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US 2003/0119162 A1Published application
Structural basis of quorum sensing signal generation and methods and therapeutic agents derived therefrom
OwnerMair E.A. Churchill, Susanne B. von Bodman +5inventors
InventorsMair E.A. Churchill, Susanne B. von Bodman, Herbert P. Schweizer, Ty A. Gould, Tung Thanh Hoang, Frank V. Murphy IV, William T. Watson
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Abstract
The three dimensional structure of acyl-homoserine lactone synthases, and particularly EsaI and LasI, and uses thereof. Novel acyl-homoserine lactone synthases from mycobacteria, nucleic acid molecules encoding such synthases, recombinant molecules and host cells, and uses thereof.
Claims82
A method of structure-based identification of compounds which potentially bind to an AHL synthase, comprising:
- a. obtaining atomic coordinates that define the three dimensional structure of an AHL synthase, said atomic coordinates being selected from the group consisting of:
- i. atomic coordinates determined by X-ray diffraction of a crystalline EsaI or a crystalline LasI;
- ii. atomic coordinates selected from the group consisting of:
- (1) atomic coordinates represented in any one of Tables 2-5;
- (2) atomic coordinates that define a three dimensional structure having an average root-mean-square deviation (RMSD) of equal to or less than about 1.7 Å over the backbone atoms in secondary structure elements of at least 50% of the residues in a three dimensional structure represented by said atomic coordinates of (1);
- wherein said structure has an amino acid sequence comprising at least three of eight conserved amino acid residues corresponding to the following residues in SEQ ID NO:
- 1:
- Arg24, Phe28, Trp34, Asp45, ASp48, Arg68, Glu97, or Arg100 or to the following residues in SEQ ID NO:2:
- Arg23, Phe27, Trp33, Asp44, ASP47, Arg70, Glu101 or Arg104;
- and
- wherein said structure has an amino acid sequence comprising at least three regions having detectable sequence homology with the following three regions in SEQ ID NO:
- 1:
- amino acid residues 19 through 56, amino acid residues 63-83, and amino acid residues 90-101;
- or with the following three regions in SEQ ID NO:2:
- amino acid residues 18-55, 65-85 and 95-105;
- and
- (3) atomic coordinates in any one of Tables 2-5 defining a portion of said AHL synthase, wherein the portion of said AHL synthase comprises sufficient structural information to perform step (b);
- and
- iii. atomic coordinates defining the three dimensional structure of EsaI molecules arranged in a crystalline manner in a space group p43 so as to form a unit cell having approximate dimensions of a=b=66.40, c=47.33;
- iv. atomic coordinates defining the three dimensional structure of EsaI molecules arranged in a crystalline manner in a space group p43 so as to form a unit cell having approximate dimensions of a=b=66.99, c=47.01;
- v. atomic coordinates defining the three dimensional structure of LasI molecules arranged in a crystalline manner in a space group F23, so as to form a unit cell having approximate dimensions of a—b=c=154.90 Å;
- and
- b. selecting candidate compounds for binding to said AHL synthase by performing structure based drug design with said structure of (a), wherein said step of selecting is performed in conjunction with computer modeling.
20 more elements
A method to produce an AHL synthase homologue that catalyzes the synthesis of AHL compounds having antibacterial biological activity, comprising:
- a. obtaining atomic coordinates that define the three dimensional structure of an AHL synthase, said atomic coordinates being selected from the group consisting of:
- i. atomic coordinates determined by X-ray diffraction of a crystalline EsaI or a crystalline LasI;
- ii. atomic coordinates selected from the group consisting of:
- (1) atomic coordinates represented in any one of Tables 2-5;
- (2) atomic coordinates that define a three dimensional structure having an average root-mean-square deviation (RMSD) of equal to or less than about 1.7 Å over the backbone atoms in secondary structure elements of at least 50% of the residues in a three dimensional structure represented by said atomic coordinates of (1);
- wherein said structure has an amino acid sequence comprising at least three of eight conserved amino acid residues corresponding to the following residues in SEQ ID NO:
- 1:
- Arg24, Phe28, Trp34, Asp45, Asp48, Arg68, Glu97, or Arg100 or to the following residues in SEQ ID NO:2:
- Arg23, Phe27 Trp33, Asp44 Asp47, Ar70, Glu101 or Arg104;
- and
- wherein said structure has an amino acid sequence comprising at least three regions having detectable sequence homology with the following three regions in SEQ ID NO:
- 1:
- amino acid residues 19 through 56, amino acid residues 63-83, and amino acid residues 90-101;
- or with the following three regions in SEQ ID NO:2:
- amino acid residues 18-55, 65-85 and 95-105;
- and
- (3) atomic coordinates in any one of Tables 2-5 defining a portion of said AHL synthase, wherein the portion of said AHL synthase comprises sufficient structural information to perform step (b);
- and
- iii. atomic coordinates defining the three dimensional structure of EsaI molecules arranged in a crystalline manner in a space group p43 so as to form a unit cell having approximate dimensions of a=b=66.40, c=47.33;
- iv. atomic coordinates defining the three dimensional structure of EsaI molecules arranged in a crystalline manner in a space group p43 so as to form a unit cell having approximate dimensions of a=b=66.99, c=47.01;
- v. atomic coordinates defining the three dimensional structure of LasI molecules arranged in a crystalline manner in a space group F23, so as to form a unit cell having approximate dimensions of a—b=c=154.90 Å;
- a. performing computer modeling with said atomic coordinates of (a) to identify at least one site in said AHL synthase structure that is predicted to modify the biological activity of said AHL synthase;
- b. producing a candidate AHL synthase homologue that is modified in said at least one site identified in (b);
- and
- c. determining whether said candidate AHL synthase homologue of (c) catalyzes the synthesis of AHL compounds having antibacterial biological activity.
22 more elements
A method to produce an AHL synthase homologue with modified biological activity as compared to a natural AHL synthase, comprising:
- a. obtaining atomic coordinates that define the three dimensional structure of an AHL synthase, said atomic coordinates being selected from the group consisting of:
- i. atomic coordinates determined by X-ray diffraction of a crystalline EsaI or a crystalline LasI;
- ii. atomic coordinates selected from the group consisting of:
- (1) atomic coordinates represented in any one of Tables 2-5;
- (2) atomic coordinates that define a three dimensional structure having an average root-mean-square deviation (RMSD) of equal to or less than about 1.7 Å over the backbone atoms in secondary structure elements of at least 50% of the residues in a three dimensional structure represented by said atomic coordinates of (1);
- wherein said structure has an amino acid sequence comprising at least three of eight conserved amino acid residues corresponding to the following residues in SEQ ID NO:
- 1:
- Arg24, Phe28, Trp34, Asp45, Asp48, Arg68, Glu97, or Arg100 or to the following residues in SEQ ID NO:2:
- Arg23, Phe27, Trp33, Asp44, Asp47, Arg70, Glu101 or Arg104;
- and
- wherein said structure has an amino acid sequence comprising at least three regions having detectable sequence homology with the following three regions in SEQ ID NO:
- 1:
- amino acid residues 19 through 56, amino acid residues 63-83, and amino acid residues 90-101;
- or with the following three regions in SEQ ID NO:2:
- amino acid residues 18-55, 65-85 and 95-105;
- and
- (3) atomic coordinates in any one of Tables 2-5 defining a portion of said AHL synthase, wherein the portion of said AHL synthase comprises sufficient structural information to perform step (b);
- and
- iii. atomic coordinates defining the three dimensional structure of EsaI molecules arranged in a crystalline manner in a space group p43 so as to form a unit cell having approximate dimensions of a=b=66.40, c=47.33;
- iv. atomic coordinates defining the three dimensional structure of EsaI molecules arranged in a crystalline manner in a space group p43 so as to form a unit cell having approximate dimensions of a=b=66.99, c=47.01;
- V. atomic coordinates defining the three dimensional structure of LasI molecules arranged in a crystalline manner in a space group F23, so as to form a unit cell having approximate dimensions of a=b=c=154.90 Å;
- a. using computer modeling of said atomic coordinates in (a) to identify at least one site in said AHL synthase structure that is predicted to contribute to the biological activity of said AHL synthase;
- and
- b. modifying said at least one site in an AHL synthase protein to produce an AHL synthase homologue which is predicted to have modified biological activity as compared to a natural AHL synthase.
21 more elements
A method to construct a three dimensional model of an AHL synthase, comprising:
- a. obtaining atomic coordinates that define the three dimensional structure of a first AHL synthase, said atomic coordinates being selected from the group consisting of:
- i. atomic coordinates determined by X-ray diffraction of a crystalline EsaI or a crystalline LasI;
- ii. atomic coordinates selected from the group consisting of:
- (1) atomic coordinates represented in any one of Tables 2-5;
- (2) atomic coordinates that define a three dimensional structure having an average root-mean-square deviation (RMSD) of equal to or less than about 1.7 Å over the backbone atoms in secondary structure elements of at least 50% of the residues in a three dimensional structure represented by said atomic coordinates of (1);
- wherein said structure has an amino acid sequence comprising at least three of eight conserved amino acid residues corresponding to the following residues in SEQ ID NO:
- 1:
- Arg24, Phe28, Trp34, Asp45, Asp48, Arg68, Glu97, or Arg100 or to the following residues in SEQ ID NO:2:
- Arg23, Phe27, Trp33, Asp44, ASP47, Arg70, Glu101 or Arg104;
- and
- wherein said structure has an amino acid sequence comprising at least three regions having detectable sequence homology with the following three regions in SEQ ID NO:
- 1:
- amino acid residues 19 through 56, amino acid residues 63-83, and amino acid residues 90-101;
- or with the following three regions in SEQ ID NO:2:
- amino acid residues 18-55, 65-85 and 95-105;
- and
- (3) atomic coordinates in any one of Tables 2-5 defining a portion of said AHL synthase, wherein the portion of said AHL synthase comprises sufficient structural information to perform step (b);
- and
- iii. atomic coordinates defining the three dimensional structure of EsaI molecules arranged in a crystalline manner in a space group p43 so as to form a unit cell having approximate dimensions of a=b=66.40, c=47.33;
- iv. atomic coordinates defining the three dimensional structure of EsaI molecules arranged in a crystalline manner in a space group p43 so as to form a unit cell having approximate dimensions of a=b=66.99, c=47.01;
- v. atomic coordinates defining the three dimensional structure of LasI molecules arranged in a crystalline manner in a space group F23, so as to form a unit cell having approximate dimensions of a—b=c=154.90 Å;
- and
- a. performing computer modeling with said atomic coordinates of (a) and an amino acid sequence of a second AHL synthase to construct a model of a three dimensional structure of said second AHL synthase.
20 more elements
A crystal comprising an AHL synthase, wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates of the AHL synthase to a resolution of greater than 3.2 Å, and wherein said crystal has a space group p43 so as to form a unit cell having approximate dimensions of a=b=66.40, c=47.33.
A crystal comprising an AHL synthase, wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates of the AHL synthase to a resolution of greater than 3.2 Å, and wherein said crystal has a space group p43 so as to form a unit cell having approximate dimensions of a=b=66.99, c=47.01.
A crystal comprising an AHL synthase, wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates of the AHL synthase to a resolution of greater than 3.2 Å, and wherein said crystal has a space group F23, so as to form a unit cell having approximate dimensions of a=b=c=154.90 Å.
A therapeutic composition comprising a compound that inhibits the biological activity of an AHL synthase, said compound being identified by the method comprising:
- a. obtaining atomic coordinates that define the three dimensional structure of an AHL synthase, said atomic coordinates being selected from the group consisting of:
- i. atomic coordinates determined by X-ray diffraction of a crystalline EsaI or a crystalline LasI;
- ii. atomic coordinates selected from the group consisting of:
- (1) atomic coordinates represented in any one of Tables 2-5;
- (2) atomic coordinates that define a three dimensional structure having an average root-mean-square deviation (RMSD) of equal to or less than about 1.7 Å over the backbone atoms in secondary structure elements of at least 50% of the residues in a three dimensional structure represented by said atomic coordinates of (1);
- wherein said structure has an amino acid sequence comprising at least three of eight conserved amino acid residues corresponding to the following residues in SEQ ID NO:
- 1:
- Arg24, Phe 28, Trp34, Asp45, Asp48, Arg68, Glu97, or Arg100 or to the following residues in SEQ ID NO:2:
- Arg23, Phe27, Trp33, Asp44, Asp47, Arg70, Glu101 or Arg104;
- and
- wherein said structure has an amino acid sequence comprising at least three regions having detectable sequence homology with the following three regions in SEQ ID NO:1:
- amino acid residues 19 through 56, amino acid residues 63-83, and amino acid residues 90-101;
- or with the following three regions in SEQ ID NO:2:
- amino acid residues 18-55, 65-85 and 95-105;
- and
- (3) atomic coordinates in any one of Tables 2-5 defining a portion of said AHL synthase, wherein the portion of said AHL synthase comprises sufficient structural information to perform step (b);
- and
- iii. atomic coordinates defining the three dimensional structure of EsaI molecules arranged in a crystalline manner in a space group p43 so as to form a unit cell having approximate dimensions of a=b=66.40, c=47.33;
- iv. atomic coordinates defining the three dimensional structure of EsaI molecules arranged in a crystalline manner in a space group p43 so as to form a unit cell having approximate dimensions of a=b=66.99, c=47.01;
- v. atomic coordinates defining the three dimensional structure of LasI molecules arranged in a crystalline manner in a space group F23, so as to form a unit cell having approximate dimensions of a=b=c=154.90 Å;
- and
- b. selecting candidate compounds for binding to said AHL synthase by performing structure based drug design with said structure of (a), wherein said step of selecting is performed in conjunction with computer modeling;
- c. synthesizing said candidate compound selected in (b);
- and
- d. further selecting candidate compounds that inhibit the biological activity of said AHL synthase.
22 more elements
An isolated protein comprising a mutant AHL synthase, wherein said protein comprises an amino acid sequence that differs from the amino acid sequence of a naturally occurring AHL synthase by at least one amino acid modification that results in a mutant AHL synthase that catalyzes the production of a different AHL product as compared to the naturally occurring AHL synthase.
An isolated protein comprising a mutant EsaI protein, wherein said protein comprises an amino acid sequence that differs from SEQ ID NO:1 by at least one modification including at least one amino acid substitution selected from the group consisting of:
- a non-arginine amino acid residue at position 24, a non-phenyalanine amino acid residue at position 28, a non-tryptophan amino acid residue at position 34, a non-aspartate amino acid residue at position 45, a non-aspartate amino acid residue at position 48, a non-arginine amino acid residue at position 68, a non-glutamate amino acid residue at position 97, a non-serine amino acid residue at position 99, a non-arginine amino acid residue at position 100;
- and a non-threonine amino acid residue at position 140;
- wherein said mutant EsaI protein has modified biological activity as compared to a wild-type EsaI protein.
An isolated AHL synthase comprising an amino acid sequence selected from the group consisting of:
- a. an amino acid sequence that is at least about 70% identical to an amino acid sequence selected from any of SEQ ID NOs:67 or SEQ ID NOs:83-100, wherein said amino acid sequence has AHL synthase activity;
- and
- b. a fragment of an amino acid sequence of (a), wherein said fragment has AHL synthase activity.
An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of:
- a. a nucleic acid sequence that encodes an amino acid sequence that is at least about 70% identical and less than 100% identical to an amino acid sequence selected from any of SEQ ID NOs:67 or SEQ ID NOs:83-100, wherein said amino acid sequence has AHL synthase activity;
- b. a nucleic acid sequence encoding a fragment of said amino acid sequence of (a), wherein said fragment has AHL synthase activity;
- c. a nucleic acid sequence that is a probe or primer that hybridizes under high stringency conditions to a nucleic acid sequence of (a) or (b);
- and
- d. a nucleic acid sequence that is a complement of any of the nucleic acid sequences of (a)-(c).
2 more elements
An isolated AHL synthase comprising an amino acid sequence selected from the group consisting of:
- a. an amino acid sequence that is at least about 30% identical to SEQ ID NO:67, wherein said amino acid sequence comprises at least three amino acid residues corresponding to amino acid residues of SEQ ID NO:67 selected from:
- Arg9, Phe13, Phe19, Asp32, Asp35, Arg56, Glu89 and Arg92, and wherein said amino acid sequence has AHL synthase activity;
- and
- b. a fragment of an amino acid sequence of (a), wherein said fragment has AHL synthase activity.
1 more element
A method of identifying a compound that regulates quorum sensing signal generation, comprising:
- a. contacting an AHL synthase or biologically active fragment thereof with a putative regulatory compound, wherein said AHL synthase comprises an amino acid sequence that is at least about 70% identical to an amino acid sequence selected from any of SEQ ID NOs:67 or SEQ ID NOs:83-100, or a biologically active fragment thereof, wherein said amino acid sequence has AHL synthase activity;
- b. detecting whether said putative regulatory compound increases or decreases a biological activity of said AHL synthase as compared to in the absence of contact with said compound;
- wherein compounds that increases or decreases activity of the AHL synthase, as compared to in the absence of said compound, indicates that said putative regulatory compound is a regulator of said AHL synthase.
A method to inhibit quorum sensing signal generation in a population of microbial cells, comprising contacting a population of microbial cells that express an AHL synthase with an antagonist of said AHL synthase, wherein said antagonist decreases the biological activity of said AHL synthase, and wherein said AHL synthase comprises an amino acid sequence that is at least about 70% identical to an amino acid sequence selected from any of SEQ ID NOs:67 or SEQ ID NOs:83-100.
Dependencies are inferred from the claim text.