Pharmacogenic therapies targeting the metal-ion transcriptional regulation machinery in bacteria
View Patent ↗The subject invention pertains to methods of identifying compounds that bind to a binding pocket on metal ion binding proteins of the ArsR/SmtB family and modulate their regulatory activity by increasing the propensity of the proteins to remain in a conformationally trapped form such as the metal ion bound conformation or disulfide-linked conformation, the DNA bound conformation, or the apo-conformation. The invention provides methods of identifying such compounds using computer software programs. The invention also provides bioassays for identifying such compounds. The invention also provides pharmaceutical compositions containing compounds that modulate metalloregulatory activities of the metal ion binding proteins of the ArsR/SmtB family.
1. A method of identifying a candidate compound that binds to a binding pocket of a protein of the ArsR/SmtB family of transcriptional factors, wherein, the regulatory protein exists in a first allosteric form, a second allosteric form, and an apo-form, wherein the first allosteric form is a conformationally restricted form (metal ion bound form or di-sulfide bound form) and the second allosteric form is a DNA bound form, and the apo-form is devoid of either the metal or the DNA,
the method comprising:
providing a computer program with information comprising:
a) structural information about a binding pocket of a protein of ArsR/SmtB family or metal ion transcriptional factors having similar allosteric mechanisms,
b) structural information of the compound, and
c) a set of rules and instructions about binding interactions between the compound and the binding pocket of the a protein belonging to the ArsR/SmtB family or metal ion transcriptional factors having similar allosteric mechanisms,
wherein, based on the information and the instructions provided to the computer program, the computer program predicts whether the binding of the compound to the metal ion binding protein of ArsR/SmtB family or the metal ion transcriptional factor having similar allosteric mechanisms increases the propensity of the protein to remain in the metal ion bound form, the DNA bound form, or the apo-form, and
selecting the compound if the propensity of the regulatory protein or the metal ion transcriptional factor to remain in the metal ion bound conformation, the DNA bound conformation, or the apo-conformation is higher when bound to the compound than the propensity of the protein or the metal ion transcriptional factor to remain in the metal ion bound conformation (or similar conformationally locked state), the DNA bound conformation, or the apo-conformation when not bound to the compound.
2. The method of claim 1 , wherein the computer program predicts whether the binding of the compound to the metal ion binding protein of ArsR/SmtB family or the metal ion transcriptional factor having similar allosteric mechanisms increases the propensity of the protein to remain in the metal ion bound form.
3. The method of claim 1 , wherein the computer program predicts whether the binding of the compound to the metal ion binding protein of ArsR/SmtB family or the metal ion transcriptional factor having similar allosteric mechanisms increases the propensity of the protein to remain in the DNA bound form.
4. The method of claim 1 , wherein the computer program predicts whether the binding of the compound to the regulatory protein of ArsR/SmtB family or the metal ion transcriptional factor having similar allosteric mechanisms increases the propensity of the protein to remain in the apo-form.
5. The method of claim 1 , wherein, the metal ion is a heavy metal ion.
6. The method of claim 1 , wherein in the metal ion binding protein of ArsR/SmtB family is selected from the group consisting of Staphylococcus aureus CzrA, Staphylococcus aureus p1258 CadC, Mycobacterium tuberculosis CsoR, Mycobacterium tuberculosis NmtR, Synechococcus spp. SmtB or a metal ion transcriptional factor having similar allosteric mechanisms selected from Steptococcus pneumonia AdcR of the MarR family of proteins, Escherichia coli CsoR and Helicobacter pylori NikR.
7. The method of claim 1 , wherein the computer programs are Glide and AMBER.
8. The method of claim 1 , the method further comprising modifying the selected compound wherein the propensity of the protein to remain in the metal ion bound conformation, the DNA bound conformation, or the apo-conformation is higher when bound to the modified compound than the propensity of the protein to remain in the metal ion bound conformation, the DNA bound conformation, or the apo-conformation when bound to the compound.
9. The method of claim 8 wherein the modifications comprise one or more of the following: changing a side chain, a ring structure, a functional group, the stereoisomeric conformation, and/or the ionic charge of a compound.
10. The method of claim 1 , wherein said method further comprises contacting a bacterial cell with said selected compound and:
a) growing a bacterial culture in the presence or absence of said selected compound, comparing the growth rates of the bacterial culture in the presence or absence of said selected compound, and determining if the growth rate of the bacterial culture is substantially reduced in the presence of the compound; and/or
b) growing a bacterial culture in the presence or absence of said selected compound, comparing the rate of influx or efflux of isotopic heavy metal ions between bacterial cells treated with a candidate compound and untreated microorganisms.
11. A compound identified to bind to the binding pocket of a protein of ArsR/SmtB family by the method of claim 1 .
12. A composition comprising the compound of claim 11 and pharmaceutically acceptable excipients.
13. A method of inhibiting bacterial growth by the contacting the bacteria with the compound of claim 11 .
14. A method of treating a bacterial infection by administering a pharmaceutically effective amount of the compound of claim 11 to a subject suffering from the bacterial infection.