CRYSTAL STRUCTURE OF THE LARGE RIBOSOMAL SUBUNIT FROM S. AUREUS

• US 20200131232A1
• Filed: 12/24/2019
• Published: 04/30/2020
• Est. Priority Date: 01/29/2015
• Status: Active Grant

• Current Assignee: Yeda Research and Development Co. Ltd. (Weizmann Institute of Science)
• Original Assignee: Yeda Research and Development Co. Ltd. (Weizmann Institute of Science)
• Inventors: YONATH, Ada, EYAL, Zohar, MATZOV, Donna, ROZENBERG, Haim, ZIMMERMAN, Ella, BASHAN, Anat
• Granted Patent: US 10,723,770 B2
• Time in Patent Office: Days
• CPC Class Codes: C07D 211/54 Sulfur atoms
• CPC Class Codes: C07D 263/20 attached in position 2
• CPC Class Codes: C07H 17/08 Hetero rings containing eight or more ring members, e.g. erythromycins
• CPC Class Codes: C07K 14/31 from Staphylococcus (G)
• CPC Class Codes: C07K 2299/00 Coordinates from 3D structures of peptides, e.g. proteins or enzymes
• CPC Class Codes: G16B 15/00 ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
• CPC Class Codes: G16B 15/20 Protein or domain folding
• CPC Class Codes: G16B 15/30 Drug targeting using structural data; Docking or binding prediction
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search
• CPC Class Codes: G16B 35/00 ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides
• CPC Class Codes: G16C 20/50 Molecular design, e.g. of drugs
• CPC Class Codes: G16C 20/60 In silico combinatorial chemistry
• CPC Class Codes: G16C 20/62 Design of libraries

Abstract

A composition-of-matter comprising a crystallized form of a large ribosomal (50S) subunit of a pathogenic bacterium, and the atomic coordinates of the three-dimensional structure thereof are provided herein, as well as methods for crystallizing the same, and using the atomic coordinates of the same to design de novo ligands with high specificity thereto.

Claims

1. A method producing a ligand having an affinity to a binding site of a large ribosomal subunit of a pathogenic bacterium, the method comprising:

(a) obtaining positioning data indicative of atomic coordinates of at least one binding site determined from an electron density map having a resolution of at least 4 Å

calculated from X-rays diffraction data obtained using at least one of a composition-of-matter that comprises a crystallized Staphylococcus aureus 50S large ribosomal subunit, wherein the crystallized 50S large ribosomal subunit effectively diffracts X-rays for calculating an electron density map and determination of atomic coordinates to a resolution of at least 4 Å and

forms in a hexagonal space group with unit cell dimensions of a=279.6±

10 Å

, b=279.6±

10 Å

, c=872.7±

10 Å

, α

=90, β

=90, γ

=120;

(b) calculating a molecular surface of the binding site;

(c) computationally constructing a model of a chemically feasible ligand having a molecular surface that match the molecular surface of the binding site; and

synthesizing the ligand based on said model.

2. The method of claim 1, wherein said Staphylococcus aureus is capable of developing a resistance to an antibacterial agent.

3. The method of claim 2, wherein said Staphylococcus aureus is selected from the group consisting of a methicillin-resistant Staphylococcus aureus (MRSA), an oxacillin-resistant Staphylococcus aureus (ORSA), a vancomycin-resistant Staphylococcus aureus (VRSA) and a vancomycin intermediate Staphylococcus aureus (VISA).

4. The method of claim 2, characterized by the atomic coordinates deposited at the Protein Data Bank under accession number PDB ID:

4WCE.

5. The method of claim 1, wherein a ligand is bound to said large ribosomal subunit to form a crystallized complex of the subunit and said ligand.

6. The method of claim 5, wherein said ligand is selected from the group consisting of linezolid, BC-3205, telithromycin, lefamulin and lincomycin.

7. The method of claim 6, wherein said ligand is linezolid and the composition is characterized by the atomic coordinates deposited at the Protein Data Bank under accession number PDB ID:

4WFA.

8. The method of claim 6, wherein said ligand is BC-3205 and the composition is characterized by the atomic coordinates deposited at the Protein Data Bank under accession number PDB ID:

4WFB.

9. The method of claim 6, wherein said ligand is telithromycin and lincomycin and the composition is characterized by the atomic coordinates deposited at the Protein Data Bank under accession number PDB ID:

4WF9.

10. The method of claim 6, wherein said ligand is lefamulin and the composition is characterized by the atomic coordinates deposited at the Protein Data Bank under accession number PDB ID:

5HL7.

11. The method of claim 6, wherein said ligand is lincomycin and the composition is characterized by the atomic coordinates deposited at the Protein Data Bank under accession number PDB ID:

5HKV.

12. The method of claim 1, further comprising, prior to step (c):

computationally constructing a library of structures of chemically feasible ligands having a molecular surface that matches the molecular surface of the binding site.

13. The method of claim 12, further comprising:

(d) computationally determining a matching score for each of said ligands; and

(e) based on said matching score selecting at least one putative ligand having the desired affinity to the binding site of the large ribosomal subunit of a pathogenic bacterium.

14. The method of claim 13, further comprising, prior to step (d), adding to said library a plurality of structures of chemically feasible variants of pre-existing ligands.

15. The method of claim 1, further comprising, prior to step (c), calculating a molecular surface of at least a portion of the binding site of a large ribosomal subunit of a different organism.

16. The method of claim 15, wherein said different organism is selected from the group consisting of a host of the pathogenic bacterium and a benign microorganism.

17. The method of claim 16, wherein step (c) comprises computationally constructing a chemically feasible ligand having a molecular surface that matches the molecular surface of the binding site of the large ribosomal subunit of a pathogenic bacterium, and mismatches at least one feature in said molecular surface of the binding site in said of a large ribosomal subunit of said different organism.

18. The method of claim 1, wherein the active site is selected from the group consisting of a inter-subunit interface, a peptidyl transferase site, a GTPase center, an mRNA binding site, an A-site, a P-site, an E-site, a polypeptide exit tunnel, a translation initiation factor (IF1) binding site, a translation initiation factor (IF2) binding site, a translation initiation factor (IF3) binding site, an elongation factor G (EF-G) binding site, elongation factor Tu (EF-Tu) binding site, hibernation factor HPF binding site, hibernation factor RMF binding site, hibernation factor YfiA binding site, a GTP binding site and a ricin binding site.

19. The method of claim 1, wherein the ligand has a molecular weight of less than 1,500 g/mol.