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2 1 V16 4 A2 (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number r » (43) International Publication Date i 1 /i 22 December 2011 (22.12.2011) 2 1 V16 4 A2 (51) International Patent Classification: AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, G01N 33/68 (2006.01) C12Q 1/18 (2006.01) CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, G01N 33/15 (2006.01) DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, (21) International Application Number: KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, PCT/US201 1/040926 ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, (22) International Filing Date: NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, 17 June 201 1 (17.06.201 1) SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (25) Filing Language: English (84) Designated States (unless otherwise indicated, for every (26) Publication Language: English kind of regional protection available): ARIPO (BW, GH, (30) Priority Data: GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, 61/355,786 17 June 2010 (17.06.2010) ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, 61/490,295 26 May 201 1 (26.05.201 1) TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, ΓΓ, LT, LU, (71) Applicant (for all designated States except US): THE LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, JOHNS HOPKINS UNIVERSITY [US/US]; 3400 SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, North Charles Street, Baltimore, Maryland 21218 (US). GW, ML, MR, NE, SN, TD, TG). (72) Inventors; and Declarations under Rule 4.17 : (75) Inventors/ Applicants (for US only): ZHANG, Ying [US/ — as to applicant's entitlement to apply for and be granted US]; MM1, JHSPH, 615 North Wolfe Street, Baltimore, a patent (Rule 4.1 7(H)) Maryland 21205 (US). SHI, Wanliang [CN/US]; MM1, JHSPH, 615 North Wolfe Street, Baltimore, Maryland — as to the applicant's entitlement to claim the priority of 21205 (US). the earlier application (Rule 4.17(Hi)) (74) Agent: ALATHARI, Zayd; VENABLE LLP, P.O. Box — of inventorship (Rule 4.1 7(iv)) 34385, Washington, District of Columbia 20043-9998 Published: (US). — without international search report and to be republished (81) Designated States (unless otherwise indicated, for every upon receipt of that report (Rule 48.2(g)) kind of national protection available): AE, AG, AL, AM, < o o (54) Title: METHODS OF IDENTIFYING THERAPEUTIC AGENTS FOR TREATING PERSISTER AND BACTERIAL IN o FECTION (57) Abstract: The present invention relates to methods, compositions, assays and kits for identifying an antibacterial agent that decreases persister formation or survival, eliminates or reduces bacterial infection or disease and/or increases killing of a bacterial cell. METHODS OF IDENTIFYING THERAPEUTIC AGENTS FOR TREATING PERSISTER AND BACTERIAL INFECTION The research resulting in the invention described herein was supported in part by funding from the National Institutes of Health AI44063. The United States Government has certain rights in the invention. Background Pyrazinamide (PZA) is an important first-line tuberculosis (TB) drug that is most commonly used in combination with isoniazid and rifampin for the treatment of tuberculosis. PZA plays a unique role in shortening the tuberculosis treatment from previously 9-12 months to 6 months as a result of its ability to kill a population of persister M. tuberculosis bacteria that are not killed by other TB drugs. Persisters pose a significant challenge to the control of various bacterial infections, as they underlie latent infections, chronic and recurrent infections, biofilm infections, lengthy therapy of certain bacterial infections as in tuberculosis and post-treatment persistence and relapse (e.g., Zhang, Y., Persistent and dormant tubercle bacilli and latent tuberculosis. Front Biosci, 2004. 9 : p. 1136-56; McDermott, W., Microbial persistence. The Yale J Biol Med, 1958. 30: p. 257-91; and Lewis, K., Persister cells, dormancy and infectious disease. Nat Rev Microbiol, 2007. 5(1): p. 48-56). Persister bacteria pose enormous public health problems. The persister tubercle bacilli (TB) present a tremendous challenge for effective TB control and underlie the lengthy TB therapy. This makes patient compliance very difficult and is in part responsible for the increasing emergence of drug resistant TB such as the recently reported extreme drug resistant TB (XDR-TB) (J. Cohen, Science 313, 1554 (2006). Identifying how drugs like PZA that kill persister bacteria is key to finding new generation of persister antibiotics. Moreover, most strains of M. tuberculosis that are resistant to PZA are due to mutations in the gene pncA encoding pyrazinamidase/nicotinamidase that is involved in conversion of PZA to pyrazinoic acid (POA), but a few PZA-resistant M. tuberculosis strains do not have pncA mutations, suggesting new mechanism of resistance. Despite the importance of PZA in shortening the treatment of TB, its mechanism of action is the least understood of all tuberculosis drugs, and the target of PZA or its active metabolite, pyrazinoic acid (POA), remains elusive. Previous attempts to identify the mode of PZA action by genetic approaches have so far been unsuccessful. This invention identified a number of drug targets of PZA which can be used for identifying new antibiotics for treatment of TB infection and other bacterial infections and for identifying PZA-resistant TB bacteria. There is a need for more effective sterilizing drugs, methods and compositions for treating persistent bacterial infections such as tuberculosis. Brief Description of the Drawings Figure 1. Mycobacterial lysates were loaded onto the POA-linked and control columns and the proteins that bound to POA (A) and the control column (B) were analyzed by SDS- PAGE. Lane M, protein ladder; Lane 1, whole cell lysate; 2, flow-through fraction; 3, washing fraction; 4, elution fraction. The band indicated by the red arrow is RpsA. Figure 2. Concentration-dependent inhibition of trans-translation by POA with M. tuberculosis ribosome and DHFR template with rare codon cluster. This bar graph represents densitometry scan of band intensities of similar experiments in Figure 4C performed 5 times (P value <0.024, n=5). Figure 3. Structures of POA derivative (5-hydroxyl-2-pyrazinecarboxylic acid) (A) and the control compound ethanolamine (B) coupled to Sepharose 6B column for the identification of POA binding proteins from M. tuberculosis. Figure 4. RpsA alignment and isothermal titration calorimetry (ITC) titration of RpsA and POA. (A) Alignment of RpsA from M. tuberculosis H37Rv, M. tuberculosis PZA-resistant strain DHM444 and M. smegmatis. Rl to R4 represent the four homologous RNA-binding domains in RpsA. Colored vertical lines in gray boxes indicate sequence variations in the highly conserved RpsA sequences compared with the wild type M. tuberculosis sequence. The expanded region shows the variability in amino acid sequence in the C-terminus of RpsA among mycobacterial species. The red arrow at position 438 amino acid residue indicates the deletion of alanine in the C-terminal region of the mutant RpsA. ITC binding studies indicate POA bound to the M. tuberculosis H37Rv RpsA (WT) (B, inset VI), but not DHM444 RpsA (Mutant)(Inset, IV), and only weakly with the M. smegmatis RpsA (M. smeg) (Inset II). PZA did not bind to wild type RpsA (Inset V) or mutant RpsA (Inset III). The lower panel of the Figure 2B shows the typical molar ratio saturation plot of POA with wild type Mtb RpsA. Figure 5. (A) Concentration-dependent inhibition of tmRNA binding to wild type M. tuberculosis RpsA by POA (Lanes 2-7). tmRNA from M. tuberculosis was used as RNA alone control (Lane 1). The wild type RpsA interaction with tmRNA was not affected by PZA (200 g/ml) (Lane 8) or INH ( 1 g/ml) (Lane 9). (B) tmRNA had impaired binding to the mutant RpsA (Lane 2), and POA at different concentrations did not inhibit the interaction of the DHM444 mutant RpsA with tmRNA (Lanes 3-7); The mutant RpsA interaction with tmRNA was not affected by PZA (200 g/ml) (Lane 8) or INH ( 1 g/ml) (Lane 9). (C) POA at 100, 50, and 25 µg/ml inhibited trans-translation of the DHFR product in a concentration-dependent manner in the in vitro system that contained ribosomes from M. tuberculosis, tmRNA and recombinant SmpB from M. tuberculosis, template pDHFR-8 AGG rare codons that are required for trans- translation (Lanes 1-5). Arrowheads indicate the trans-translation product DHFR was still present with low concentration of POA at 12.5 µ ηΐ (Lane 4) or in the absence of POA (Lane 5). POA at different concentrations did not inhibit canonical translation in in vitro translation system using ribosomes from M. tuberculosis, template pDHFR with stop codon (D, Lanes 6- 10), nor the trans-translation of DHFR using ribosome from M. smegmatis (E, Lanes 1-4), or using ribosome from E. coli (F, Lanes 1-4) in the trans-translation system that contained tmRNA and recombinant SmpB from M. tuberculosis, template pDHFR-8 AGG rare codons. Figure 6. A new model for the mode of action of PZA. PZA is converted to the active form POA by M. tuberculosis PZase intracellularly and inhibits targets including RpsA. Upon stress, translating ribosomes are stalled and incomplete polypeptides may be toxic to the cell. The bacterial cell resolves this problem by adding tmRNA to the stalled mRNA.
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