US-12625139-B2 - Methods for modulation of acetyltransferase activity and applications thereof including treatments

Abstract

Methods to identify modulators of histone acetyltransferases are described. Modulators of histone acetyltransferases can be used to treat individuals. In some instances, modulators of histone acetyltransferases are utilized to treat individuals having a neoplasm.

Inventors

• Joshua James Gruber
• Benjamin Geller
• Andrew Lipchik
• Michael P. Snyder

Assignees

• THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY

Dates

Publication Date

20260512

Application Date

20200422

Claims (17)

1. 1 . A method to identify modulators of histone acetyltransferase 1 activity, comprising: providing a substrate in a solution within a well, wherein the substrate is a peptide comprising a natural substrate of histone acetyltransferase 1, wherein the substrate is fixed to a surface of the well; adding to the solution a histone acetyltransferase 1, a donor molecule, and a modulator, wherein the donor molecule comprises a coenzyme A structure with an acetyl-like group, wherein the acetyl-like group comprises a carbonyl group and an alkyl extending from the carbonyl group, wherein the alkyl comprises a terminal alkyne, wherein the histone acetyltransferase 1 is extracellular and is capable of transferring the acetyl-like group of the donor molecule onto the substrate, wherein the modulator is a compound being assessed for its ability to modulate the transfer of the acetyl-like group onto the substrate; removing the histone acetyltransferase 1, the donor molecule, and the modulator from the solution, wherein the substrate remains fixed to the surface of the well; adding to the solution biotin-azide, wherein the azide group is configured to conjugate to the alkyne of the acetyl-like group to yield a biotin extending from the substrate via a transferred acetyl-like group; removing unconjugated biotin-azide from the solution, wherein the substrate remains fixed to the surface of the well; adding to the solution a reporter molecule conjugated with streptavidin, wherein the reporter molecule is configured to bind to the biotin extending from the substrate; removing unbound reporter molecule from the solution, wherein the substrate remains fixed to the surface of the well; and measuring signal of the reporter molecule bound to the substrate.
2. 2 . The method of claim 1 , wherein the histone acetyltransferase 1 is a recombinant histone acetyltransferase 1.
3. 3 . The method of claim 1 , wherein the substrate is an n-terminal peptide of histone H4.
4. 4 . The method of claim 3 , wherein the n-terminal peptide of histone H4 has a sequence consisting of SEQ ID No. 1.
5. 5 . The method of claim 3 , wherein the n-terminal peptide of histone H4 is provided at 15 μM.
6. 6 . The method of claim 1 , wherein the donor molecule is 4-pentynoyl Co-A.
7. 7 . The method of claim 1 , wherein the reporter molecule is selected from the group consisting of a fluorophore, a radioisotope, an enzyme, and an antigen binding molecule.
8. 8 . The method of claim 1 , wherein the reporter molecule is horse radish peroxidase (HRP) and a HRP substrate is utilized to visualize HRP activity.
9. 9 . The method of claim 8 , wherein the HRP substrate is 10-Acetyl-3,7-dihydroxyphenoxazine.
10. 10 . The method of claim 1 , wherein the histone acetyltransferase 1 is a recombinant histone acetyltransferase 1, the substrate is an n-terminal peptide of histone H4, the donor molecule is 4-pentynoyl Co-A, the reporter molecule is horse radish peroxidase (HRP) and 10-Acetyl-3,7-dihydroxyphenoxazine is used as a HRP substrate to visualize HRP activity.
11. 11 . The method of claim 2 , further comprising producing the recombinant histone acetyltransferase 1 in a human cell line.
12. 12 . The method of claim 11 , wherein the human cell line is HEK-293f.
13. 13 . The method of claim 11 , wherein producing the recombinant histone acetyltransferase 1 further comprises co-expressing the recombinant histone acetyltransferase 1 with a recombinant RBAP46 within the human cell line.
14. 14 . The method of claim 11 , wherein producing the recombinant histone acetyltransferase 1 further comprises adding one or more factors to the human cell line, wherein the one or more factors are selected from the group consisting of acetate, forskolin, orthovanadate, and AICAR.
15. 15 . The method of claim 1 , further comprising adding one or more reagents to the solution to promote the transfer of the acetyl-like group onto the substrate, wherein the one or more reagents are selected from the group consisting of EDTA and DTT.
16. 16 . The method of claim 1 , wherein the modulator is an agonist of histone acetyltransferase 1, wherein the agonist is selected from the group consisting of 2-(Hydroxymethyl)-6-[1,3,4-trihydroxy-1-(2-phenyltriazol-4-yl)butan-2-yl]oxyoxane-3,4,5-triol and 1-(1H-Benzimidazol-2-yl)hexane-1,2,3,4,5,6-hexol;2,3-dihydroxybutanedioic acid.
17. 17 . The method of claim 1 , wherein the modulator is an antagonist of histone acetyltransferase 1, wherein the antagonist is selected from the group consisting of nogalarol, 3-[3,4-Dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6,7-dihydroxy-6-methyl-5,7-dihydroimidazo[1,2-a]purin-9-one, daunomycin 3-oxime hydrochloride, rhodomycin A, aclacinomycin A1, cinerubin B, Methyl (1R,2R,4S)-4-[5-[5-(4,5-dihydroxy-6-methyloxan-2-yl)oxy-4-hydroxy-6-methyloxan-2-yl]oxy-4-(dimethylamino)-6-methyloxan-2-yl]oxy-2-ethyl-2,5,7,10-tetrahydroxy-6,11-dioxo-3,4-dihydro-1H-tetracene-1-carboxylate, nagalomycin C, N,N-Dibenzyldaunorubicin, and pyrromycin.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a national stage of PCT Patent Application No. PCT/US2020/029395, entitled “Methods for Modulation of Acetyltransferase Activity and Applications Thereof Including Treatments” to Joshua James Gruber et al., filed on Apr. 22, 2020, which claims priority to U.S. Provisional Patent Application No. 62/837,086, entitled “Methods for Modulation of Histone Acetyltransferase Activity and Applications Thereof Including Treatments” to Joshua James Gruber et al., filed on Apr. 22, 2019, the disclosures of which are incorporated herein by reference in their entireties. REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB The instant application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 22, 2020, is named “06132PCT_ST25.txt” and is 3612 bytes in size. FIELD OF THE INVENTION The invention is generally directed to processes to modulate acetyltransferases and applications thereof, and more specifically to methods for identifying modulators of acetyltransferase activity including activity of histone acetyltransferase 1 (HAT1) and treatments utilizing histone acetyltransferase modulators. BACKGROUND Acetyltransferases are enzymes that transfer an acetyl functional group from a donor molecule onto a biomolecule. Acetyltransferase enzymes include (but are not limited to) histone acetyltransferases, choline acetyltransferase, chloramphenicol acetyltransferase, serotonin N-acetyltransferase, and N-terminal acetyltransferases. A common acetylation reaction performed by acetyltransferases is the transfer of the acetyl group from acetyl coenzyme A onto its substrate. For example, choline acetyltransferase (ChAT) transfers an acetyl group from the donor acetyl CoA onto a choline molecule yielding acetylcholine, a functional molecule for neural signaling. Another common example is the class of histone acetyltransferases (HATs), which transfer an acetyl group from acetyl CoA onto histones, which are a class of proteins that help facilitate DNA chromatin formation and regulation of gene expression. Four histones (H2A, H2B, H3 and H4) are combined to form a histone core. Each of these four histone proteins can be acetylated. Acetylation of histones occurs on particular lysines of the protein. This reaction is portrayed in FIG. 1. The acetyl group of acetyl-CoA is transferred onto the amino group of lysine. Histone deacetylases (HDACs) remove acetyl groups from histones and back onto acetyl CoA. HAT and HDAC enzymatic activity results in particular acetylation patterns, conferring gene expression regulation. Generally, histone cores with high acetylation yields higher levels of gene expression and vice versa. Histone acetyltransferase 1 (HAT1) is an enzyme that acetylates the histone H4, and particularly on lysines 5 and 12. However, the role of di-acetylation of H4 remains enigmatic. SUMMARY OF THE INVENTION Many embodiments are directed to methods of assaying for acetyltransferase modulators and treatments of neoplasms based upon discovery of modulators of acetyltransferase activity. In many of these embodiments, an assay is performed with putative modulators to determine their ability to modulate acetyltransferase activity. Various embodiments are also directed towards utilizing histone acetyltransferase 1 (HAT1) antagonists for cancer treatment. In an embodiment to identify modulators of acetyltransferase activity, a substrate is provided. The substrate has available functional groups for acetylation. An acetyltransferase, a donor molecule having an acetyl-like group with an alkyne handle, and a modulator is added to the substrate such that the acetyltransferase is capable of transferring the acetyl-like group with the alkyne handle of the donor molecule onto the available functional groups of the substrate. The modulator is a compound being assessed for its ability to modulate the transfer of the acetyl-like group onto the available functional groups of the substrate. Biotin-azide is added to the substrate such that the azide group is capable of conjugating to the acetyl-like groups with alkyne handle that have been transferred onto the substrate such that the biotin from the biotin-azide is extended from the acetyl-like groups. A reporter molecule conjugated with streptavidin is added to the substrate such that the reporter molecule binds to the biotin that is extended from the acetyl-like groups. The reporter molecule enables a measurement of the transfer of the acetyl-like group with the alkyne handle of the donor molecule onto the available functional groups of the substrate. In another embodiment, the acetyltransferase is a histone acetyltransferase, a choline acetyltransferase, a chloramphenicol acetyltransferase, a serotonin N-acetyltransferase, or a N-terminal acetyltransferase. In yet another embodiment, the acetyltransfera