# US 2025/0154579 A1 · System and method for high throughput screening of small molecule-protein interactions

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- Number: US 2025/0154579 A1
- Document ID: US20250154579A1
- Status: Published application
- Kind: A1
- Application number: 18/388,850
- Filed: 2023-11-12
- Published: 2025-05-15
- Earliest claimed: 2023-11-12
- Assignees: Interplay Bio
- Applicants: Interplay Bio
- Inventors: Pavan Kommareddi
- Web page: https://patentgrep.com/patent/US20250154579A1

## CPC

- C12Q 1/6869: Methods for sequencing
- C12N 15/1037: {Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display}
- C12N 15/1096: {cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR}
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A system and method for the high throughput screening of small molecule-protein interactions includes first immobilizing selected small molecules on a solid matrix. Immobilization is preferably made by photo affinity labelling in which a linker used to bind the small molecule to the matrix. A cDNA library is established from a broad array of human tissues. The tissue mRNA is converted to cDNA and transferred to a phage genome to make a phage display library which is mixed with small molecules coupled to the beads and incubated. The incubation mixture is washed to remove unbound phage including non-specific and weak binders. The bound phage are amplified and are used in the next round of biopanning. The biopanning cycles are repeated followed by PCR of individual plaques selected from the final biopanning round. The PCR products are barcoded from each small molecule experiment and are pooled followed by Nextgen sequencing.

## Claims

### Claim 1

A method for high throughput screening of small molecule-protein interactions comprising:
selecting a tissue;
selecting small molecules for screening;
forming a matrix;
immobilizing the selected molecules on a matrix;
converting the tissue mRNA to cDNA;
establishing a cDNA library;
transferring the cDNA to a phage genome to make a phage display library;
mixing the phage library with the selected small molecules coupled to the matrix;
incubating the mixture for different time points based on the particular experiment;
washing the incubated mixture to remove unbound phage as non-specific and weak binders;
amplifying the bound phage;
biopanning using the amplified bound phage;
repeating biopanning;
undertaking PCR of the individual plaques selected from the final biopanning round;
barcoding the PCR products from each small molecule experiment;
pooling the barcoded PCR products;
subjecting the pooled PCR products to sequencing;
correlating the resulting protein-small molecule interactions identified following screening with literature to identify disease pathways involved with the protein; and
validating the protein-small molecule interactions.

### Claim 2 (depends on 1)

The method of claim 1, wherein the matrix is formed from styrene, silica beads, glass, and magnetic beads.

### Claim 3 (depends on 1)

The method of claim 1, wherein the magnetic beads have aminated surfaces.

### Claim 4 (depends on 2)

The method of claim 2, wherein the magnetic beads are oligo d(T25) magnetic beads.

### Claim 5 (depends on 1)

The method of claim 1, wherein immobilization is made by photo affinity labeling.

### Claim 6 (depends on 1)

The method of claim 1, wherein a linker is used to bind the small molecule to the matrix.

### Claim 7 (depends on 6)

The method of claim 6, wherein the linker is a photocrosslinker.

### Claim 8 (depends on 7)

The method of claim 7, wherein the photocrosslinker is a diazirine.

### Claim 9 (depends on 1)

The method of claim 1, wherein amplification of the bound phage is achieved by infecting them with E. coli.

### Claim 10 (depends on 1)

The method of claim 1, wherein sequencing is Nextgen sequencing.

### Claim 11 (depends on 1)

The method of claim 1, wherein conversion of the mRNA to cDNA is through the use of directional cloning random primer and oligo dT primer.

### Claim 12 (depends on 1)

The method of claim 1, wherein the tissue is selected from the group consisting of animal and human tissue.

### Claim 13 (depends on 1)

The method of claim 1, including subjecting the cDNA ends to flushing and phosphorylating.

### Claim 14 (depends on 1)

The method of claim 1, wherein the linkers are directional EcoRI/HindIII linkers.

### Claim 15 (depends on 14)

The method of high throughput screening according to claim 14, wherein the EcoRI and HindIII sites on the ligated linkers are enzymatically digested.

### Claim 16

A method for high throughput screening of small molecule-protein interactions comprising:
selecting a tissue;
selecting small molecules for screening;
forming a matrix;
binding the small molecule to a matrix using a linker resulting in immobilization of the small molecule;
converting the tissue mRNA to cDNA;
establishing a cDNA library;
making a phage display library;
mixing the phage library with the selected small molecules coupled to the matrix;
incubating the mixture;
washing the incubated mixture to remove unbound phage;
amplifying the bound phage;
biopanning using the bound phage;
repeating biopanning;
undertaking PCR of the individual plaques selected from the final biopanning round;
barcoding the PCR products;
pooling the barcoded PCR products;
subjecting the pooled PCR products to Nextgen sequencing;
correlating the resulting protein-small molecule interactions identified following screening with literature to identify disease pathways involved with the protein; and
validating the protein-small molecule interactions.

### Claim 17 (depends on 16)

The method of claim 16, wherein the linker is a photocrosslinker.

### Claim 18 (depends on 17)

The method of claim 17, wherein the photocrosslinker is a diazirine.

### Claim 19 (depends on 16)

The method of claim 16, wherein conversion of the mRNA to cDNA is through the use of directional cloning random primer and oligo dT primer.

### Claim 20

A method for high throughput screening of small molecule-protein interactions comprising:
selecting a tissue;
selecting small molecules for screening;
forming a matrix;
immobilizing the small molecule on the matrix;
converting the tissue mRNA to cDNA;
establishing a cDNA library;
making a phage display library;
mixing the phage library with the immobilized small molecules;
incubating the mixture;
washing the incubated mixture to remove unbound phage;
amplifying the bound phage;
biopanning using the bound phage;
repeating biopanning;
undertaking PCR of the individual plaques selected from the final biopanning round;
barcoding the PCR products;
pooling the barcoded PCR products; and
sequencing the pooled PCR products.