Crystal structure of the large ribosomal subunit from s. aureus

Patent metadata

Field	Value
Publication number	US-2018009853-A1
Application number	US-201615547499-A
Country	US
Kind code	A1
Filing date	Jan 26, 2016
Priority date	Jan 29, 2015
Publication date	Jan 11, 2018
Grant date	—

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.

First claim

1 . A composition-of-matter comprising a crystallized large ribosomal subunit of a pathogenic bacterium, wherein the pathogenic bacterium is:
a pathogenic Gram positive bacterium; and/or a pathogenic bacterium exhibiting a degree of 23S rRNA sequence identity of at least 80% compared to rRNA of Staphylococcus aureus ; and/or a pathogenic bacterium exhibiting a degree of 23S rRNA sequence identity of less than 99.9% compared to rRNA of Escherichia coli , and the crystallized 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.

2 - 4 . (canceled)

5 . The composition of claim 1 , wherein said Gram positive pathogenic bacterium is a Staphylococcus aureus that is capable of developing a resistance to an antibacterial agent.

6 . The composition of claim 5 , 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).

7 . The composition of claim 5 , characterized by the atomic coordinates deposited at the Protein Data Bank under accession number PDB ID: 4WCE.

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

9 . The composition of claim 8 , wherein said ligand is selected from the group consisting of linezolid, BC-3205, telithromycin, lefamulin and lincomycin.

10 . The composition of claim 9 , wherein said ligand is linezolid and the composition characterized by the atomic coordinates deposited at the Protein Data Bank under accession number PDB ID: 4WFA.

11 . (canceled)

12 . The composition of claim 9 , wherein said ligand is BC-3205 and the composition characterized by the atomic coordinates deposited at the Protein Data Bank under accession number PDB ID: 4WFB.

13 . (canceled)

14 . The composition of claim 9 , wherein said ligand is telithromycin and lincomycin and the composition characterized by the atomic coordinates deposited at the Protein Data Bank under accession number PDB ID: 4WF9.

15 . (canceled)

16 . The composition of claim 9 , wherein said ligand is lefamulin and the composition characterized by the atomic coordinates deposited at the Protein Data Bank under accession number PDB ID: 5HL7.

17 . (canceled)

18 . The composition of claim 9 , wherein said ligand is lincomycin and the composition characterized by the atomic coordinates deposited at the Protein Data Bank under accession number PDB ID: 5HKV.

19 - 22 . (canceled)

23 . A method for designing a putative 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 the composition-of-matter of claim 1 ; (b) calculating a molecular surface of the binding site; and (c) computationally constructing a chemically feasible ligand having a molecular surface that match the molecular surface of the binding site.

24 . The method of claim 23 , further comprising, prior to step (b):
determining the binding site in said large ribosomal subunit using said positioning data, wherein said atomic coordinates define at least a portion of a ligand bound to said large ribosomal subunit the binding site being in association with said ligand.

25 - 30 . (canceled)

31 . The method of claim 23 , wherein the active site is selected from the group consisting of an 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.

32 - 34 . (canceled)

35 . A ligand having an affinity to a molecular surface of at least a portion of a binding site of a large ribosomal subunit of a pathogenic bacterium designed by the method of claim 23 .

36 . The ligand of claim 35 , being a protein synthesis inhibitor that comprises:
a first binding moiety having a molecular surface that mimics or duplicates a molecular surface of a first pre-existing ligand that binds to a first binding site in a large ribosomal subunit; and at least one second binding moiety having a molecular surface that mimics or duplicates a surface of a second pre-existing ligand that binds with a second binding site in said ribosomal subunit, wherein: said first pre-existing ligand is different than said second pre-existing ligand; said first binding site is different than said second binding site; said first binding moiety is attached to said second binding moiety via a linking moiety so as to permit both the first moiety and the second moiety to bind simultaneously each with its respective binding site thereby disrupting protein synthesis in a ribosomal subunit.

37 - 39 . (canceled)

40 . The ligand of claim 36 , wherein each of said first pre-existing ligand and said second pre-existing ligand is selected from the group consisting of linezolid, BC-3205, telithromycin, lefamulin and lincomycin.

41 . A ligand comprising:
a first binding moiety having a molecular surface that mimics or duplicates a molecular surface of a first pre-existing ligand that binds to a first binding site in a large ribosomal subunit; and a second binding moiety having a molecular surface that mimics or duplicates a surface of a second pre-existing ligand that binds with a second binding site in said ribosomal subunit, wherein: said first pre-existing ligand is not said second pre-existing ligand; said first binding site is not said second binding site; said first binding moiety is attached to said second binding moiety via a linking moiety so as to permit both the first moiety and the second moiety to bind simultaneously each with its respective binding site thereby disrupting protein synthesis in a ribosomal subunit, wherein a positioning data of said first binding moiety relative to a positioning data of said second binding moiety, is determined according to atomic coordinates indicative of a positioning data of said first binding moiety and said second binding moiety obtained from an electron density map having a resolution of at least 4 Å calculated from X-rays diffraction data obtained using the composition-of-matter of claim 1 .

42 - 44 . (canceled)

45 . The ligand of claim 41 , wherein each of said first pre-existing ligand and said second pre-existing ligand is selected from the group consisting of linezolid, BC-3205, telithromycin, lefamulin and lincomycin.

46 - 50 . (canceled)

Assignees

• Yeda Res & Dev

Inventors

• Yonath Ada
• Eyal Zohar
• Matzov Donna
• Rozenberg Haim
• Zimmerman Ella
• Bashan Anat

Classifications

• G16C20/60

  In silico combinatorial chemistry · CPC title

• G16B30/00

  ICT specially adapted for sequence analysis involving nucleotides or amino acids · CPC title

• G16B35/00

  ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides · CPC title

• C07K2299/00

  Coordinates from 3D structures of peptides, e.g. proteins or enzymes · CPC title

• C07D211/54

  Sulfur atoms · CPC title