransferase-binding moiety binds to the methyltransferase protein indirectly. For example, the methyltransferase-binding moiety may be configured to bind a methyltransferase fusion protein (e.g., comprising methyltransferase and a protein tag). Alternatively, or in addition to the aforementioned embodiments, the target protein-binding moiety may bind directly to the POI in some embodiments, or indirectly in other embodiments, for example, by binding to a protein tag on the POI.

[0014] One of the unique and inventive technical features of the present invention is that targeted intracellular degradation of POIs can be accomplished with the lysosome, rather than the proteasome, which allows for the degradation of proteins that are otherwise not possible utilizingReference No.: 2024-9AV-1 , UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025 only the proteasome. Another of the unique and inventive technical features of the present invention is that the POI is brought into proximity with the methyltransferase by the small molecule, allowing methylation of the POI. This obviates the need for the methyltransferase to enter close proximity with the lysosome, which helps maintain global levels of methyltransferase.

[0015] Another of the unique and inventive technical features of the present invention is that utilizing the proteasome requires use of the UPP, and the UPP involves El (Ubiquitin-activating enzyme), E2 (ubiquitin carrier or conjugation protein), and E3 (ubiquitin protein ligase), which greatly limits the number of POIs that may be targeted with the UPP system. Conversely, the methylation-dependent pathways of the present invention do not suffer from this technical limitation and rely on only a single enzy me, thus allowing targeting of a far greater number of POIs. Furthermore, E3 ligases commonly used in PROTACs are expressed in every' tissue in the human body, which limits specific targeting using the UPP, while specific methyltransferases are expressed in different tissues of the body, allowing greater tissue-specific targeting than prior approaches. Without wishing to limit the invention to any theory or mechanism, it is believed that the technical feature of the present invention also advantageously provides for the use of the microautophagy' pathw ay, which is more direct and less limited in its potential application than previous approaches, which have utilized macroautophagy (involving autophagosomes). Autophagosome production is only stimulated under certain cellular conditions, further limiting its potential applications. Conversely, and without wishing to limit the invention to any theory' or mechanism, it is believed that the present invention relies on microautophagy and thereby avoids these limitations.

[0016] Furthermore, the present invention may' be used to screen POIs that are compatible with the small molecule methylation-inducing system of the present invention through indirect proximity with HaloTag and SNAP-tag. After identify ing compatible POIs, small molecules may be synthesized to evaluate the success of a small molecule methylation-inducing system of the present invention via direct POEPRMTl targeting methods. This includes, but is not limited to, small molecules for PRMTl :GSK3b and PRMTfcMyc. The downstream efficacy of a small molecule methylation-inducing system of the present invention to induce methylation of POIs may be tested in vitro and in vivo with POI-specific readouts.

[0017] Moreover, the prior references teach away from the present invention. For example, unlike previous approaches, which have been limited to regulating the inhibition of target methylation, the present invention uniquely activates protein methylation.Reference No.: 2024-9AV-1 , UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025

[0018] None of the presently known prior references or works have the unique inventive technical feature of the present invention.

[0019] Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0020] The features and advantages of the present invention will become apparent from a consideration of the following detailed description presented in connection with the accompanying drawings in which:

[0021] FIG. 1 shows a schematic representation of both direct and indirect embodiments of the present invention. In direct embodiments, a small molecule of the present invention directly binds both a methyltransferase (for example. PRMT1) and the POL In indirect embodiments, a methyltransferase protein is bound to a first fusion protein; and a target protein (i.e., a POI) is bound to a second fusion protein, and both the first fusion protein and the second fusion protein bind to a small molecule of the present invention.

[0022] FIGs 2A-2I show protein levels of Snap-GSK3P degradation in HeLa cells (FIGs 2A-2B) and HEK293T (FIGs 2C-2D), and Snap-cMyc degradation in HEK293T cells (FIGs 2E-2F). Indirect PRMTLPOI targeting experiments have shown successful induction of methylation on multiple POIs and resulting biological effects using the Hal oTag: SNAP -tag dimerization system. Biochemical analyses demonstrate methylation-dependent degradation of multiple POIs (GSK3P and c-Myc) upon proximity with PRMT1. Degradation of GSK3P via induced methylation increases proliferation (FIG. 2G). whereas degradation of c-Myc via induced methylation decreases proliferation (FIG. 2H). The drug had no effect on proliferation when fusion proteins were not expressed (FIG. 21). Small molecules may directly target PRMTLPOI with GSK3P as a first exemplary POI and cMyc as a second exemplary POI.

[0023] FIG. 3A-3N shows MrTAC mediates proximity with PRMT1 for target degradation. FIG. 3A shows a scheme of MrTAC inducing proximity between a protein of interest (POI) and protein arginine methyltransferase 1 (PRMT1). FIG. 3B shows the structure of MrTACHaXS8. FIGs 3C-3E show an immunoblot analysis of HEK293T cells co-expressing GSK3P-SNAP (70 kDa) and HaloTag-empty (30 kDa). in a 3 hour MrTACHaXS8 dose curve. Protein levelsReference No.: 2024-9AV-1 , UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025(GSK3[3) line shows linear regression, complex formation rate shows proportion of GSK3(3 in complex to total GSK3 . FIGs 3F-3H show immunoblot analysis of HEK293 cells stably expressing Halo-PRMTl (70 kDa) and mCherry -tagged SNAP-GSK3P (100 kDa) in a 24 hour MrTACHaXS8 dose curve. FIGs 3I-3K show immunoblot analysis of HEK293 cells stably- expressing Halo-PRMTl and mCherry -tagged SNAP-GSK3P in a 3 hour MrTACHaXS8 dose curve. FIG. 3L shows confocal microscopy of GSK3P-SNAP+ HEK293T cells detected by FLAG antibody following MrTACHaXS8 treatment (10 pM, 3 hrs). FIG. 3M shows bars that are a fraction of GSK3p-expressing cells (n=513 cells / condition over 3 fields of view), *P<0.0124 by unpaired two-sided t-test. Scale bar is 50 pm, arbitrary unit (au). FIG. 3N shows live-cell imaging of GFP-tagged GSK3P-SNAP during MrTAC treatment (0.1 pM, 1 hr) in HeLa cells. Scale bar is 5 pm. For FIGs 3F-3K, DC50 values are by sigmoidal regression and % remaining values are indicated for blots depicted. Immunoblots are representative of three independent experiments. Data are represented as means ± SEM.

[0024] FIGs 4A-4L show MrTAC targeting GSK3P to lysosomes for degradation. FIGs 4A-4C show live-cell imaging of mCherry -tagged GSK3P-SNAP during MrTACHaXS8(0.5 pM, 40 min) treatment in stably expressing HEK293 cells. Lysosomes are stained with Lysotracker. Arrow-s mark colocalization. FIGs 4D-4F show- a diagram and microscopy of proximity ligation assay (PLA) for GSK3P-SNAP delivery into lysosomes in MrTAC -treated cells (10 pM, 1 hr). FLAG antibody detects GSK3p. LAMP-1 antibody recognizes a luminal LAMP-1 epitope. PLA of IgG:FLAG and IgG:LAMP-l control for nonspecific interactions. Bars are number of PLA puncta / cell per field of view (n=l 14 DMSO, 110 MrTAC, 111 FLAG, 113 LAMP-1 cells), ****P<0.0001 by unpaired two-sided t-test. FIGs 4G-4H show- immunoblot analysis of complex levels in MrTAC -treated cells (10 pM, 24 hr) in the presence of bafilomycin (Baf, 100 nM), bortezomib (Bort, 50 nM), or MG132 (5 pM) (n=3). Blue arrowheads mark MrTAC complex. *P<0.0335 by one-way ANOVA with Dunnett’s multiple comparisons (*P<0.0382 drug effect). FIG. 41 shows immunoblot analysis of MrTACHaXS8degradation in HEK293T cells transiently expressing Halo-PRMT1 / SNAP-GSK3(3 with siRNA against Vps4a, Lamp2a, or Atg7 (10 pM, 24 hrs) (n=3). FIGs 4J-4L show a model of microautophagy of MrTAC complex via vacuolar protein sorting 4 (VPS4) and immunofluorescent analysis of MrTAC degradation of SNAP-GSK3(3 in control or siVps4a HEK293T cells with a LAMP-1 costain (10 pM, 2 hrs). Scale bars are 10 pm. Immunoblots are representative of three independent experiments. Data are represented as means ± SEM.

[0025] FIGs 5A-5H show arginine methylation of the protein target underscoring MrTACReference No.: 2024-9AV-1, UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025 degradation. FIGs 5A-5B show a proximity ligation assay (PLA) detection of GSK3(3-SNAP methylation in MrTACHaXS 8 -treated HEK293T cells (10 pM, 1 hr). FLAG antibody detects GSK3(3-SNAP, asymmetric dimethylated arginine (ADMA) antibody detects methylation. PLA of IgG:FLAG and IgG:ADMA control for nonspecific interactions. Scale bar is 10 pm. bars are number of PLA puncta per cell per field of view (n=120 DMSO, 113 MrTAC, 120 FLAG, 116 ADMA cells), **P<0.0069 by unpaired two-sided t-test. FIG. 5C shows conservation of GSK3P by Consurf, arrows denote R4 and Rill. FIGs 5D-5F show an immunoblot analysis of GSK3P-SNAP in MrTAC -treated cells (10 pM, 3 hr) in the presence of GSK3368715 (10 pM) (n=3), ****P<0.0001 by unpaired two-sided t-test. FIGs 5G-5H show an immunoblot analysis of GSK3P-SNAP over a 24 hour MrTAC dose curve in HEK293 cells stably expressing catalytically dead PRMTl-Halo (VLD to AAA) with an mCherry tagged GSK3|B-SNAP (n=3), ns by one-way ANOVA with Dunnett’s multiple comparisons. Immunoblots are representative of three independent experiments. Data are represented as means ± SEM.

[0026] FIGs 6A-6H show MrTAC degradation recapitulating loss-of-function phenotypes. FIGs 6A-6B show immunoblot analysis of GSK.30 levels over a 3 day MrTAC treatment in HEK293 cells stably expressing PRMTl-Halo with an mCherry -tagged GSK3P-SNAP (n=3). **PDAYI:O. <O.OO42, **PD1:1<0.0045, **PD2:01<0.0086, **PD2;1<0.0038, *PD3;0, <0.0103,**PD3 1<0.0061 by two-way ANOVA with Dunnetfs multiple comparisons, b, Immunoblot analysis of (3-catenin and GSK3(3-SNAP in MrTAC-treated HEK293T cells (1. 10 pM, 4 days) (n=2). FIGs 6C-6D show mRNA levels of c-Myc and Birc5 following treatment with DMSO or MrTACHaXS8 (0.5 pM, 7 days) (n=3 experiments) in cells expressing CRISPR-integrated PRMTl-Halo with GSK30-SNAP, **PBirc5<0.0012, **Pc.Myc<0.0059 by unpaired two-sided t-test. FIGs 6E-6F show proliferation following MrTAC treatment (0.5 pM) in HEK293 cells expressing CRISPR-integrated PRMTl-Halo with GSK30-SNAP over three and six days (n=3 experiments), **P<0.0079 drug effect, *PD6<0.0245 by two-way ANOVA with Bonferroni’s multiple comparisons. FIG. 6G shows Immunoblot analysis of c-Myc-SNAP levels in MrTACHaXS8-treated HEK293 cells (24 hrs) expressing PRMTl-Halo (n=3). FIG. 6H shows proliferation of MrTACHaXS8-treated HeLa cells expressing PRMTl-Halo with c-Myc-SNAP over seven days (0.5 pM, n=4 experiments), *P<0.0182 drug effect, *PD7<0.0490 at day 7 by two-way ANOVA with Bonferroni’s multiple comparisons. Immunoblots are representative of at least two independent experiments. Data are represented as means ± SEM.

[0027] FIGs 7A-7L show induced degradation of endogenous substrates. FIGs 7A shows MrTACJQ1targeting endogenous bromodomain containing protein 4 (BRD4). FIG. 7B-7C showReference No.: 2024-9AV-1, UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025 immunoblot analysis of BRD4 levels following treatment with MrTACJQ1or Control1'21(JQ 1 -PEG2-NH) over 4 hours (n=3). FIGs 7D-7E show immunofluorescent staining of BRD4 levels following MrTACJQl treatment (0.5 pM, 4 hrs), *P<0.0139 by unpaired two-sided t-test (MFI, mean fluorescent intensity, n=7 fields of view). FIG. 7F shows co-immunoprecipitation of BRD4 by Myc pull-down of PRMTl-Halo-Myc following MrTACJQl treatment (2 hrs) co-treated with Bafilomycin (400 nM) and MG132 (10 pM). FIG. 7G shows MrTACSAHAtargeting endogenous Histone Deacetylase 6 (HDAC6). FIGs 7H-7I show immunoblot analysis of HDAC6 following treatment with MrTACSAHAor ControlSAHAligand over 4 hours (n=4 MrTAC'"'" '. n=3 ControlSAHA). FIGs 7J-7K show immunofluorescent staining of HDAC6 following MrTACSAHAtreatment (4 hrs, 0.5 pM). ****P<0.0001 by unpaired two-sided t-test (n=6 fields of view). FIG. 7L shows c-immunoprecipitation of HDAC6 by Myc pull-down of PRMTl-Halo-Myc following MrTACSAHA treatment (2 hrs) co-treated with Bafilomycin (400 nM) and MG132 (10 pM) (n=3 experiments). Assays performed in HEK293 cells stably expressing PRMTl-Halo-Myc. Immunoblots are representative of at least three independent experiments. Data are represented as means ± SEM.

[0028] FIGs 8A-8J show MrTAC regulating cell function via endogenous degradation. FIG. 8A shows mRNA levels of Brd4, Bcl2, Cdknla, and Birc3 in cells treated with DMSO or MrTAC (1 pM, 24 hrs) (n=3), *PBcl2<0.0155, **Pcdknla<0.0023. *PBirc3<0.0319 by two-way ANOVA with Bonferronrs multiple comparisons. FIG. 8B shows cell proliferation after 6 days of MrTACJQ1or ControlJQ1treatment relative to DMSO (1 pM, n=3 experiments), **P<0.0051 by unpaired two-sided t-test. FIGs 8C-8D show’ immunoblot analysis of K40-acetylated a-tubulin following 3 days of MrTACSAHA treatment relative to total a-tubulin (n=3), **P<0.0081 by unpaired two-sided t-test. FIG. 8E shows cell survival over a 3 day treatment of MrTACSAHA in HEK293 cells stably expressing PRMTl-Halo with a non-transfected control by CCK-8 assay (n=6 technical replicates). FIG. 8F show’s untargeted proteomic analysis of cells treated with MrTAC"'1"' or DMSO (1 pM, 4 hrs), P-values by unpaired two-sided t-test (n=6 technical replicates). FIG. 8G shows immunoblot analysis of HDAC6 levels in MrTACSAHA-treated HEK293 cells (1 pM, 4 hrs) stably expressing wild type or catalytically dead (VLD to AAA) PRMTl-Halo (n=3). FIGs 8H-8J show immunofluorescent staining of HDAC6 levels following MrTACSAHAtreatment in HEK293 cells stably expressing wild ty pe or catalytically dead (VLD to AAA) PRMT1 (0.5 pM, 4 hrs), *P<0.0477 by one-way ANOVA with Bonferroni’s multiple comparisons (nWT= 2 experiments with 80nMSOand 79MrTACpooled cells, nnead=3 experiments over 98DMSOand 89NfrTACpooled cells). Assays performed in HEK293 cells stably expressingReference No.: 2024-9AV-1, UCI 24.12 PCTInventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025PRMTl-Halo-Myc unless otherwise specified. Immunoblots are representative of three independent experiments. Data are represented as means ± SEM.DETAILED DESCRIPTION OF THE INVENTION

[0029] For purposes of summarizing the disclosure, certain aspects, advantages, and novel features of the disclosure are described herein. It is to be understood that not necessarily all such advantages may be achieved in accordance with any particular embodiments of the disclosure. Thus, the disclosure may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other advantages as may be taught or suggested herein.

[0030] Additionally, although embodiments of the disclosure have been described in detail, certain variations and modifications will be apparent to those skilled in the art. including embodiments that do not provide all the features and benefits described herein. It will be understood by those skilled in the art that the present disclosure extends beyond the specifically disclosed embodiments to other alternative or additional embodiments and / or uses and obvious modifications and equivalents thereof. Moreover, while a number of variations have been shown and described in varying detail, other modifications, which are within the scope of the present disclosure, will be readily apparent to those of skill in the art based upon this disclosure. It is also contemplated that various combinations or sub-combinations of the specific features and aspects of the embodiments may be made and still fall within the scope of the present disclosure. Accordingly, it should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for one another in order to form varying modes of the present disclosure. Thus, it is intended that the scope of the present disclosure herein disclosed should not be limited by the particular disclosed embodiments described herein.

[0031] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which a disclosed invention belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "comprising" means that other elements can also be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation. Stated another way, the term "comprising" means "including principally, but not necessary solely". Furthermore, variation of the word "comprising", such as "comprise" and "comprises", have correspondingly the same meanings. In one respect, the technology ioReference No.: 2024-9AV-1 , UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025 described herein related to the herein described compositions, methods, and respective component(s) thereof, as essential to the invention, yet open to the inclusion of unspecified elements, essential or not ("comprising").

[0032] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes. In case of conflict, the present specification, including explanations of terms, will control.

[0033] Although methods and materials similar or equivalent to those described herein can be used to practice or test the disclosed technology, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0034] As used herein, "methyl transferase" and "methylase" may be used interchangeably and refer to an enzyme that promotes the transfer of a methyl group from one compound to another.

[0035] Referring now to FIGs. 1-8J, the present invention features compounds (e.g., heterobifunctional compounds) effective for the targeted, induced methylation of specific intracellular proteins of interest (POIs) to promote lysosomal degradation and other downstream protein modifications. The invention also provides methods for inducing such methylation using the described compounds.

[0036] The present invention features a compound effective for targeted intracellular methylation of a protein of interest (POI). In some embodiments, the compound may comprise a methyltransferase-binding moiety' configured to bind a methyltransferase protein, a target protein-binding moiety configured to bind the POI, and a linker moiety connecting the methyltransferase-binding moiety and the target protein-binding moiety. In some embodiments, the methyltransferase-binding moiety binds directly to the methyltransferase protein. In other embodiments, the methyltransferase-binding moiety binds to the methyltransferase protein indirectly. For example, the methyltransferase-binding moiety may be configured to bind a methyltransferase fusion protein (e.g., comprising methyltransferase and a protein tag). In some embodiments, the methyltransferase-binding moiety may be configured to bind the protein tag on the methyltransferase fusion protein Alternatively, or in addition to the aforementioned embodiments, the target protein-binding moiety' may bind directly to the POI in some embodiments, or indirectly in other embodiments, for example by binding to a protein tag on the fusion POI.Reference No.: 2024-9AV-1, UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025

[0037] In some embodiments, the methyltransferase-binding moiety covalently reacts with a hyperreactive cysteine residue (e.g., C 101) that is largely unique to the methyltransferase, such as PRMT1, without impairing the catalytic activity of the enzyme. This covalent binding confers selectivity by targeting cysteine residues that are unique to a specific methyltransferase, thereby distinguishing it from other PRMT family members. Notably, the methyltransferase binding moiety remains covalently attached to the methyltransferase following binding, as the cysteine-reactive group is integrated within the ligand (i.e., the binding moiety) itself. This persistent covalent interaction enhances specificity and functional modulation of the targeted methyllransferase.

[0038] In other embodiments, the compound effective for targeted intracellular methylation of a POI comprises a methyltransferase-binding moiety7configured to bind a methyltransferase fusion protein, a target protein-binding moiety configured to bind the POI, and a linker moiety connecting the methyltransferase-binding moiety and the target protein-binding moiety. In some embodiments, the methyltransferase fusion protein comprises methyltransferase and a protein tag. In certain embodiments, the protein tag is genetically encoded as a fusion with the methyltransferase.

[0039] In further embodiments, the compound effective for targeted intracellular methylation of a POI comprises a methyltransferase-binding moiety configured to bind a methyltransferase fusion protein, a target protein-binding moiety configured to bind a POI fusion (i.e., indirectly bind the POI). and a linker moiety connecting the methyltransferase-binding moiety and the target protein-binding moiety. In some embodiments, the methyltransferase fusion protein comprises methyltransferase and a protein tag. In certain embodiments, the protein tag is genetically encoded as a fusion with the methyltransferase. In some embodiments, the methyllransferase-binding moiety is configured to bind to the protein tag on the methyltransferase fusion protein. In some embodiments, the target protein-binding moiety binds to a protein tag on the POI fusion, e.g., the POI may be genetically encoded together with the protein tag. In some embodiments, the target protein-binding moiety7is configured to bind to the protein tag on the POI fusion.

[0040] In some embodiments, the compounds described herein are for targeted intracellular methylation (e.g., arginine methylation) of a target protein (e.g., a POI).

[0041] In some embodiments, the present invention comprises a compound effective for targetedReference No.: 2024-9AV-1, UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025 intracellular methylation of a target protein, wherein the methyltransferase binding site is configured to bind a methyltransferase protein; wherein the methyltransferase protein is protein methyltransferase 1 (PRMT1) or PRMT8.

[0042] In some embodiments, the present invention comprises a compound effective for targeted intracellular methylation of a target protein, wherein the methyltransferase binding site is configured to bind to protein methyltransferase 1 (PRMT1).

[0043] In some embodiments, the present invention comprises a compound effective for targeted intracellular methylation of a target protein, wherein the methyltransferase binding site is configured to bind to protein methyltransferase 8 (PRMT8).

[0044] In some embodiments, the methyltransferase protein, or the methyltransferase within a fusion protein, is an arginine methyltransferase (PRMT). In some embodiments, the PRMT is a type I PRMT including PRMT1, PRMT2, PRMT3, PRMT4, PRMT6, PRMT8, or any combination thereof. In other embodiments, the PRMT is a type II PRMT including PRMT5, PRMT9, or both PRMT5 and PRMT9. In other embodiments, the PRMT is a type III PRMT and includes PRMT7.

[0045] In some the linker moiety comprises polyethylene glycol (PEG). Additional non-limiting examples of linkers that may be used include, but are not limited to, alkane, alkyne, cycloalkyl, spirocylic, piperidine, piperazine, triazole linkers.

[0046] In some embodiments, the present invention comprises a compound effective for targeted intracellular methylation of a target protein, wherein the target protein binding moiety is configured to bind to a target protein; wherein the target protein is GSK-3. In some embodiments, the present invention comprises a compound effective for targeted intracellular methylation of a target protein, wherein the target protein binding moiety is configured to bind to Glycogen synthase kinase 3 (GSK-3).

[0047] Non-limiting examples of POIs that may be targeted by the target protein-binding moiety include but are not limited to glycogen synthase kinase 3 (GSK-3), bromodomain-containing protein 4 (BRD4), or MYC. However, the target protein-binding moiety may target any suitable protein (e.g.. POI), and thus the present invention is not limited to the specific POI examples described herein.Reference No.: 2024-9AV-1, UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025

[0048] Non-limiting examples of protein tags that may be genetically encoded with either the methyltransferase or the POI include, but are not limited to, a HALOtag, a SNAP-tag, a CLIP-tag, a FKBP12 (F36V) tag, a BromoTag, BromoCatch, a Spy Tag, SpyCatcher, a SnoopTag, SnoopCatcher, an eGFP, a vhhGFP, an ALFA, a nbALFA, or a NanoBiT tag. In some embodiments, two different protein tags are used — one encoded with the methyltransferase and another with the POI — while in certain embodiments, the same protein tag is encoded with both.

[0049] In some embodiments, the present invention comprises a compound effective for targeted intracellular methylation of a target protein, wherein when the methyltransferase binding moiety binds to a methyltransferase protein and the target protein binding moiety binds to a target protein (e.g., the POI), the methyltransferase protein methylates at least one arginine on the target protein (e.g., the POI). In some embodiments, when the methyltransferase binding moiety binds to a methyltransferase protein and the target protein binding moiety binds to a target protein (e.g., the POI). the methyltransferase protein methylates one arginine on the target protein (e.g., the POI). In other embodiments, when the methyltransferase binding moiety binds to a methyltransferase protein and the target protein binding moiety’ binds to a target protein (e.g., the POI), the methyltransferase protein methylates two arginine on the target protein (e.g., the POI).

[0050] In some embodiments, the present invention comprises a compound effective for targeted intracellular methylation of a target protein (e.g. a POI), wherein the compound is effective to induce degradation of the target protein (e g., the POI) in a non-proteas omal mediated manner.

[0051] In some embodiments, the present invention comprises a compound effective for targeted intracellular methylation of a target protein (e.g. a POI), wherein the compound is effective to induce lysosomal degradation of the target protein (e.g., the POI).

[0052] In some embodiments, the present invention comprises a compound effective for targeted intracellular methylation of a target protein (e.g. a POI), wherein the compound is effective to induce microautophagy of the target protein (e.g., the POI).

[0053] In some embodiments, the present invention comprises a compound effective for targeted intracellular methylation of a target protein (e.g. a POI), wherein the compound is effective to prevent the target protein (e.g., the POI) from entering a nucleus of a cell.

[0054] In some embodiments, the present invention comprises a compound effective for targeted intracellular methylation of a target protein (e.g. a POI), wherein the compound is effective toReference No.: 2024-9AV-1 , UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025 bring the target protein (e.g., the POI) into sufficiently close proximity to a methyltransferase, thereby facilitating methylation of the target protein (e g., the POI) by the methyltransferase.

[0055] The present invention may also comprise a method of screening compatibility between the methyltransferase protein and a target protein (e.g., a POI). In some embodiments, the method comprises administering to an experimental biological system any of the compounds effective for targeted intracellular methylation as described herein. For example, the compound may comprise a methyltransferase-binding moiety configured to bind a methyltransferase protein, a target protein-binding moiety configured to bind the POI, and a linker moiety connecting the methyltransferase-binding moiety and the target protein-binding moiety. In some embodiments, the methyltransferase-binding moiety binds directly to the methyltransferase protein. In other embodiments, the methyltransferase-binding moiety binds to the methyltransferase protein indirectly. For example, the methyltransferase-binding moiety may be configured to bind a methyltransferase fusion protein (e.g., comprising methyltransferase and a protein tag). Alternatively, or in addition to the aforementioned embodiments, the target protein-binding moiety' may bind directly to the POI in some embodiments, or indirectly in other embodiments, for example by binding to a protein tag on the POI. In some embodiments, when the methyltransferase-binding moiety’ binds to the methyltransferase and the target protein binding moiety’ binds the POI; the methyltransferase protein methylates at least one arginine on the POI.

[0056] In some embodiments, compatibility is indicated when the pairing of the methyltransferase protein and the POI results in one or more of the following: protein-protein interactions are induced (e.g., either using purified proteins in vitro or within cells); methylation of at least one arginine residue on the POI (e.g., using purified proteins in vitro or within cells); or degradation of the POI by 20% to 100% compared to a control system lacking the compound. In certain embodiments, compatibility is indicated when the pairing of the methyltransferase protein and the POI induces degradation of the POI in an amount ranging from 20% to 100% compared to a system without the compound.

[0057] In some embodiments, the present invention comprises an in vitro method of inducing targeted intracellular methylation of a target protein in a subject in need thereof, comprising: administering to the subject any of the compounds effective for targeted intracellular methylation as described herein. For example, the compound may comprise a methyltransferase-binding moiety configured to bind a methyltransferase protein, a target protein-binding moiety configuredReference No.: 2024-9AV-1 , UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025 to bind the POI. and a linker moiety connecting the methyltransferase-binding moiety and the target protein-binding moiety. In some embodiments, the methyltransferase-binding moiety binds directly to the methyltransferase protein. In other embodiments, the methyltransferase-binding moiety binds to the methyltransferase protein indirectly. For example, the methyltransferase-binding moiety may be configured to bind a methyltransferase fusion protein (e.g., comprising methyltransferase and a protein tag). Alternatively, or in addition to the aforementioned embodiments, the target protein-binding moiety may bind directly to the POI in some embodiments, or indirectly in other embodiments, for example by binding to a protein tag on the POI. In some embodiments, when the methyltransferase-binding moiety binds to the methyltransferase and the target protein binding moiety binds the POI; the methyltransferase protein methylates at least one arginine on the POI.

[0058] In some embodiments, the present invention comprises a method of inducing targeted intracellular methylation of a target protein in a subject in need thereof, comprising: contacting a cell with a compound effective for targeted intracellular methylation of a target protein as described herein.

[0059] In some embodiments, the present invention comprises a method of inducing targeted intracellular methylation of a target protein as part of a screening protocol, comprising: administering to an experimental biological system at least two compounds effective for targeted intracellular methylation of a target protein, the at least two compounds comprising: a methyltransferase protein bound to a first fusion protein; and a target protein bound to a second fusion protein; wherein the first fusion protein is configured to bind to the second fusion protein, thereby linking the methyltransferase protein to the target protein, wherein the method is effective to screen for compatibility' between the methyltransferase protein and the target protein. In some embodiments, compatibility between a given methyltransferase protein and a given target protein is determined to exist w here pairing of the given methyltransferase protein and the given target protein results in 20-100% degradation of the given target protein.

[0060] EXAMPLE

[0061] The following is a non-limiting example of the present invention. It is to be understood that said example is not intended to limit the present invention in any way. Equivalents or substitutes are w ithin the scope of the present invention.

[0062] A paradigm shift in drug development is the discovery’ of small molecules that harness theReference No.: 2024-9AV-1, UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025 ubiquitin-proteasomal pathway to eliminate pathogenic proteins. Here, a modality for targeted protein degradation in lysosomes is provided. The present invention allows for the exploitation of an endogenous lysosomal pathway whereby protein arginine methyltransferases (PRMTs) initiate substrate degradation via arginine methylation. The present experiment was directed to heterobifunctional small molecules. Methyl arginine TArgeting Chimera (MrTAC), that recruit PRMT1 to a target protein for induced degradation in lysosomes. MrTAC compounds degraded substrates across cell lines, timescales, and doses. MrTAC degradation required target protein methylation for subsequent lysosomal delivery via microautophagy. A library' of MrTAC molecules exemplified the generality of MrTAC to degrade known targets and neo-substrates: GSK3 , cMYC, BRD4. and HDAC6. MrTAC selectively degraded target proteins and drove biological loss-of-function phenotypes in survival, transcription, and proliferation. Collectively, MrTAC demonstrates the utility of endogenous lysosomal proteolysis in the generation of a new class of small molecule degraders.

[0063] This work evaluated synthetically inducing arginine methylation targets intracellular proteins for lysosomal degradation. This novel degradation pathway was harnessed to develop a general chemical tool termed Methyl arginine TArgeting Chimera (MrTAC), which employs heterobifunctional molecules that engage PRMT1 and a target protein. It was shown that MrTACs induced target protein methylation and degradation in the nanomolar range across diverse pathogenic targets and endogenous proteins. Together, this work provided evidence for a generalized degrader modality’ for intracellular proteins that is ubiquitin-independent and offers translational potential by exploiting a naturally occurring modification for lysosomes.

[0064] In physiologic contexts, PRMT1 catalyzed arginine methylation (MrDegron) modifications on proteins that led to their subsequent delivery into lysosomes. The present experiment evaluated whether arginine methylation could be synthetically induced and exploited for targeted lysosomal degradation. Towards this goal, a heterobifunctional MrTAC small molecule was developed that simultaneously bound PRMT1 and a protein-of-interest (POI) (FIG. 3 A). Ligand selection of endogenous proteins represented a major challenge in degrader development. Given this, a system was engineered to circumvent ligand screening for initial studies by generating a PRMTl-HaloTag fusion protein and a POLSNAP-tag fusion protein. HaloTag and SNAP -tag were dimerized by HaXS8 through chloroalkane and benzylguanine ligands, respectively, which were repurposed as a MrTAC surrogate (denoted MrTACHaXS8).

[0065] It was hypothesized that recruiting PRMT1 with MrTAC would induce POI degradation.Reference No.: 2024-9AV-1 , UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025Glycogen synthase kinase 30 (GSK.30) was selected as an initial POI given its native regulation by PRMT129 and its central role in tissue regenerative medicine, cancer, and neurodegenerative disease. The first aim was to validate that covalent interactions between SNAP -tag and HaloTag could be induced by MrTACHaXS8. A GSK30-SNAP-tag fusion protein was generated and co-transfected with an HaloTag-Empty Vector control (lacking PRMT1) in HEK293T cells. Cells were treated with MrTACHaXS8and GSK30 was evaluated by immunoblotting. In control treatments, GSK30-SNAP was expressed at the expected monomeric molecular weight of 70 kDa (FIG. 3C, lane 1). Upon MrTACHaXS8 addition, GSK30-SNAP shifted into -110 kDa protein-protein complexes with HaloTag-Empty. Complex formation gradually increased and was most efficient at 1 pM MrTACHaXS 8 104 (FIG. 3C). Doses above 1 pM showed decreased complex formation, which was consistent with the hook effect, a phenomenon whereby the binary complex outcompetes the formation of a ternary complex. Importantly, total levels of GSK30 (monomer and complex) remained constant across all MrTACHaXS 8 doses (FIG. 3C), which validated that MrTACHaXS8induced Halo / SNAP proximity in the engineered cell system without degradative effects.

[0066] Robust protein expression of GSK30-SNAP and PRMTl-Halo in stably expressing HEK293 cells was confirmed by microscopy and immunoblot. Next, DMSO or MrTACHaXS8treatments were performed and protein levels were assessed after 24 hours. GSK30 was entirely monomeric in DMSO treated cells and shifted into complexes with PRMT1 with 0.1 nM MrTACHaXS8. Strikingly, GSK30 degradation was observed with 1 nM MrTACHaXS8. The half maximal degradation (DC50) of GSK30 was 11.3 nM MrTACHaXS8(FIGs 3F-3H). In contrast, protein levels of endogenous and Halo-tagged PRMT1 remained constant across MrTACHaXS8treatment (FIGs 3F-3H). To evaluate degradation kinetics. MrTAC dose curves were performed at shorter time points. GSK30 was robustly degraded by MrTACHaXS8after 3 hours with a DC50 of 94.0 nM (FIG. 3I-3K). The hook effect was observed at 50 pM MrTAC 120 HaXS8 with increasing monomeric GSK30 protein levels (FIGs 3F-3K). The higher DC50 for GSK30 at 3 hours could be attributed to the improved efficiency of target degradation at 24 hours. As seen with HEK293 stable lines, GSK30 was recruited and degraded by MrTACHaXS 8 in transiently transfected HEK293T and HeLa cells (FIG. 3N). GSK30 degradation was similarly evident by fixed microscopy analyses comparing DMSO and MrTACHaXS8treatments for 3 hours (FIGs 3L-3M). Next, MrTAC degradation of GSK30 was visualized in real time using live-cell microscopy. Cells expressing a GFP-tagged GSK30-SNAP showed strong expression before treatment that was reduced by MrTACHaXS8after 30 minutes, supporting rapid GSK30Reference No.: 2024-9AV-1, UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025 degradation (FIG. 3N). Thus. MrTAC dose responses induce GSK3P degradation across timeframes and cell lines.

[0067] Next, MrTAC induced proteolytic mechanisms were evaluated. Live-cell imaging showed that GSK3P reorganized into cytosolic puncta following treatment of MrTACHaXS8(FIG. 3N). To evaluate whether these puncta were in fact representative of lysosomes, degradation was visualized in live cells that were stained with LysoTracker. MrTACHaXS8treatment drove stably expressed GSK3P-mCherry into puncta that overlapped with lysosomes within 25 minutes and over time, GSK3P fluorescence decreased (FIGs 4A-4C). Similar lysosomal delivery and degradation was observed with GSK3P-GFP upon MrTACHaXS8incubation. Colocalization immunofluorescence was performed to evaluate MrTACHaXS8trafficking of GSK3P into lysosomal puncta marked by lysosomal associated membrane protein 1 (LAMP-1) in fixed cells. Proximity ligation assays (PLA) were used as an orthogonal approach to evaluate substrate internalization into lysosomes. This assay monitored fluorescent signals that were generated from polymerase-driven rolling circle amplification when two protein targets were within 40-100 nm40 (FIGs 4D-4F). Cells were stained with anti-FLAG antibody to recognize GSK3P and co-stained with a luminal LAMP-1 antibody to report lysosomal internalization. Compared to DMSO treatments, MrTACHaXS8increased PLA signal in GSK3P:LAMP-1 cells while IgG antibody control pairs were negative (FIGs 4D-4F). These analyses using live imaging, fixed imaging, and PLA showed that MrTAC spatially redirected cytosolic GSK3P into lysosomes.

[0068] During cell growth, lysosomal delivery of methylated GSK30 occurred within the minute timescale. Given that the induced kinetics of GSK.3P trafficking into lysosomes by MrTACHaXS8similarly occurred within minutes, the mechanisms of the synthetically induced GSK3P proteolytic process were observed. To ensure that GSK3P was specifically degraded in lysosomes, cells were treated with MrTACHaXS8for 24 hours in the presence of lysosomal or proteasomal inhibitors. Importantly, the lysosomal inhibitor bafilomycin significantly stabilized the MrTAC complex 4-fold (compare lanes 2 and 4) while proteasomal inhibitors, bortezomib or MG132, had no effect (FIG. 4G). This evidence supported that MrTACHaXS8induced lysosomal GSK3P proteolysis.

[0069] To further study the mechanism of MrTAC proteolysis, a genetic approach was employed to disrupt lysosomal degradation. Proteins were delivered into lysosomes via three distinct forms of autophagy: (macro)autophagy, chaperone-mediated autophagy (CMA), and microautophagy. Previous work demonstrated that native delivery of methyl-substrates via MrDegronReference No.: 2024-9AV-1 , UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025 modifications occurred through microautophagy, which was coordinated by endosomal sorting complexes required for transport (ESCRT) signaling. During microautophagy, vacuolar protein sorting 4 (VPS4) mediated the final step of invagination of the lysosomal membrane. To evaluate the role of microautophagy in MrTAC degradation, colocalization analyses were performed with GSK30 and VPS4 in cells expressing either wildtype- or dominant negative (DN)-VPS4. MrTACHaXS8led to colocalization of GSK.30 and WT-VPS4-GFP across cell-types. However, GSK30 entry into lysosomes was ablated by expression of a dominant negative (DN)-VPS4-GFP. To evaluate the necessity of microautophagy' for MrTAC degradation, MrTACHaXS8GSK30 degradation assays were performed in cells treated with siRNA against Vps4a. Cells treated with siVps4a showed a complete rescue of GSK.30 protein levels during MrTACHaXS8treatment (FIG. 41). In contrast, knockdown of macroautophagy or CMA with siRNA against Atg7 or Lamp2a, respectively, were unable to rescue MrTACHaXS8degradation of GSK3P (FIG. 41). Further, MrTACHaXS8treated cells lacked GSK3P localization with lysosomes in siVPS4a cells compared to control treatment (FIGs 4J-4L). Rescue by either pharmacologic- or genetic-based targeting of lysosomal activity supported that MrTAC co-opted endogenous degradation machinery, representing the first application of microautophagy for targeted protein degradation.

[0070] In naturally occurring methyl-driven proteolysis, PRMT1 catalyzes asymmetric dimethyl-arginine modifications to mark substrates with MrDegrons for lysosomal delivery. It was examined whether synthetically induced PRMT1 proximity by MrTAC led to GSK3P methylation. To first detect substrate methylation within native cellular environments, PLA assays were performed using antibodies recognizing the FLAG tag on GSK3P-SNAP and asymmetric dimethyl arginine (ADMA). Compared to DMSO controls, MrTACHaXS8treatment resulted in a 5-fold increase in PLA signal, demonstrating induced methylation (FIGs 5A-5B). PLA signal was absent in cells incubated with IgG control antibody pairs. To identify the arginine methylation sites, liquid chromatography-mass spectrometry (LC-MS) was used to evaluate GSK3P from in vitro reactions with or without purified PRMT1 protein. GSK.30 was methylated at conserved arginine residues within an N-terminal intrinsically disordered region (IDR) and within surface-exposed arginine residues 96, 102, and 111 (FIG. 5C). Next, LC-MS was used to study GSK30 arginine methylation in cells treated with MrTACHaXS8by isolating GSK30-MrTAC-PRMTl complexes from cultured HEK293 cells. Cells were treated with MrTACHaXS8and complexes were immunoprecipitated using magnetic anti-FLAG beads that recognized GSK.30. By LC-MS, PRMT1 and GSK.30 peptides were both present, confirming successful protein-protein interaction. Consistent with the in vitro assays, GSK.30 was methylatedReference No.: 2024-9AV-1 , UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025 at multiple arginine residues, including native sites identified in vitro and at new sites. These data indicated that inducing PRMT1 proximity with MrTAC is sufficient for GSK.30 methylation.

[0071] It was hypothesized that MrTAC induced protein methylation to drive targeted lysosomal proteolysis. To confirm that PRMT1 methyltransferase activity was required for MrTAC degradation, MrTACIIaXS8treatments were performed in the presence of GSK3368715, a selective type I-PRMT inhibitor. MrTACHaXS8induced GSK3[3 degradation at expected doses while co-incubation of MrTACHaXS8with GSK3368715 had no effect, suggesting that GSK3P methylation precedes degradation (FIGs 5D-5F). To further substantiate these data, a genetic approach was employed using a catalytically dead PRMT1 mutant, which has been comprehensively characterized to disrupt enzymatic activity, by replacing residues in the s-adenosyl methionine (SAM) binding site (VLD to AAA). Stable cell lines were generated to express a Halo-tagged fusion of the catalytically dead PRMT1 (denoted PRMTlDead) and MrTACHaXS8assays were performed. GSK3P protein levels were unaffected by MrTACHaXS8treatments in PRMTlneadstable cells (FIGs 5G-5H) as seen with methyl-inhibitor analyses. Together, pharmacologic- or genetic-based inhibition of methyltransferase activity' rescued the effects of MrTAC and supports a model that methylation drove MrTAC degradation.

[0072] GSK3P regulated canonical Wnt signaling during cell growth, embryogenesis, and tissue regeneration. Upon stimulation. Wnt ligands bind to membrane receptors, and cytosolic GSK3P becomes sequestered into lysosomes, which allows P-catenin to accumulate and initiate transcriptional activity. Given this, it was evaluated whether synthetically induced GSK3P degradation recapitulated physiologic Wnt signaling. Immunoblotting confirmed that MrTACHaXS8degradation of GSK3P-SNAP was maintained across multiple days (FIGs 6A-6B) and was sufficient to stabilize total levels of P-catenin compared to DMSO (FIG. 6C). Using cells that stably express GSK3P-SNAP by CRISPR integration, the downstream consequences of targeted GSK3P degradation were evaluated by directly comparing control and MrTACHaXS8treatments. In these cells, MrTACHaXS8significantly increased Wnt target genes cMyc and Birc5 relative to DMSO treatment (FIG. 6D). Finally, cell growth assays were performed to test whether induced GSK3P degradation would be sufficient to increase proliferation as seen during Wnt signaling. Compared to DMSO treatment, MrTACHaXS8significantly increased rates of proliferation in cells co-expressing PRMTl-Halo and GSK3P-SNAP (FIGs 6E-6F). In contrast, no significant changes were observed during drug treatment in control cells, which included non-transfected cells and cells expressing Halo-empty / SNAP-empty vectors. PRMT1 proteinReference No.: 2024-9AV-1, UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025 levels were stable across treatments. These data, directly comparing DMSO and MrTACIIaXS8using stably integrated cells, suggested that MrTAC reduction of global GSK3 protein levels promoted grow th phenotypes.

[0073] The next aim was to extend MrTAC as a proof-of-concept degrader modality by targeting proteins that were classically degraded in proteasomes. Myelocytomatosis (c-MYC) is a nuclear transcription factor that regulates myriad growth processes and is the most frequently upregulated oncogene in cancer. Despite intensive efforts and drug campaigns, c-MYC is recalcitrant to small molecules and considered undruggable. This prompted the team to evaluate whether human c-MYC could be targeted specifically to lysosomes via MrTAC (FIG. 6G). Human c-MYC was fused with SNAP-tag and expressed in HEK293 cells containing CRISPR-integrated PRMTl-Halo. It was first confirmed that MrTACHaXS8could induce monomeric c-MYC (70 kDa) into complexes with PRMTl-Halo (150 kDa) at 0.01 pM MrTACHaXS8(FIG. 6G). MrTAC doses above 0.1 pM MrTACHaXS8corresponded with decreased c-MYC protein levels after 24 hours indicative of degradation. Immunofluorescence analyses were performed to evaluate the cellular distribution of c-MYC. DMSO-treated cells displayed c-MYC localization in nuclear and cytosolic cellular compartments. In contrast, MrTACHaXS8treatment led to the colocalization of c-MYC with an arginine methylation antibody and lysosomes marked by LAMP-1. These data indicate that MrTAC could degrade and relocalize SNAP-tagged c-MYC.

[0074] To confirm that lysosomal activity was responsible for c-MYC proteolysis, short-term MrTAC assays were performed in the presence of lysosomal or proteasomal inhibitors. Assays were performed at short time points using cycloheximide, which inhibits protein synthesis, to enable sensitive analysis of protein turnover. During MrTACHaXS8treatment. c-MYC was readily incorporated into ternary complexes within 30 minutes. The lysosome inhibitor bafilomycin stabilized the ternary complex while the proteasome inhibitor MG132 had no stabilizing effect, supporting that induced c-MYC degradation was mediated through lysosomes. Given the role of c-MYC in cell growth, the engineered system was next utilized to test whether inducing degradation of c-MYC with MrTACHaXS8would be sufficient to decrease proliferation rates. DMSO or MrTACHaXS8treatments were performed in HeLa cells co-expressing c-MYC-SNAP and PRMTl-HaloTag constructs and cell numbers were counted across 7 days. Direct comparisons between engineered cell lines showed MrTACHaXS8treatments were associated with significantly decreased cell density after seven days compared to DMSO (FIG. 6H). Together, these assays hinted at the tractability’ of MrTAC as a generalizable degrader towardsReference No.: 2024-9AV-1 , UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025 neo-substrates.

[0075] Degrader therapeutics rely on endogenous cellular machinery to mediate target protein degradation. The proof-of-concept degradation of multiple target proteins using SNAP -tag and MrTACHaXS8suggested that endogenous, untagged proteins could also be targeted for degradation by replacing the SNAP ligand with an endogenous protein binder. Bromodomain containing protein 4 (BRD4) is an essential protein in mammalian biology that has been strongly linked to cancer and inflammatory diseases. BRD4 is widely used as a model substrate in developing degrader modalities as highlighted by ARV-825, A-1874, dBETl, and MZ-1. BRD4 has a well-established ligand, JQ1, with a binding affinity of 42 nM (IC50) to the first bromodomain. Given disease relevance and a defined ligand, BRD4 was nominated as the first candidate to evaluate endogenous protein degradation using MrTAC. 3-D molecular modeling was performed using the first bromodomain of BRD4 co-crystalized with JQ1 (PDB ID: 3MXF) with HaloTag (PDB ID: 6U32) to develop a MrTAC compound that could engage BRD4 and PRMTl-Halo without steric or electrostatic clash. From this, a chloroalkane-derivatized JQ1 warhead was synthesized using a polyethylene glycol (PEG) linker length of 2 units, which was denoted MrTACJQ1(FIG. 7A). To evaluate target degradation, dose-responses of MrTACJQ1or ControlJQ1(JQ1-PEG2-NH) treatments were performed in HEK293 cells stably expressing PRMTl-Halo and BRD4 levels were measured by immunoblotting. Compared to control treatment, BRD4 protein levels were significantly reduced at 100 nM MrTACJQ1where it also reached its Draax. PRMT1 protein levels were constant in control or MrTACJQ1treatments. Similarly, BRD4 protein levels were reduced in immunofluorescence microscopy analyses of MrTACJQ1treated cells (FIGs 7D-7E). Co-immunoprecipitation of PRMT1 showed BRD4 interactions were induced by MrTACJQ1(FIG. 7F). It was noted that cells were incubated with proteolytic inhibitors to resolve protein-protein interactions, as in previous studies.

[0076] Next, a target from a different protein family was evaluated. Histone deacetylase 6 (HDAC6) is a critical enzyme that is found in the cytoplasm and associates with microtubule cytoskeletal components. Given the central role of HDAC6 in several human diseases, extensive drug discovery efforts have been devoted to developing HDAC6 ligands. Vorinostat, also known as suberoylanilide hydroxamic acid (SAHA), is an FDA approved therapeutic and a potent ligand of HDAC6 with 40.9 nM (IC50). A previously synthesized chloroalkane derivatized-SAHA molecule was repurposed to enable PRMT1 recruitment to HDAC6, denoted MrTACSAHA(FIG. 7G). Dose-response assays with MrTACSAHAor ControlSAHA(SAHA ligand) were performed inReference No.: 2024-9AV-1 , UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025 the aforementioned HEK293 stable line and protein levels were evaluated by immunoblotting (FIGs 7H-7I). While HDAC6 levels were unchanged in cells treated with ControlSAHA, a striking reduction of HDAC6 was observed in 100 nM MrTACSAHAtreatments with a Dmaxoccurring at 10 pM MrTACSAHA(FIGs 7H-7I). PRMT1 protein levels and localization were unaffected by MrTAC5'" '. Further, HDAC6 was also degraded in HeLa cells expressing PRMTl-Halo with a Dmax at 1 pM MrTAC "'1' and a hook effect occurred above 5 pM. Finally, microscopy immunofluorescence staining of endogenous HDAC6 further demonstrated protein levels were reduced by MrTACSAHA in HEK293 (FIGs 7J-7K) or HeLa cells. Co-immunoprecipitation assays confirmed PRMT1 interactions were selectively induced with HDAC6 in cells treated with MrTACSAHA(FIG. 7L). HDAC6 is typically degraded in the proteasome, which suggests that MrTAC enabled endogenous neo-substrate activity by inducing HDAC6 lysosomal proteolysis. To confirm this possibility, degradation experiments were performed in the presence of proteasome or lysosome inhibitors. In MrTACSAHAtreated cells, lysosomal inhibition stabilized HDAC6 levels while proteasomal inhibition was unable to prevent degradation. Hence, these data support a model whereby methyl-induced proteolysis by MrTAC has generalizability towards endogenous proteins.

[0077] To further confirm the selectivity of target degradation, it was next determined whether downstream responses mimicked loss-of-function phenotypes with target proteins. Cells treated with MrTACJQ1reported Bcl2 and Birc3 downregulation and Cdknla upregulation (FIG. 8A), consistent with previous BRD4 loss-of-function studies. Brd4 transcript levels were unaffected, as expected. BRD4 has established roles in cell growth and proliferation. Consistent with this, cell proliferation rates were significantly reduced following 6 days of treatment with MrTAC JQ1 compared to ControlJQ1compound (FIG. 8B). To extend HDAC6 degradation studies, next whether MrTACSAHAwas sufficient to recapitulate classic HDAC6 loss-of-function phenotypes was examined. HDAC6 is a deacetylase enzyme and is known to promote proliferation across multiple cell types and tissues including HEK293 cells used here. Given this, it was first evaluated whether targeted degradation of HDAC6 was sufficient to impact acetylation status of a known substrate, tubulin. MrTACSAHAtreatments led to a 15-fold increase in acety lated tubulin levels relative to total tubulin (FIGs 8C-8D). To determine whether targeted HDAC6 degradation would similarly recapitulate biological responses after longer time points, cell survival was evaluated. MrTACSAHAtreated cells significantly reduced survival compared to cells not expressing PRMTl-Halo (FIG. 8B). PRMT1 protein levels were unaffected by MrTAC""''"'' treatments. In HDAC6-depleted cells, levels of other HDAC family members were unaffected,Reference No.: 2024-9AV-1, UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025 confirming the selectivity of MrTAC (FIG. 8F). These data suggest that selective depletion of endogenous proteins by MrTAC recapitulates loss-of-function phenotypes on cell function.

[0078] The model supported a mechanism whereby methylation is induced by proximity with PRMT1 to enable lysosomal degradation. To evaluate methylation requirements for endogenous degradation, MrTAC in PRMTlDeador PRMT1WT stable cell lines were evaluated. While HDAC6 was reduced by MrTACSAHAin PRMT1WT cells, protein levels in PRMTlDeadcells were unchanged consistent across treatments (FIGs 8G-8J). These data further support that methylation drives targeted degradation by MrTAC of endogenous proteins in living cells. Collectively, MrTAC harnesses an entirely natural proteolytic pathway and opens a new class of degraders for basic research and clinical development.

[0079] MrTAC is a small molecule that induces the proximity of PRMT1 with a target protein. With the recent discovery of the naturally occurring methyl arginine degron (MrDegron), it was evaluated whether synthetically inducing protein methylation could enable targeted proteolysis. It was demonstrated that MrTAC degrades diverse substrates with distinct structural features, turnover kinetics, and subcellular locations. It was also shown that MrTAC is amenable towards both canonical MrDegron targets and neo-substrates, which are typically degraded in proteasomes. Engineering a dual-covalent MrTAC system enabled step-wise tracking of (i) complex formation by immunoblot, (ii) target protein methylation by LC-MS, (iii) lysosomal delivery by live-cell imaging, (iv) degradation by immunoblot / microscopy, and (v) functional effects of protein depletion. Finally, use of a catalytic MrTAC to engage endogenous proteins led to the lysosomal proteolysis of two substrates with well-defined ligands: BRD4 and HDAC6.

[0080] The lysosome is a fundamental organelle across all tissues and is responsible for the catabolism of diverse protein families and macromolecular structures. Additionally, lysosomes degrade proteins that are inaccessible to proteasomes such as aggregates or targets with extended repeat motifs. Extracellular and membrane proteins can be degraded by endocytosis-lysosomal TPDs such as Lysosomal Targeting Chimeras (LYTACs), Bispecific Aptamer Chimera, AbTAC, GlueTAC, and MoDE-A19. Within cells, macroautophagy sequesters proteins into autophagosomes that fuse onto lysosomes during fasting and can be exploited by autophagy-targeting chimeras (AUTACs) that induce target K63-ubiquitination. Subsequent strategies ATTEC and AUTOTAC have since emerged, which tether substrates to autophagosomes. To combat cell-type variability of basal macroautophagy activity. AUTOTAC deployed a two-step mechanism that tethers substrates to p62 and stimulates autophagosomeReference No.: 2024-9AV-1, UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025 biogenesis. Microautophagy is constitutively active and delivers proteins via ESCRT machinery proteins with VPS4 operating at the critical final step. MrTAC synthetically triggers methyl-driven proteolysis via microautophagy7, which may have benefits by circumventing autophagosomes or ubiquitination.

[0081] PROTACs use natural degradative tags to selectively eliminate pathogenic proteins and over 25 PROTACs have entered clinical trials. MrTAC utilized a natural but entirely distinct proteolytic pathway in lysosomes, and an alternative degradative tag, arginine methylation. Acquired resistance to PROTACs has been linked to lysine mutations that block ubiquitin proteasomal targeting. MrTAC was independent of ubiquitin and ligases, which have cell-type specific expression profiles, and would not be impacted by lysine mutations. The chemical biology field has widely adopted the use of encoded degron tags that respond to small molecules, like dTAGs, that enable targeted degradation of genetically modified proteins. It was show n that MrTAC similarly degraded proteins that lack endogenous ligands by incorporating fusion proteins for lysosomal proteolysis, which opens new possibilities for functional dissection of biological pathways in engineered living systems. Previously, HaloTag was used in PROTAC studies with ligands towards Von Hippel Ligase (VHL) and cereblon (CRBN). The study extended this by incorporating HaloTag and SNAP-tag together to study a new7degrader modality, given the absence of an established silent PRMT1 ligand recmiter. The dual covalent approach enabled feasible detection of ternary complex dynamics, which offered insight into MrTAC kinetics and mechanism. This approach could be readily extended to other degrader modalities like PROTAC to study complex features like substrate PROTAC tability and kinetics.

[0082] Methylation orchestrates fundamental biological processes by modifying diverse substrates such as proteins, nucleic acids, metabolites, and lipids. A proximity-based small molecule inducer of methylation could offer a valuable resource across a spectrum of research settings but has yet to be developed. In this study, a first-in-class proximity inducer of protein methylation was presented, which was applied for targeted degradation. A growing body highlights proximity inducers of protein phosphorylation and acetylation modifications with phosphorylation-inducing chimeric small molecules (PHICS) and AceTAG, respectively. Inducing methylation now7broadens the range of tools available and could be used across a spectrum of fields. Given the benefits of sub-stoichiometric mechanisms-of-action for chimeric small molecules, future MrTACs would benefit from non-covalent, silent PRMT1 ligand recruiters.Reference No.: 2024-9AV-1, UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025

[0083] Antibodies used for western blots, immunofluorescence and PLA were as follows: anti-FLAG (Sigma Aldrich, 1804), anti-SNAP-tag (New England BioLabs, P9310S), anti-Actin (Sigma Aldrich, A1978), anti-LAMP-1 (D2D11, Cell Signaling Technology, 9091), anti-GSK3p (Enzo Life Sciences, ADI-KAP-ST002), anti-PRMTl (Sigma Aldrich, SAB2702290), anti-ADMA (21C7, Abeam. Ab413), anti-Lamp2a (EPR4207(2), Abeam. abl25068), anti-Atg7 (D12B11, Cell Signaling Technology, 8558), anti-Vps4 (Sigma-Aldrich, SAB4200025), anti-H3 (96C10, Cell Signaling Technology, 3638), anti-HDAC6 (D2E5, Cell Signaling Technology, 7558), anti-Acetyl-a-Tubulin (Lys40) (D20G3, Cell Signaling Technology7, 5335), anti-a-Tubulin (DM1A, Cell Signaling Technology, 3873), anti-BRD4 (ThermoFisher, A301-985A-T). Antibodies were used at 1 :300 dilutions for immunofluorescence with exception of ADMA (1:50), BRD4 (1 :200) and HDAC6 (1:200). Antibodies were used for immunoblot at 1 : 1,000.

[0084] All cells were obtained from ATCC. Cells were cultured in DMEM supplemented with 10% heat-inactivated FBS (GIBCO), 1% penicillin / streptomycin, 0.25 mg / mL Plasmocin (Invivogen, ant-mpp). and 1% glutamine. The cells were maintained at 37°C and 5% CO2. Media was replenished every 2-3 days and passaged with 0.25% trypsin when cells reached -75% confluency. Cells were routinely tested with the Invivogen MycoStrip Mycoplasma Detection Kit (Invivogen, rep-mys). Cells were transfected using Lipofectamine 2000 as per manufacturer instructions. Stable HEK293 cell lines expressing wild type or catalytically dead (VLD92-95AAA 715 ) PRMTl-HaloTag were generated by plasmid transfection and selection for 2 weeks in 500 pg / mL G418. Cells co-expressing GSK3|3-SNAP-tag-mCherry-FLAG were generated by plasmid transfection and selection for 2-3 weeks in 100 pg / mL zeocin. Cells were maintained in 0.5x selection media. For HEK293T cell lines with CRISPR insertion of either Halo-PRMTl or Halo-PRMT1 / GSK3[3-SNAP. homology-independent targeted insertion was used as previously described. HEK293T cells were transfected with Halo-PRMTl or Halo-PRMT1 / GSK3P-SNAP constructs, Cas9, and gRNA targeting a safe harbor in Chromosome 19 using Lipofectamine 3000 as per manufacturer instructions. Cells were in selection for 4 weeks in 100 pg / mL zeocin. Cells were sorted and maintained in 100 pg / mL zeocin for 1 week post-sort to ensure high stable expression.

[0085] All primers were obtained from IDT. Halo-PRMTl -My c vector was generated by ligating HaloTag (Addgene, 154144) into the PRMT1 plasmid (Origene, RC224239) via EcoRI / KpnI. Catalytically dead PRMT1 was generated using the QuikChange site-directed mutagenesis kit (Agilent, 200523) according to manufacturer’s instructions. SNAP-GSK3P-FLAG vector wasReference No.: 2024-9AV-1, UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025 generated by replacing PRMTl(Origene RC224239) with GSK3P using KpnI / Notl. The SNAP -tag gene (Addgene, 101135) was inserted to the GSK3 C-terminus via EcoRI / KpnI. Halo-PRMTl and SNAP-GSK3P (GGS)2 linkers were added with 5’-phospho PCR ligation. 5 ’-phospho PCR ligation was performed again to remove unneeded affinity tags. The Snap-GSK30-eGFP and mchenyconstructs were generated using a backbone encoding eGFP or mCherry under the human PGK promoter. Snap-GSK.3(3 was amplified and inserted into these plasmids using KpnI / EcoRI. Snap-c-MYC plasmid was generated from the Snap-GSK3|3 plasmid using BamHI / Notl with the c-MYC (Addgene, 101135) primers. Plasmids for CRISPR / Cas9 homology -independent targeted insertion (HITI) encoded Halo-PRMTl or Halo-PRMTl-T2A-SNAP-GSK3P-mCherry. By Gibson Assembly, the Halo-PRMTl gene was inserted into a parental HITI plasmid. By Gibson Assembly, the T2A-SNAP-GSK3P-mCherry was then inserted.

[0086] Cultured cells were lysed in 2x Laemmli sample buffer (4% SDS. 20% Glycerol, 120 mM Tris-Cl pH 6.8, 0.02% bromophenol blue) with lOmM fresh DTT. After a 10 minute incubation at 95 °C, lysates were loaded on a 10% SDS-PAGE gel. Following separation, proteins on the gel were transferred onto nitrocellulose membranes at 10V for 75-130 minutes. Membranes were then blocked in milk and probed overnight with primary' antibodies. Signal was detected using ECL (Sigma Aldrich, WBLUR0100) after a 1 hour incubation in secondary antibody (mouse 1 : 10,000 Invitrogen 61-6520, or rabbit 1:20,000 Prometheus 20-303). Images were acquired using the iBright FL750 imaging system. Bands were quantified using ImageJ software and normalized to the individual loading controls prior to normalization to the control sample. In some experiments, a treatment eliciting high ternary complex formation was quantified relative to the amount of ternary complex in a higher, degradative dose.

[0087] Cells were plated and grown on glass coverslips. For HEK293 / T cells, glass coverslips were coated in Poly-L-Lysine (Newcomer Supply, 1339A) prior to plating. Following indicated treatments, cells were washed twice in PBS and fixed for 20 min in 4% paraformaldehyde (PFA). Fixed cells were washed 2x 5 min in PBS. permeabilized in 0.2% Triton X-100 for 10 min, washed 2x 5 min in PBS, and blocked in 0.5% BSA before an overnight incubation in primary antibody. The following day, coverslips were washed 3x 7 min in PBS, incubated in secondary antibody (1:2,000-5,000), washed 5x 5 min in PBS, and mounted with or without DAPI (ThermoFisher, P36931 and P36930).

[0088] The proximity ligation assay (PLA) of HEK293T cells was performed using the SigmaReference No.: 2024-9AV-1 , UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025Aldrich PLA kit (DU092004, DU092002) as per manufacturer’s instructions. Briefly. HEK293T cells growing on Poly-L-Lysine coated glass coverslips were treated with 10 pM HaXS8 for 60 minutes, fixed with 4% PFA, and prepared for staining with primary antibodies as described above. Anti-FLAG, anti-LAMP-1, and anti-IgG were used at 1:300 dilutions and anti-ADMA was used at 1:50 dilutions. Anti-IgG was used as a negative control. The following day, specimens were incubated with anti-mouse and anti-rabbit secondary antibodies conjugated to oligonucleotides. Close proximity of target antigens allows hybridization of the complementary secondary' antibody nucleotide sequences. After a 30 minute 37°C incubation with ligase and additional oligonucleotides, a closed DNA circle is formed. A subsequent step involving polymerase-driven rolling circle amplification incorporates fluorescently labeled nucleotides. Fluorescent nucleotides used in this experiment fluoresce in the red channel upon excitation. Protein-protein interactions between FLAG-LAMP- 1 or FLAG-ADMA were visualized with confocal microscopy and quantified with ImageJ (NIH, http: / / imagej.nih.gov / ij / ). Data were normalized by applying the same brightness / contrast profile and threshold values. The ImageJ analyze particles feature was utilized to quantify PLA signals over 113-120 cells for each condition. Raw values for each paired experiment (FLAG-LAMP- 1, FLAG-ADMA) were then converted to fold change with respect to the appropriate control.

[0089] HEK293T cells were seeded in 6-wells and transfected with lOOnM siRNA using Lipofectamine RNAiMax (Invitrogen, 13778100) according to the manufacturer’s protocol. ON-TARGETplus SMARTpool siRNA targeting Lamp2a (L-011715-00-0005), Atg7 (L-020112-00-0005), or Vps4a (L-013092-00-0005) were obtained from Dharmacon. After 24 hours of siRNA treatment, cells were split into 12- well dishes. The following day (day 2 of siRNA knockdown), cells were transfected with PRMTl-Halo and GSK3(3-SNAP for 24 hours. On day 3 of siRNA knockdown, cells were treated for 24 hours with DMSO or 10 pM MrTACHaXS8. Lysates were collected for immunoblot analysis and cells were processed for immunofluorescence assays as described above.

[0090] HEK293 cells stably expressing Halo-PRMTl-Myc were cultured in 10 cm dishes with DMSO or 1 pM MrTACSAHAfor 2 hours in the presence of 400 nM Bafilomycin and 10 pM MG132. Cells were lysed by scraping in lysis buffer (50 mM Tris pH 7.9, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 10 pM leupeptin, 100 pM AEBSF, and 2 mM PMSF) and clarified by centrifugation at 16,000 x g for 5 minutes at 4°C. Clarified lysates were incubated with anti-Myc magnetic beads (Thermofisher, 88842) (equilibrated with 3x 10 vol / vol TBSReference No.: 2024-9AV-1, UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025 washes) for 3 hours on a rotator at 4°C. Following incubation, beads were washed twice with TBS and eluted in 4x Laemmli Buffer followed by incubation at 95°C for 20 minutes. Lysates and immunoprecipitated samples were analyzed by immunoblot as described above.

[0091] For dose curve and rescue experiments, variable doses of HaXS8 (MrTAC) were added to cell culture media to a final concentration of 0.1% DMSO (v / v). Cells were treated 1 day post-transfection. The following compounds were used at the indicated concentrations and pre-treatments with a final concentration of 0.1% DMSO (v / v): bafilomycin (100-400 nM, Cayman Chemicals, 11038), bortezomib (50 nM, AvaChem Scientific, 2164), MG132 (5-10 pM, AdooQ Bioscience, A11043), GSK3368715 (10 pM, 24 hr pre-treatment, MedChemExpress, HY- 128717 A), SAHA chloroalkane T1 (GLPBio, GC34772), and SAHA (Thomas Scientific, C835T10). For stably integrated GSK30 proliferation experiments, HEK293 cells stably expressing Halo-PRMTl with SNAP-GSK3P-mCherry were seeded at 50,000 cells / well in 12-wells and treated with DMSO or 0.5 pM MrTACHaXS8on days 0, 1, 2, and 3. Live cells were counted on days 3 and 6 using a Trypan Blue stain with a Countess 2 Automated Cell Counter. At indicated time points, cells were lysed in 2x Laemmli buffer for immunoblot analysis.

[0092] For transient GSK3(3 and c-Myc proliferation experiments, HeLa cells were transfected in 10 cm dishes overnight and were subsequently plated in 12-wells with 50,000 cells / well. Treatment with DMSO or 0.5 pM HaXS8 began on the same day of plating (day 0). Live cells were counted on days 1 , 3, 5 and 7 using a Trypan Blue stain with a Countess 2 Automated Cell Counter. At indicated time points, cells were lysed in 2x Laemmli, processed for qPCR, or stained with crystal violet. For crystal violet staining, cells were grown on coverslips, fixed in 4% PFA for 20 minutes, washed twice with PBS and stained with 50 pg / mL crystal violet for 30 minutes before imaging. For proliferation assays of endogenous BRD4, HEK293-PRMT1-Halo stable cells were seeded onto 12-well dishes at 50,000 cells / well. The following day, cells were treated in triplicate with 1 pM of MrTACJQ1or JQ1-PEG2-NH control. Cells were refreshed with media and drug treatments every day. After five days, wells were counted as described above. The fold changes in cell number were calculated based on the seeding density, followed by normalization against the control, where the control was always set as 1. For the cell survival assay, HEK293-PRMT1-Halo stable cells and non-transfected HEK293 cells were seeded onto 96-well dishes at 25,000 cells per well. The following day, cells were treated with a MrTACSAHAdose curve (100. 50. 10. 1, 0.5, 0.1. 0 pM) for 72 hours. Cell survival was measured using a Cell Counting Kit-8 (Dojindo, CK04) according to manufacturer's instructions after a 2-hourReference No.: 2024-9AV-1 , UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025 incubation with the CCK8 reagent.

[0093] For experiments involving gene expression analysis by quantitative real time PCR (qRT-PCR), cells were plated at 50,000 cells / well in 12-well plates in complete DMEM. Cells were treated with MrTACJQ1or DMSO and lysed at indicated timepoints. Then, RNA was isolated using the Direct-zol RNA Miniprep Kit (Zymo, R2050). cDNA was synthesized using 500 ng RNA per reaction using the High Capacity RNA-to-cDNA Kit (Invitrogen 4387406). qRT-PCR was performed using a Roche LightCycler480 and PowerUp SYBR Green qPCR Master Mix (Applied Biosystems A25742). 0.5ug / ml primers were used for each reaction. Each biological replicate was plated in technical triplicate. Fold change related to the control group was calculated using 2 ACP method with Rpll3a as the reference gene.

[0094] Sequence-based GSK3(3 conservation was modeled using Consurf software. Briefly, human GSK3( protein (Uniprot ID P49841) was aligned against 150 sequences using the HHMER search algorithm and the UniREF-90 protein database. The following settings were used: 1 iteration. E-value cutoff 0.0001. 95% maximal identity between sequences, 35% minimal identity for homologs, MAFFT-L-INS-i alignment, Bayesian calculation method, and Best Model evolutionary substitution model.

[0095] In vitro, 1 pg His-GSK3(3-GST (Cayman Chemicals, 33738) and 200 pM S-adenosyl methionine (SAM. Sigma Aldrich, A4377) were incubated in the presence or absence of 1 pg GST-PRMT1 (Cayman Chemical, 10350) in 50 pL reaction buffer (50 mM Tris-HCl, pH 8.0, 20 mM KCI, 5 mM DTT, 4 mM EDTA), and incubated at 37°C for 1 hr. The reaction was reduced with DTT (10 mM, 80°C, 20 min) and alkylated (30 mM iodoacetamide, RT, dark, 1 hr) prior to digestion with sequencing grade trypsin (Promega. 1 / 50 (w / w). 37°C, 18 h). Samples were analyzed by LC-MS using an ACQUITY UPC H-class LC coupled to an Xevo Q-ToF (Waters Corp). Observed m / z were mapped to an in silico digestion of GSK3(3 using Biopharmalynx (Waters Corp, v 1.3.5, 30 ppm, fixed carbamidomethyl Cys, variable oxidation of Met, variable Arg methylation). In silico digestion of PRMT1 was also used to exclude PRMT1 peptides in analysis. Peptides were injected onto a reverse phase C4 column (ACQUITY UPLC Protein BEH C4 Column, 300A, 1.7 pm, 2.1 mm X 50 mm, Waters Corp) in a buffer stream of 3% ACN in 0.1% formic acid (0.3 mL / min) and desalted for 0.5 m before gradient elution from 3-60% ACN over 1.5 m. The Xevo Z-spray source was operated with a capillary voltage of 3000 V and a cone voltage of 40 V (NaCsi calibration, Leu749 enkephalin lock-mass). Nitrogen was used as the desolvation gas at 350°C and a total flow of 800 L / hr.Reference No.: 2024-9AV-1 , UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025

[0096] HEK293 cells cultured in 10 cm dishes were transiently transfected with SNAP-GSK3f>-FLAG and Halo-PRMTl for 24 hours before treatment with 10 (iM MrTACHaXS8 896 for 2 hours in the presence of 400 nM bafilomycin and 10 pM MG132. Cells were lysed by scraping in lysis buffer (50 mM Tris pH 7.9, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 10 pM leupeptin, 100 pM AEBSF, and 2 mM PMSF) and clarified by centrifugation at 16,000 x g for 5 minutes at 4°C. Clarified lysates were incubated with anti-FLAG M2 magnetic beads (Sigma- Aldrich, M8823) (equilibrated with 3x 10 vol / vol TBS washes) for 3 hours on a rotator at 4°C. Following incubation, beads were washed three times with TBS and eluted in 1% formic acid. Immunoprecipitated samples were loaded for LC-MS analysis as described above.

[0097] HEK293 cells stably expressing PRMTl-Halo were cultured in 6-well plates with either DMSO or MrTACSAHA(1 pM, 4 hrs) before lysis by scraping in lysis buffer (50 mM Tris pH 7.9, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 10 pM leupeptin, 100 pM AEBSF, and 2 mM PMSF). Lysates were clarified by centrifugation at 16,000 x g for 10 min and snap-frozen in liquid nitrogen before processing. Samples were reduced with 10 mM dithiothreitol (DTT) for 30 minutes at room temperature. Next, samples were alky lated with 30 mM iodoacetamide for 30 minutes at room temperature in the dark. Samples were pelleted for 30 minutes at maximum speed at 4°C and resuspended in ice cold methanol for two washes and left to air dry. Once dried, samples were digested in 1% LysC in 8 M urea for 4 hours at 37°C followed by digestion with 2% trypsin in 1.5 M urea overnight at 37°C. The following day, samples were acidified in 1% trifluoroacetic acid (TFA), desalted using Sep-Pak C18 cartridges (Waters, 186000308), and eluted in 50% acetonitrile. Digested peptides were separated and analyzed by LCMS / MS using an LUtiMate 3000 RSLC liquid chromatograph (Thermo Fisher Scientific) coupled with in-line to a Thermo Fusion Lumos mass spectrometer (Thermo Fisher Scientific).

[0098] Peptides were separated by reversed-phase chromatography on a Thermo Pepmap Acclaim column (50 cm x 75 pm) over a 90 min submission comprising a 60 min gradient from 4% to 22% mobile buffer B (mobile buffer A: H2O, 0.1% formic acid, mobile buffer B: ACN, 0.1% formic acid). For MS / MS analysis of peptides, each duty consisted of one FTMS scan (375-1,800 m / z, resolution of 120,000) followed by data-dependent MS / MS scans acquired in the ion trap with higher-collisional dissociation (normalized energy 30%) for 3 seconds at top speed in rapid mode. MS / MS data were subjected to database searching using MaxQuant (2.0.3.0) against a database obtained from SwissProt (2024.01.12) containing 20,428 humanReference No.: 2024-9AV-1, UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025 protein entries. The mass tolerances for parent ions and fragment ions were set as default, trypsin / P was set as the enzyme with two maximum missed cleavages allowed, and protein N-terminal acetylation and methionine oxidation were selected as variable modifications; cysteine carbamidomethylation was specified as a fixed modification. PSM FDR, protein FDR, and site decoy fraction were all reported at 0.01. Protein abundances for each sample were measured using iBAQ, and following export were normalized across samples by total ion current (TIC) prior to data analysis.

[0099] All experiments were repeated independently three times unless otherwise specified. All stated values represent the mean ± SEM, values ± 2*SD were excluded as outliers. Sample size is described in figure captions. Sample size n refers to independent biological experiments unless otherwise specified. GraphPad Prism 10 software was used for statistical analyses. Two-sided, unpaired student’s t-test was used to compare variables between two groups (degrees of freedom = nl+n2 - 2). One-way ANOVA was used to compare variables between more than two groups (degrees of freedom = n - 1). Two-way ANOVA was used for multi -day, multi-dose experiments testing multiple variables. Dose curve regressions were performed as described in figure legends where DC50 values were calculated. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 were considered to be statistically significant. Image quantification was performed as described previously. For microscopy experiments, samples were analyzed across indicated cell counts / fields of view in 1 biological replicate unless otherwise specified.

[0100] As used herein, the term ‘"about” refers to plus or minus 10% of the referenced number.

[0101] Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase “comprising” includes embodiments that could be described as “consisting essentially of’ or “consisting of’, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting essentially of’ or “consisting of’ is met.

Claims

Reference No.: 2024-9AV-1 , UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025WHAT IS CLAIMED IS:

1. A compound effective for targeted intracellular methylation of a protein of interest (POI), comprising: a) a methyltransferase-binding moiety configured to bind a methyltransferase protein; b) a target protein-binding moiety configured to bind the POI; and c) a linker moiety connecting the methyltransferase-binding moiety and the target protein-binding moiety.

2. The compound of claim 1. wherein the intracellular methylation is arginine methylation.

3. The compound of claim 1 or claim 2, wherein the methyltransferase-binding moiety binds directly to the methyltransferase protein.

4. The compound of claim 1 or claim 2, wherein the methyltransferase-binding moiety binds indirectly to the methyltransferase protein.

5. The compound of claim 4, wherein the methyltransferase protein comprises a protein tag.

6. The compound of claim 5, wherein the protein tag is genetically encoded with the methyltransferase to generate a methyltransferase fusion protein.

7. A compound effective for targeted intracellular methylation of a protein of interest (POI), compnsing: a) a methyltransferase-binding moiety configured to bind a methyltransferase fusion protein, wherein the methyltransferase fusion protein comprises methyltransferase and a protein tag; b) a target protein-binding moiety configured to bind the POI; and c) a linker moiety' connecting the methyltransferase-binding moiety and the target protein-binding moiety7.

8. The compound of claim 6 or claim 7, wherein the methyltransferase-binding moiety is configured to bind to the protein tag on the methyltransferase fusion protein.

9. The compound of any one of claims 5-8, wherein the protein tag comprises a HaloTAG, a SNAP-tag, a CLIP-tag, a FKBP12 (F36V) tag, a BromoTag, BromoCatch, a Spy Tag, SpyCatcher, a SnoopTag, SnoopCatcher, an eGFP, a vhhGFP, an ALFA, a nbALFA, or a NanoBiT tag.

10. The method of any one of claims 1-9. wherein the target protein-binding moiety binds directly to the POLReference No.: 2024-9AV-1 , UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 202511. The method of any one of claims 1-10, wherein the target protein-binding moiety binds indirectly to the POI.

12. The compound of claim 11, wherein the POI comprises a protein tag.

13. The compound of claim 12, wherein the protein tag is genetically encoded as a fusion with the POI to generate a POI fusion.

14. A compound effective for targeted intracellular methylation of a protein of interest (POI), comprising: a) a methyltransferase-binding moiety configured to bind a methyltransferase fusion protein, wherein the methyltransferase fusion protein comprises methyltransferase and a protein tag; b) a target protein-binding moiety configured to bind a POI fusion, wherein the POI fusion comprises the POI and a protein tag; and c) a linker moiety connecting the methyltransferase-binding moiety and the target protein-binding moiety.

15. The compound of claim 14, wherein the methyltransferase-binding moiety is configured to bind to the protein tag on the methyltransferase fusion protein.

16. The compound of claim 14 or claim 15, wherein the target protein-binding moiety is configured to bind to the protein tag on the POI fusion.

17. The compound of any one of claims 14-16, wherein the protein tag compnses a HaloTAG, a SNAP -tag, a CLIP-tag, a FKBP12 (F36V) tag, a BromoTag, BromoCatch, a Spy Tag, SpyCatcher, a SnoopTag, SnoopCatcher, an eGFP, a vhhGFP, an ALFA, a nbALFA, or a NanoBiT tag.

18. The compound of any one of claims 1-17. wherein the methyltransferase protein is an arginine methyltransferase (PRMT).

19. The compound of claim 18, wherein the PRMT is a type I PRMT.

20. The compound of claim 19, wherein the PRMT is PRMT1, PRMT2, PRMT3, PRMT4, PRMT6, PRMT8, or any combination thereof.

21. The compound of claim 18, wherein the PRMT is a type II PRMT.

22. The compound of claim 21, wherein the PRMT is PRMT5, PRMT9, or both PRMT5 and PRMT9.

23. The compound of claim 18, wherein the PRMT is a type III PRMT.

24. The compound of claim 23, wherein the PRMT is PRMT7.Reference No.: 2024-9AV-1, UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 202525. The compound of any one of claims 1-24, wherein the linker moiety comprises polyethylene glycol (PEG).

26. The compound of any one of claims 1-24, wherein the linker moiety7comprises an alkane linker, an alkyne linker, a cycloalkyl linker, a spirocylic linker, a piperidine linker, a piperazine linker, or a triazole linker.

27. The compound of any one of claims 1-26, wherein the POI is glycogen synthase kinase 3 (GSK-3).

28. The compound of any one of claims 1-26, wherein the POI is bromodomain-containing protein 4 (BRD4).

29. The compound of any one of claims 1-26. wherein the POI is MYC.

30. The compound of any one of claims 1-29, wherein when the methyltransferase-binding moiety binds to the methyltransferase and the target protein binding moiety binds the POI; the methyltransferase protein methylates at least one arginine on the POI.

31. The compound of any one of claims 1-29. wherein when the methyltransferase-binding moiety binds to the methyltransferase and the target protein binding moiety binds the POI; the methyltransferase protein methylates one arginine on the POI.

32. The compound of any one of claims 1-29, wherein when the methyltransferase-binding moiety binds to the methyltransferase and the target protein binding moiety binds the POI; the methyltransferase protein methylates two arginines on the POI.

33. The compound of any one of claims 1-32, wherein the compound is effective to induce degradation of the POI in a non-proteas ome mediated manner.

34. The compound of any one of claims 1-32, wherein the compound is effective to induce lysosomal degradation of the POI.

35. The compound of any one of claims 1-32, wherein the compound is effective to induce microautophagy of the POI.

36. The compound of any one of claims 1-32, wherein the compound is effective to prevent the POI from entering a nucleus of a cell.

37. The compound of any one of claims 1-32, wherein the compound is effective to bring the POI into sufficiently close proximity’ to the methyltransferase, thereby facilitating methylation of the POI by the methyltransferase.

38. A method for screening compatibility between the methyltransferase protein and a POI, the method comprising; administering to an experimental biological system a compound according to any one of claims 1-37;Reference No.: 2024-9AV-1 , UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025 wherein when the methyltransferase-binding moiety binds to the methyltransferase and the target protein binding moiety binds the POI; the methyltransferase protein methylates at least one arginine on the POI; wherein compatibility is indicated when the pairing of the methyltransferase protein and the POI results in one or more of the following: i) protein-protein interactions are induced; ii) methylation of at least one arginine residue on the POI; or iii) degradation of the POI by 20% to 100% compared to a control system lacking the compound.

39. A method for screening compatibility' between the methyltransferase protein and a POI, the method comprising: administering to an experimental biological system a compound comprising: a) a methyltransferase-binding moiety configured to bind a methyltransferase protein; b) a target protein-binding moiety configured to bind the POI; and c) a linker moiety’ connecting the methyltransferase-binding moiety and the target protein-binding moiety; wherein when the methyltransferase-binding moiety binds to the methyltransferase and the target protein binding moiety' binds the POI; the methyltransferase protein methylates at least one arginine on the POI; wherein compatibility is indicated when the pairing of the methyltransferase protein and the POI results in one or more of the following: i) protein-protein interactions are induced; ii) methylation of at least one arginine residue on the POI; or iii) degradation of the POI by 20% to 100% compared to a control sy stem lacking the compound.

40. The method of claim 38 or claim 39, wherein the compatibility is measured in vitro using purified protein.

41. The method of claim 40, wherein the compatibility' is indicated when the pairing of the methyltransferase protein and the POI results in one or more of the following: i) protein-protein interactions are induced using purified proteins in vitro; ii) methylation of at least one arginine residue on the POI using purified protein in vitro; or iii) degradation of the POI by 20% to 100% compared to a control system lacking the compound.

42. The method of claim 38 or claim 39, wherein the compatibility is measured in cells.

43. The method of claim 40, wherein the compatibility is indicated when the pairing of the methyltransferase protein and the POI results in one or more of the following: i)Reference No.: 2024-9AV-1 , UCI 24.12 PCT Inventor’s last name: Albrecht, Seabrook Document Date: August 11 , 2025 protein-protein interactions are induced in cells; ii) methylation of at least one arginine residue on the POI in cells; or iii) degradation of the POI by 20% to 100% compared to a cell lacking the compound.

44. An in vitro method of inducing targeted intracellular methylation of a POI, the method comprising: contacting a cell with a compound according to any one of claims 1-37; wherein, when the methyltransferase-binding moiety binds to the methyltransferase and the target protein binding moiety binds the POI; the methyltransferase protein methylates at least one arginine on the POI.

45. A method of inducing targeted intracellular methylation of a target protein in a subject in need thereof, the method comprising: administering to the subject a compound according to any one of claims 1-37; wherein when the methyltransferase-binding moiety binds to the methyltransferase and the target protein binding moiety binds the POI; the methyltransferase protein methylates at least one arginine on the POI.

46. An in vitro method of inducing targeted intracellular methylation of a POI, the method comprising: contacting a cell with a compound comprising: a) a methyltransferase-binding moiety configured to bind a methyltransferase protein; b) a target protein-binding moiety configured to bind the POI; and c) a linker moiety connecting the methyltransferase-binding moiety and the target protein-binding moiety; wherein, when the methyltransferase-binding moiety binds to the methyltransferase and the target protein binding moiety binds the POI; the methyltransferase protein methylates at least one arginine on the POI.

47. A method of inducing targeted intracellular methylation of a target protein in a subject in need thereof, the method comprising: administering to the subject a compound effective for targeted intracellular methylation of a target protein, said compound comprising: a) a methyltransferase-binding moiety configured to bind a methyltransferase protein; b) a target protein-binding moiety configured to bind the POI; and c) a linker moiety connecting the methyltransferase-binding moiety and the target protein-binding moiety;Reference No.: 2024-9AV-1, UCI 24.12 PCTInventor’s last name: Albrecht, SeabrookDocument Date: August 11 , 2025 wherein when the methyltransferase-binding moiety binds to the methyltransferase and the target protein binding moiety binds the POI; the methyltransferase protein methylates at least one arginine on the POI.

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