(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2016/123368 Al 4 August 2016 (04.08.2016) P O P C T

(51) International Patent Classification: AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, A61K 31/28 (2006.01) A61K 39/04 (2006.01) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, A61K 31/70 (2006.01) A61P 31/04 (2006.01) DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, A61K 31/135 (2006.01) A61P 31/06 (2006.01) HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, (21) International Application Number: MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PCT/US20 16/0 15409 PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, (22) International Filing Date: SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, 28 January 2016 (28.01 .2016) TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (25) Filing Language: English (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (26) Publication Language: English GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, (30) Priority Data: TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, 62/109,447 29 January 2015 (29.01.2015) U S TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, (71) Applicant: THE CALIFORNIA INSTITUTE FOR LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, BIOMEDICAL RESEARCH [US/US]; 11119 North SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, Torrey Pines Road, Suite 100, La Jo11a, California 92037 GW, KM, ML, MR, NE, SN, TD, TG). (US). Declarations under Rule 4.17 : (72) Inventors: WANG, Feng; 615 1 Citracado Circle, Carls — as to applicant's entitlement to apply for and be granted a bad, California 92009 (US). HARBUT, Michael; 201 1 W . patent (Rule 4.1 7(H)) California St., San Diego, California 921 10 (US). SCHULTZ, Peter G.; 1650 La Jolla Rancho Road, La — as to the applicant's entitlement to claim the priority of the Jolla, California 92037 (US). earlier application (Rule 4.1 7(in))

(74) Agent: HARDT, Ingo H.; Wilson Sonsini Goodrich & Published: Rosati, 650 Page Mill Road, Palo Alto, California 94304 — with international search report (Art. 21(3)) (US). — before the expiration of the time limit for amending the (81) Designated States (unless otherwise indicated, for every claims and to be republished in the event of receipt of kind of national protection available): AE, AG, AL, AM, amendments (Rule 48.2(h))

(54) Title: METHODS OF TREATMENT WITH METAL-THIOLATE COMPLEXES

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(57) Abstract: The present invention provides methods of treating bacterial infections with metal thiolate complexes, such as auran- ofin. Disclosed herein are methods of treating a bacterial infection in a subject, comprising administering a metal thiolate complex, wherein the metal thiolate complex is cytotoxic to a . The bacteria may be a gram-positive bacteria. The gram-positive bac teria may be of a Mycobacterium genus. The gram-positive bacteria may be of a Bacillus genus. The gram-positive bacteria may be of an genus. The gram-positive bacteria may be Mycobacterium tuberculosis. The gram-positive bacteria may be Ba cillus subtilis. The gram-positive bacteria may be selected from Enterococcusfaecium and Enterococcusfaecalis. The gram-positive bacteria may be aureus. The may be resistant to a drug. The drug may be an antibiotic agent selected from and linezolid. METHODS OF ANTIBIOTIC TREATMENT WITH METAL-THIOLATE COMPLEXES

FEDERALLY SPONSORED RESEARCH

[001] This invention was made in part during work supported by N H grant R01 AI097548 from the National Institutes of Health. The government may have certain rights to this invention.

BACKGROUND OF THE INVENTION

[002] The emergence and spread of antibiotic-resistant bacterial infections is an ongoing and growing public health concern, and has resulted in a growing urgency to identify new compounds with novel mechanisms of action that are active against antibiotic-resistant pathogenic bacteria. Drug-resistance has a profound clinical significance; including increased hospitalizations and excess healthcare costs and higher rates of mortality. Outbreaks of -resistant S. aureus (MRSA), previously confined to hospitals now have expanded due to community-associated spreading. For tuberculosis (TB), the lengthy course of drug treatment persisters has contributed to multidrug- resistant M tuberculosis (MDR-MTB). Costs for a drug course for multidrug-resistant TB (MDR- TB) can be thousands of times more than for regular treatment, and treatment success rates for individuals with extensively drug resistant TB are less than 50%.

SUMMARY OF THE INVENTION

[003] Disclosed herein are methods of treating a bacterial infection in a subject, comprising administering a metal thiolate complex, wherein the metal thiolate complex is cytotoxic to a bacteria. The bacteria may be a gram-positive bacteria. The gram-positive bacteria may be of a Mycobacterium genus. The gram-positive bacteria may be of a Bacillus genus. The gram-positive bacteria may be of an Enterococcus genus. The gram-positive bacteria may be Mycobacterium tuberculosis. The gram-positive bacteria may be Bacillus subtilis. The gram-positive bacteria may be selected from Enterococcusfaecium and Enterococcusfaecalis. The gram-positive bacteria may be Staphylococcus aureus. The Staphylococcus aureus may be resistant to a drug. The drug may be an antibiotic agent selected from vancomycin and linezolid. The drug may be methicillin. The bacteria may express a thioredoxin and a thioredoxin reductase. The bacteria may be Staphylococcus aureus and the thioredoxin may be TrxC. The bacteria may be Mycobacterium tuberculosis and the thioredoxin reductase may be TrxB2. The bacteria may not express a glutathione reductase. The bacteria may be a dormant bacteria. The metal thiolate complex may be a salt. The metal thiolate complex may be a gold complex. The metal thiolate complex may be an organogold compound. The metal thiolate complex may be selected from , , disodium aurothiomalate, aurothiosulfate, and pharmaceutically acceptable derivatives thereof. The metal thiolate complex may be selected from auranofin and pharmaceutically acceptable derivatives thereof. The method may further comprise administering an agent selected from paraquat, diamide, and a combination thereof.

[004] Further disclosed herein are methods of inhibiting a thioredoxin reductase or substrate thereof in a bacteria comprising contacting the bacteria with a metal thiolate complex. The contacting may comprise administering the metal thiolate complex to a subject infected with the bacteria. The bacteria may not express a glutathione reductase. The metal thiolate complex may comprise a compound selected from auranofin and pharmaceutically acceptable derivatives thereof. The thioredoxin reductase may be a first enzyme and the method further comprises administering a therapeutic agent that inhibits at least one additional enzyme. The at least one additional enzyme may comprise an enzyme bearing a reactive cysteine residue. The at least one additional enzyme may be selected from a cysteine protease and a sulfurtransferase. The at least one additional enzyme may be an oxidoreductase. The oxidoreductase may be an NADPH-dependent oxidoreductase. The oxidoreductase may be a mycothione reductase. The bacteria may be a gram-positive bacteria. The gram-positive bacteria may be selected from Mycobacterium tuberculosis and Bacillus subtilis. The gram-positive bacteria may be a drug-resistant strain of bacteria. The drug-resistant strain of bacteria may be a strain of bacteria selected from Enterococcusfaecium and Enterococcusfaecalis. The drug-resistant strain of bacteria may be a strain of Staphylococcus aureus. The strain of Staphylococcus aureus may be resistant to an antibiotic agent selected from methicillin, vancomycin, and linezolid. The metal thiolate complex may be a gold salt. The metal thiolate complex may be a gold complex. The metal thiolate complex may be an organogold compound. The metal thiolate complex may be selected from auranofin, aurothioglucose, disodium aurothiomalate, sodium aurothiosulfate, sodium aurothiomalate and pharmaceutically acceptable derivatives thereof. The metal thiolate complex may be selected from auranofin and pharmaceutically acceptable derivatives thereof.

[005] Further disclosed herein are methods for preventing a bacterial infection in a subject, comprising administering a metal thiolate complex. The bacterial infection may be an infection with a gram-positive bacterium. The bacterial infection may be an infection with a bacterium that expresses thioredoxin reductase and does not express glutathione reductase. The metal thiolate complex may be a gold salt. The metal thiolate complex may be a gold complex. The metal thiolate complex may be an organogold compound. The metal thiolate complex may be selected from auranofin, aurothioglucose, disodium aurothiomalate, sodium aurothiosulfate, sodium aurothiomalate and pharmaceutically acceptable derivatives thereof. The metal thiolate complex may be selected from auranofin and pharmaceutically acceptable derivatives thereof. The method may further comprise administering an agent selected from paraquat, diamide, and a combination thereof. [006] Further disclosed herein are methods of treating tuberculosis in a subject comprising administering a therapeutic agent to the subject, wherein the therapeutic agent modulates an activity of a thioredoxin reductase or a substrate thereof. The thioredoxin reductase may be TrxB2. The therapeutic agent may inhibit the activity of the thioredoxin reductase.

[007] Further disclosed herein are methods of treating a Staphylococcus aureus infection in a subject comprising administering a therapeutic agent to the subject, wherein the therapeutic agent modulates a thioredoxin reductase or substrate thereof. The thioredoxin reductase may be TrxC.

[008] Further disclosed herein are compositions comprising auranofin and a therapeutic agent. The therapeutic agent may be selected from an antibiotic agent and an anti-inflammatory agent. The therapeutic agent may be a thiol oxidizing agent. The thiol oxidizing agent may be diamide. The therapeutic agent may be paraquat. The antibiotic agent may be rifampicin.

BRIEF DESCRIPTION OF THE DRAWINGS

[009] Figure 1A depicts the chemical structure of auranofin. [010] Figure IB shows auranofin is a potent inhibitor of cell viability under non-replicating conditions. [011] Figure 1C shows auranofin has potent bactericidal activity against non-replicating M. tuberculosis. [012] Figure ID shows additive interactions of auranofin during co-treatment with rifampicin to inhibit M. tuberculosis growth. [013] Figure 2A shows auranofin inhibits bacterial thioredoxin reductase in tuberculosis. [014] Figure 2B shows auranofin inhibits bacterial thioredoxin reductase in S. aureus. [015] Figure 3A shows auranofin depletes intracellular thiols and sensitizes S. aureus to oxidizing agents. [016] Figure 3B shows auranofin depletes intracellular thiols in tuberculosis. [017] Figure 3C shows combination treatment of S. aureus with auranofin and diamide has synergistic antimicrobial activities. [018] Figure 3D shows combined treatment of S. aureus with auranofin and paraquat produces synergistic antimicrobial activity. [019] Figure 3E shows loss of glutathione synthesis sensitizes E. coli to auranofin. [020] Figure 4 shows efficacy of auranofin in a murine peritonitis MRSA infection model. [021] Figure 5 shows purification of thioredoxin reductase (TrxR) and thioredoxin (Trx). [022] Figure 6 shows inhibition of mycothione reductase. [023] Figure 7 shows Auranofin toxicity on HepG2 cells. DETAILED DESCRIPTION OF THE INVENTION I. Introduction

[024] Infections caused by antibiotic-resistant bacteria are a growing public health threat and make the identification of new a priority. One strategy may be selecting agents that target essential cellular metabolic and signaling pathways in the bacteria. In many organisms, glutathione and glutathione reductase (GSH and GR) function in parallel with thioredoxin and thioredoxin reductase (Trx and TrxR) to provide the cell with a source of reducing equivalents. In these organisms, the two systems appear to be redundant, as only double mutants of the pathways are nonviable. In contrast, some gram-positive bacteria, including M tuberculosis (and certain other Actinobacteria), S. aureus, certain Bacillus, and the gram-negative Helicobacterpylori species lack the conventional redox couple GSH-GR, which is normally found in millimolar quantities intracellularly in most of gram-negatives. Thus, TrxR maintains the thiol-redox balance and protects against reactive oxidative species in these bacteria, making the Trx-TrxR system essential and presenting potential bactericidal targets.

[025] Recognizing the therapeutic potential of targeting the essential Trx-TrxR system in many gram-positive bacteria for the treatment of infections, methods of treating and/or preventing such infections have been developed and are disclosed herein. The present application describes biochemical studies that show the organogold compound, auranofin, inhibits the bacterial thioredoxin reductase, an orally bioavailable FDA-approved anti-rheumatic drug, Furthermore, it is shown that auranofin has bactericidal activity in a murine model of methicillin-resistant S. aureus (MRSA) infection. Importantly, auranofin may be used as a bactericidal agent in the treatment of widespread and often life-threatening infections caused by additional gram-positive bacteria, including Mycobacterium tuberculosis (M. tuberculosis) and Bacillus subtilis (B. subtilis), as well as drug-resistant strains of Enterococcusfaecium andfaecalis (E.faecium and E.faecalis) and Staphylococcus aureus (S. aureus).

[026] Auranofin has is an FDA-approved organogold compound with a well-established safety profile. Auranofin was originally indicated as a disease modifying anti-rheumatic drug. More recently, it has been demonstrated to have activities against a variety of parasitic pathogens and possess antineoplastic properties, and is currently in clinical trials for treatment of amoebiasis and chronic lymphatic lymphoma.

II. Methods of Treatment

[027] Disclosed herein are methods of treating a subject infected with a bacteria, comprising administering a metal thiolate complex, wherein the metal thiolate complex is cytotoxic to the bacteria. Further disclosed herein are methods for preventing a bacteria infection in a subject, comprising administering a metal thiolate complex. Also disclosed herein, are methods of treating a condition in a subject in need thereof, comprising inhibiting a redox and/or a substrate thereof in a bacteria comprising administering a metal thiolate complex. The bacteria may be a gram- positive bacteria. The bacteria may be a gram-negative bacteria. The bacteria expresses a thioredoxin reductase. The bacteria may not express a glutathione reductase. The metal thiolate complex may comprise a compound selected from auranofin and pharmaceutically acceptable derivatives thereof.

A. Metal Thiolate Complexes

[028] Disclosed herein are methods of treating a subject infected with a bacteria, comprising administering a metal thiolate complex. The metal thiolate complex may be thiol-reactive. The metal thiolate complex may have an affinity for redox-active cysteines. The metal thiolate complex may be a gold complex, also referred to herein as an organogold compound. The organogold compound is a complex of a protein/peptide with one or more gold atoms. The metal thiolate complex may be a gold salt. The metal thiolate complex may be selected from auranofin, aurothioglucose, disodium aurothiomalate, sodium aurothiosulfate, sodium aurothiomalate and pharmaceutically acceptable derivatives thereof. The methods may comprise treating the subject in need thereof with a compound selected from auranofin and pharmaceutically acceptable derivatives thereof.

B. Combination Therapies

[029] Disclosed herein are methods of treating a subject infected with a bacteria, comprising administering a metal thiolate complex in combination with a therapeutic agent. Disclosed herein are methods of killing bacteria, comprising administering a metal thiolate complex and a therapeutic agent. The therapeutic agent may be referred to herein as an additional therapeutic agent. The methods may comprise administering a combination (two or more) therapeutic agents. The therapeutic agent may be a thiol oxidizing agent. The therapeutic agent may be paraquat. (Paraquat is usually considered a toxic compound to humans, but, in very low doses, may be useful in the treatment of conditions disclosed herein). The therapeutic agent may be diamide. Also disclosed herein, are methods of treating a condition in a subject in need thereof, comprising administering a metal thiolate complex that inhibits a redox protein and administering at least one additional therapeutic agent. The therapeutic agent may be an anti-inflammatory agent. The anti-inflammatory agent may be an agent that reduces inflammatory activity and/or symptoms. Inflammatory activity and/or symptoms, by way of non-limiting example, may be selected from pain, swelling, redness, and mobilization of immune cells. The anti-inflammatory agent may be selected from a steroidal anti-inflammatory agent (e.g., dexamethasone) and a non-steroidal anti-inflammatory agent (e.g., aspirin, ibuprofen and naproxen). The therapeutic agent may be an antibiotic agent. The antibiotic may be a bactericidal antibiotic agent. The antibiotic may be a bacteriostatic antibiotic agent. [030] The antibiotic agent may be of a class of antibiotics selected from aminoglycosides, ansamycins, , , , glygopeptides, lincosamides, lipopeptide, macrolides, , nitrofurnas, oxazolidinonens, , quinolones, sulfonamides and tetracyclines. The antibiotic agent may comprise a peptide.

[031] The antibiotic agent may be selected from amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, spectinomycin, gledanamycin, herimycin, rifaximin, , , , , , , , , cefalothin, , , , , , , , , , , , cefpodosime, , , , , , , , , vancomycin, , , , clindamycin, lincomycin, , azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, spiramycin, , furazolidone, nitrofurantoin, linezolid, posizolid, radezolid, torezolid, , , , , , , , , methicillin, , , G, penicillin V, , , , amoxicillin, amoxicillin/clavulanate, ampicillin, ampicilin/, piperacillin/, ticarcillin, ticarcillin/clavulanate, , , b, ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin ,trovafloxacin, grepafloxacin, sparfloxacin, temafloxacin, mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilimide, , sulfisoxazole, trimethoprim-sulfamethoxazole, sulfonamidochrysoidine, demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline, clofazimine, dapsone, capreomycin, , ethambutol, ethionamide, , pyrazinamide, rifampicin, rifabutin, rifapentine, streptomycin, arsphenamine, chloramphenicol, , fusidic acid, metronidazole, mupirocin, platensimycin, quinupristin, quiinupristin/dalfopristin, thiamphenicol, tigecycline, tinidazole, teixobactin, and trimethoprim.

[032] Combination therapy is a cornerstone of modern TB treatment, and any novel drug must show compatibility with established anti-TB drugs (RMP and ΓΝΗ). In some embodiments, the antibiotic agent is useful in the treatment of tuberculosis and/or infection with tuberculosis and may be selected from rifampicin, TMC207, and isoniazid.

C. TargetBacteria

[033] Disclosed herein are methods of treating a condition in a subject infected with a bacteria, comprising administering a metal thiolate complex. The bacteria may be a gram-positive bacteria. [034] The gram-positive bacteria may be a bacteria of an Actinobacteria phylum. The gram- positive bacteria may be a bacteria of an order selected from and Lactobacillales. The gram-positive bacteria may be a bacteria of a Listeriaceae family. The gram-positive bacterial may be a bacteria of a genus selected from Actinomyces, Bacillus Clostridium, Corynebacterium, Enterococcus, Listeria, Nocardia, Pasteuria, Sporosarcina, Staphylococcus, Sarcina, and Streptococcus.

[035] The gram-positive bacteria may be a bacterial species selected from Actinomyces georgiae, Actinomyces gerencseriae, Actinomyces israelii, , Bacillus mojavensi,s Bacillus weihenstephanensis, Clostridium acetobutylicum, Clostridium aerotolerans, Clostridium argentinense, Clostridium autoethanogenum, Clostridium baratii, Clostridium beijerinckii, Clostridium bifermentans, Clostridium botulinum, Clostridium butyricum, Clostridium cadaveris, Clostridium cellobioparum, Clostridium cellulolyticum, Clostridium cellulovorans, Clostridium chauvoei, Clostridium clostridioforme, Clostridium colicanis, Clostridium difficile, Clostridium estertheticum, Clostridium fallax, Clostridium formicaceticum, Clostridium histolyticum, Clostridium innocuum, Clostridium kluyveri, Clostridium ljungdahlii, Clostridium novyi, Clostridium paradoxum, Clostridium paraputrificum, Clostridium perfringens, Clostridium phytofermentans, Clostridium piliforme, Clostridium ragsdalei, Clostridium ramosum, Clostridium saccharobutylicum, Clostridium saccharoperbutylacetonicum, Clostridium scatologenes, Clostridium septicum, Clostridium sordellii, Clostridium sporogenes, Clostridium stercorarium, Clostridium sticklandii, Clostridium straminisolvens, Clostridium tertium, Clostridium tetani, Clostridium thermosaccharolyticum, Clostridium tyrobutyricum, Clostridium uliginosum, Corynebacterium, Corynebacterium amycolatum, Corynebacterium bovis, Corynebacterium diphtheriae, Corynebacterium efficiens, Corynebacterium granulosum, Corynebacterium jeikeium, Corynebacterium macginleyi, Corynebacterium minutissimum, Corynebacterium renale, Desulfitobacterium dehalogenans, Fervidobacterium changbaicum, Fervidobacterium gondwanense, Fervidobacterium islandicum, Georgenia ruanii, Microbispora corallina, Nocardia asteroides, Nocardia brasiliensis, Nocardia farcinica, Nocardia ignorata, Propionibacterium acnes, Rathayibacter toxicus, Rhodococcus equi, Rothia dentocariosa, Solobacterium moorei, Sporosarcina aquimarina, Staphylococcus aureus, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcus lutrae, Staphylococcus muscae, Staphylococcus nepalensis, Staphylococcus pettenkoferi, Staphylococcus pseudintermedius, Staphylococcus saprophyticus, Staphylococcus schleiferi, Staphylococcus succinus, Staphylococcus warneri, Staphylococcus xylosus, Streptococcus agalactiae, Streptococcus anginosus, Streptococcus bovis, Streptococcus canis, Streptococcus downei, Streptococcus gordonii, Streptococcus iniae, Streptococcus lactarius, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus peroris, , Streptococcus pyogenes, Streptococcus ratti, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus sobrinus, Streptococcus suis, Streptococcus thermophilus, Streptococcus tigurinus, Streptococcus uberis, Streptococcus vestibularis, Syntrophomonas curvata, Syntrophomonas palmitatica, Syntrophomonas sapovorans, Syntrophomonas wolfei, Syntrophomonas zehnderi, and Viridans streptococci.

[036] The bacteria may be impervious to Gram staining. The bacteria may be gram-negative bacteria. For example, Helicobacter pylori is a gram-negative bacteria that lacks the conventional rdox couple GSH-GR.

[037] The bacteria may be Mycobacterium tuberculosis (M. tuberculosis). The tuberculosis may be a replicating M. tuberculosis. The tuberculosis may be a non-replicating tuberculosis.

[038] The bacteria may be Staphylococcus aureus (S.aureus). The S.aureus may be an drug- resistant S.aureus. The S.aureus may be an antibiotic-resistant S.aureus (e.g. methicillin-resistant Staphylococcus aureus (MRSA)).

[039] The bacteria may be a dormant bacteria. The bacteria may be a bacteria that was dormant before contacting a subject. The dormant bacteria may display any detectable metabolism. The dormant bacteria may be a bacteria that can survive under extreme conditions. The dormant bacteria may be or have once been in the form of an endospore. The dormant bacteria may be resistant to an antibiotic. The dormant bacteria may be characterized by an altered metabolism. The ability to target a dormant bacteria is an attractive feature in treatment of M. tuberculosis in latent TB infection or S. aureus in biofilms.

[040] In some embodiments, the gram-positive bacteria may not be of a Staphylococcus genus. In some embodiments, the gram-positive bacteria may not be Staphylococcus aureus. In some embodiments, the gram-positive bacteria may not be Staphylococcus aureus.

[041] The bacteria may express a redox protein. The redox protein may be thioredoxin (Trx). The bacteria may express a redox protein reductase. The redox protein reductase may be thioredoxin reductase (TrxR). The TrxR may share less than about 90%, less than about 80%, less than about

70% , less than about 60%>, less than about 50%>, less than about 40%>, less than about 35%>, less than about 30% , less than about 25%, less than about 20%>, less than about 15%>, less than about 10%> or less than about 5% homology with human TrxR. The TrxR may share less than about 30%> homology with human TrxR. The bacteria may not express glutathione (GSH). The bacteria may not express the glutathione reductase (GR). D. Molecular Targets

[042] Disclosed herein are methods of treating a condition in a subject in need thereof, comprising inhibiting a redox protein and/or a redox protein reductase and/or a substrate thereof in a bacteria comprising administering a metal thiolate complex. The redox protein may be thioredoxin. The redoxin protein reductase may be thioredoxin reductase. Given the thiophilic nature of the auranofin, auranofin may react with additional enzymes (beyond thioredoxin reducatase) and other and/or multiple enzymes bearing reactive cysteine residues, such as cysteine proteases and sulfurtransferases. Reactive cysteine residues may include a cysteine residue on a protein/enzyme capable of and/or known to be reactive, polarizable, redox-active, have a high affinity for metals. Reactive cysteine residues may include a cysteine residue on a protein/enzyme known to be located in a functionally important site of the protein/enzyme. A functionally important site may include, by way of non-limiting example, a site that has catalytic, regulatory, structure-stabilizing, or cofactor- binding properties. The metal thiolate complex may inhibit a redox protein that reduces and/or oxidizes a compound selected from mycothione/mycothiol (MSH) and bacillithiol (BSH). Auranofin may inhibit M. tuberculosis mycothione reductase (MTR). Mycothione reductase (MTR) is an NADPH-dependent oxidoreductase with a catalytically important redox-active disulfide that reduces mycothione to mycothiol. The potential of inhibiting multiple enzyme targets of auranofin actually works in favor of antibiotic development in that it reduces the risk of resistance emerging.

E. Modes of Action

[043] The mechanism of the metal thiolate complex (e.g. auranofin) inhibition of TrxR may be through displacement reactions of the ligands of the metal, with the gold irreversibly complexed to each of the two cysteine residues of the active site. The inhibition of TrxR may result in decreased levels of thiols, compromising the bacteria cell's ability to reduce enzymes involved in metabolizing reactive species. Such enzymes may include, but are not limited to peroxiredoxins thiol-dependent AhpC and TPx of M. tuberculosis, which may be involved in protecting against oxidative and nitrosative stress. Metal thiolate complexes (e.g. auranofin) may have bactericidal effects by inhibiting additional and/or alternative roles for thioredoxins, which include, but are not limited to, providing electrons for enzyme catalysis, such as ribonucleotide reductase and methionine sulfoxide reductase. Effects of inhibiting TrxR may be multifaceted, and may trigger cell death and/or growth arrest by impacting a variety of functions necessary for both proliferation and survival. This may account for auranofin' s potency against both replicating and non-replicating M . tuberculosis.

F. Modes of Administration

[044] Disclosed herein are methods of administering metal thiolate complexes and, in some embodiments, additional therapeutic agents. Suitable routes of administration include, but are not limited to, oral, intravenous, rectal, aerosol, parenteral, ophthalmic, pulmonary, transmucosal, transdermal, vaginal, otic, nasal, and topical administration. In addition, by way of example only, parenteral delivery includes intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intralymphatic, and intranasal injections. [045] The manner of administration can be chosen based on, for example, whether local or systemic treatment is desired, and on the area to be treated. For example, the compositions can be administered orally, parenterally (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection), by inhalation, extracorporeally, topically (including transdermally, ophthalmically, vaginally, rectally, intranasally) or the like. [046] Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained.

G. Therapeutic Doses

[047] Auranofin is approved for long-term daily dosage at 6 mg/day (for rheumatoid arthritis) and serious side effects are rare, the most common being gastrointestinal distress that is easily manageable. However, higher doses of auranofin may be suitable and safe for treatment of infections or infectious diseases. For instance, the condition may only require acute treatment, wherein a higher dose of auranofin is safe if only administered over a short period of time. In some instances, administration methods (e.g. topical), may allow for a higher dose of auranofin. In other instances, the infection may be severe, requiring a very high (e.g. life-saving) dose. The higher dose or very high dose may be greater than about 9 mg/day. The higher dose or very high dose may be greater than about 10 mg/day, greater than about 12 mg/day, greater than about 14 mg/day, greater than about 16 mg/day, or greater than about 20 mg/day. The higher dose or very high dose may be less than about 20 mg/day, 25 mg/day or 30 mg/day. The higher dose or very high dose may be administered initially and then tapered down to a maintenance dose. The maintenance dose may be less than about 9 mg/day, less than about 8 mg/day, less than about 7 mg/day, less than about 6 mg/day, less than about 5 mg/day, less than about 4 mg/day, less than about 3 mg/day, less than about 2 mg/day, less than about 1 mg/day, or less than about 0.5 mg/day. [048] The dose of auranofin, or any other metal thiolate complexes disclosed herein, may be about 1 mg/day, about 2 mg/day, about 3 mg/day, about 4 mg/day, about 5 mg/day, about 6 mg/day, about 7 mg/day, about 8 mg/day, about 9 mg/day, about 10 mg/day, about 11 mg/day, about 12 mg/day, about 13 mg/day, about 14 mg/day, about 15 mg/day, about 20 mg/day, about 25 mg/day, about 30 mg/day, about 35 mg/day, about 40 mg/day, about 45 mg/day or about 50 mg/day. The dose of auranofin, or any other metal thiolate complexes disclosed herein, may be between about 1 mg/day and about 3 mg/day, between about 1 mg/day and about 6 mg/day, between about 1 mg/day and about 9 mg/day. In some instances, the dose of auranofin may be less than 1 mg/day. [049] The methods may comprise administering the metal thiolate complex to a subject, wherein the subject is about 1 year old to about 12 years old. Auranofin is approved for subjects 1-12 years old at a dosage of O.lmg/kg/day in 1 to 2 equally divided doses. In some cases, subjects 1-12 years old receive a dose between 0.1 mg/kg/day and 0.2 mg/kg/day. In some cases (e.g. bacterial infections), it is contemplated herein, that the subject that is about 1 year old to about 12 years old would receive a higher dose or life-saving dose. The higher dose or life-saving dose may be greater than 0.2 mg/kg/day. The higher dose or life-saving dose may be greater than about 0.5 mg/kg/day, greater than about 0.8 mg/kg/day, greater than about 1 mg/kg/day, or greater than about 2 mg/kg/day. The higher dose or life-saving dose may be less than about 3 mg/kg/day, less than about 5 mg/kg/day, or less than about 10 mg/kg/day. The higher dose or very high dose may be administered initially and then tapered down to a maintenance dose. The maintenance dose may be less than about 0.2 mg/kg/day, less than about 0.1 mg/kg/day, or less than about 0.05 mg/kg/day. The maintenance dose may be greater than about 0.01 mg/kg/day. [050] The methods may comprise administering the metal thiolate complex for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week or about 1 month. The methods may comprise administering the metal thiolate complex every other day. The methods may comprise administering the metal thiolate complex for a long term, wherein the condition is chronic. The methods may comprise administering the higher dose or life-saving dose for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 12 days, or about 14 days. The methods may comprise administering the maintenance dose for about 3 days, about 5 days, about 7 days, about two weeks, about 3 weeks, about 4 weeks, about 6 weeks, or about 8 weeks.

III. Therapeutic Uses

[051] Disclosed herein are methods of treating a condition in a subject infected with a bacteria, comprising administering a metal thiolate complex. The condition may be acute. The condition may be chronic. The condition may be selected from an ulcer (gastric, duodenal), gastritis, stomach cancer, a respiratory disease, a pneumonia, walking pneumonia, a dental disease (e.g. cavities), a periodontal disease, a food poisoning, tetanus, botulism, vaginitis, a meningitis, Lyme disease, diptheria, listeriosis, leprosy, a skin infection, endocarditis, septicemia, a toxinosis, an infection associated with a prosthesis (e.g. heart valves, catheters, etc.), a cystitis, an endometritis, streptococcal pharyngitis, scarlet fever, rheumatic fever, impetigo, erysipelas, puerperal fever, and necrotizing fasciitis. [052] The condition to may be tuberculosis. Tuberculosis is exceptional among bacterial infections in that even drug-susceptible strains are difficult to treat rapidly and effectively. This is in part due to the phenomenon oiMtb persistence, a state of phenotypic drug-tolerance that is attributed to a quiescent or non-replicating population of . Long treatment regimes make compliance problematic, and lead to the emergence of drug resistant mutants. MDR (Multi-Drug-Resistant) and XDR (extensively Drug-Resistant) Mtb strains are becoming widespread resulting in high failure rates despite the use of second and third line antibiotics and longer treatment times (up to 2 years).

IV. Pharmaceutical Compositions

[053] Disclosed herein are compositions comprising metal thiolate complexes, gold complexes, , and organogold compounds. Disclosed herein are compositions comprising auranofin. The compositions may further comprise an additional therapeutic agent. The additional therapeutic agent may be an antibiotic. The additional therapeutic agent may be an anti-inflammatory agent. The therapeutic agent may be diamide. The therapeutic agent may be paraquat. Paraquat may be toxic, but given at very low doses, may be helpful in the treatment of conditions described herein. The compositions may further comprise one or more pharmaceutically acceptable salts, excipients or vehicles. Pharmaceutically acceptable salts, excipients, or vehicles for use in the present pharmaceutical compositions include carriers, excipients, diluents, antioxidants, preservatives, coloring, flavoring and diluting agents, emulsifying agents, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, tonicity agents, cosolvents, wetting agents, complexing agents, buffering agents, antimicrobials, and surfactants. [054] "Pharmaceutically acceptable" may refer to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans. "Pharmaceutically acceptable salt" may refer to a salt of a compound that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. "Pharmaceutically acceptable excipient, carrier or adjuvant" may refer to an excipient, carrier or adjuvant that may be administered to a subject, together with at least one antibody of the present disclosure, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound. Neutral buffered saline or saline mixed with serum albumin are exemplary appropriate carriers. "Pharmaceutically acceptable vehicle" may refer to a diluent, adjuvant, excipient, or carrier with which at least one antibody of the present disclosure is administered. [055] The compositions may be in liquid form or in a lyophilized or freeze-dried form. Compositions described herein may be formulated for controlled or sustained delivery in a manner that provides local concentration of the product (e.g., bolus, depot effect) and/or increased stability or half-life in a particular local environment. [056] Compositions may be suitable for parenteral administration. Exemplary compositions are suitable for injection or infusion into an animal by any route available to the skilled worker, such as intraarticular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, or intralesional routes. A parenteral formulation typically will be a sterile, pyrogen-free, isotonic aqueous solution, optionally containing pharmaceutically acceptable preservatives. [057] A pharmaceutical composition comprising an gold complex, gold salt, or organogold compound, (e.g. auranofin), disclosed herein may be formulated for inhalation, such as for example, as a dry powder. Inhalation solutions also may be formulated in a liquefied propellant for aerosol delivery. In yet another formulation, solutions may be nebulized.

EXAMPLES Example 1. Auranofin possesses activity against replicating and non-replicating M . tuberculosis

[058] To identify inhibitors with novel activity against replicating and non-replicating M . tuberculosis a cell-based screen of bioactive compounds under nutrient-deprivation conditions was performed. An M . tuberculosis strain engineered to constitutively express luciferase and used bacterial luminescence was utilized as a readout of viability. Compound efficacy after 96 hr period of starvation in PBS (also known as the Loebel model (20)), a period of time that induces transcriptional changes in response to nutrient deprivation but still provides a robust luciferase readout was assayed. Non-replicating bacteria were starved in PBS with 0.05% tyloxapol for 96 hr prior to treatment. Both cultures were treated for 5 days and then assayed for ATP content. Auranofin was identified as a potent inhibitor of reporter activity under non-replicating conditions (see FIGS. 1A & B). Indeed, auranofin appeared to have more activity under non-replicating conditions than in medium that supported growth as assayed by ATP content (450 nM versus 4.6 µΜ, respectively). Further examination of auranofin activity in growth-supporting minimal medium showed that this difference was due to the presence of bovine serum albumin (BSA) in the 7H9 medium used for assays under replicating conditions, which caused a 30-fold increase in the MIC relative to the same medium lacking BSA (Table 1). The albumin concentration corresponds to the albumin concentration found in OADC growth supplement.

[059] Auranofin MICs were determined by broth microdilution methodology according to Clinical Laboratory Standards Institute guidelines. Controls in all assays included strain-appropriate antibiotics, bacteria alone, or media alone. For non-replicating persistence assays, CFUs for respective compounds were determined against cultures that had been carbon-starved for 96 hr in PBS/tyloxapol. Assays were performed in 1 ml of culture (OD oo nm = 0.1). After 5 days of treatment all cultures were washed once in PBS and then plated as serial dilutions on 7H10 plates. Colonies were enumerated after 3 weeks.

[060] Table 1. Auranofin activity in minimal medium versus serum-containing medium.

[061] Starvation-induced non-replicating M tuberculosis was treated with auranofin, or the front line anti-tuberculosis drugs rifampicin (RMP) and isoniazid (INH), for 5 days followed by enumeration of viable colonies. Viable colonies after 5 days of auranofin treatment on non- replicating starved cultures were enumerated to ascertain whether the action of auranofin was bactericidal against non-replicating M . tuberculosis. Auranofin showed potent bactericidal activity against non-replicating M . tuberculosis, producing a 1.3 log and 3.7 log drop in viable bacteria after 5 days of treatment at 100 nM and 1.0 µΜ , respectively. (FIG. 1C). Auranofin showed potent bactericidal activity relative to INH and RMP. Under the same assay conditions and concentrations, RMP and H showed only moderate activity, each depleting viable bacteria by less than 1 log even at concentration of 1 µΜ . These results suggest that auranofin antimicrobial efficacy is not limited to bacteria with a highly active metabolic state, and that it is efficacious against dormant bacterial population with altered metabolism, which is an attractive feature in treatment of M. tuberculosis in latent TB infection or S. aureus in biofilms. [062] Combination therapy is a cornerstone of modern TB treatment, and any novel drug must show compatibility with established anti-TB drugs (25). We therefore assessed the activity of auranofin against M tuberculosis in the presence of RMP and INH. Co-treatment of tuberculosis in growth assays in 7H9 medium (supplemented with OADC) with auranofin combined with either INH or RMP showed that auranofin works in a complementary fashion with both drugs. Auranofin showed additive interactions during co-treatment with RMP (as assessed the fractional inhibitory concentration; FIC=1.00) and synergized slightly with INH ( FIC=0.90) to inhibitM tuberculosis growth (FIG. ID) suggesting it is suitable to be used with the two major front-line anti-TB drugs.

Example 2. Auranofin has differential potency against gram-positive versus gram-negative bacteria

[063] Auranofin was profiled against a panel of gram-positive and gram-negative pathogenic bacteria species, including strains resistant to methicillin, vancomycin, and linezolid. Auranofin displayed potent growth inhibition against each of the gram-positive strains tested (Table 2). In most instances it had maximal activity at a concentration at 0.5 g/mL (≥ 740 nM) and showed equal efficacy against drug-resistant and drug-sensitive strains. In contrast to results observed against the gram-positive strains, auranofin lacked potency against gram-negative bacteria.

[064] Table 2 . Auranofin displays potent activity against a panel of drug-sensitive and drug- resistant pathogenic gram-positive bacteria.

Example 3. Auranofin inhibits the essential M. tuberculosis and S. aureus thioredoxin reductase

[065] The effect of auranofin on the activity of bacterial TrxR has not yet been explored. Because of the essential nature of TrxB2 and TrxB for viability, the mechanism of action of auranofin may be at least in part mediated by inhibition of these . To assess this, both M . tuberculosis TrxB2

and Trx (TrxC) and S. aureus TrxB were cloned, expressed, and purified using E . coli for heterologous expression (FIG. 5). Activity assays of TrxR were performed in the presence of either TrxC or E . coli Trx, and the enzyme activity was assayed by utilizing a reaction that regenerates oxidized thioredoxin via the rapid intramolecular disulfide exchange reaction with 5-5'-dithiobis-(2- nitrobenzoic acid) (DT B). Purified M . tuberculosis TrxB2 was pre-incubated with NADPH and

DT B and the given concentration of auranofin for 15 minutes. Reactions were initiated with the addition of TrxC. After reduction by TrxB2, TrxC is then reoxidized by DTNB generating the TNB chromophore. The reaction is monitored at A412 nm. Reaction progress in this assay was monitored by measuring the absorbance of the liberated TNB chromophore at 412 nm. Auranofin potently inhibited TrxR of both M . tuberculosis and S. aureus in a dose-dependent fashion with IC50 of 287 nM and 90 nM against TrxB2 and TrxB, respectively (FIG. 2 A & B).

Example 4. Auranofin treatment of gram-positive bacteria results in thiol depletion and compromises defense against oxidative stress.

[066] To determine if inhibiting the function of TrxR would result in a reduction in the level of thiols relative to cells with fully functioning TrxR, thereby decreasing the reducing potential in

treated cells, free thiol content in auranofin-treated M . tuberculosis and S. aureus was assayed. Bacteria were treated for 15 min (S. aureus) or 5 hr (M tuberculosis) with the given concentrations of auranofin. Rifampicin was used as a negative control. After treatment bacteria were washed twice in PBS and then resuspended in 100 mM potassium phosphate, pH 7.4, containing 1 mM ethylenediaminetetraacetic acid, and lysed using a Precellys 24 homogenizer. Thiols were quantified using the Thiol Detection Assay Kit (Cayman Chemical, Ann Arbor, MI).

[067] S. aureus cultures treated with indicated concentrations of auranofin for 15 min show a decrease in free thiol concentration relative to untreated control. The negative control RMP was used at 50 nM and produces no decrease. M. tuberculosis cultures treated with auranofin for 5 hr at the given concentrations also show a depletion of thiols. Bacteria treated with auranofin showed a dose dependent decrease in the amount of free thiols after a short period of treatment with auranofin (FIG. 3A & B). In contrast, no increase was seen with the antibacterial RMP, which acts on RNA polymerase. These results suggest that auranofin-mediated inhibition of thioredoxin reductase severely disrupts the bacteria's thiol redox homeostasis, which may in turn, compromise a variety of functions, including protection against oxidative stress.

[068] Next, antibacterial activity of diamide, a thiol oxidizing agent, alone and in the presence of auranofin were compared to confirm that auranofin abrogates the bacteria's defense against thiol oxidation. Combination treatment of S. aureus with auranofin and diamide has synergistic antimicrobial activities. S. aureus cultures were treated for 3 hr with the indicated concentrations of diamide, 700 nM auranofin, alone or in combination. Millimolar concentrations of diamide had only

modest activity against S. aureus, producing a 1.1 log drop in CFU/mL at 10 mM (FIG. 3C). When combined with auranofin at 700 nM though, the antimicrobial activity of the two compounds was synergistic and produced a 4.4 log decrease in CFU, greater than what would be expected with dose- wise additivity (FIG. 3C), suggesting that auranofin inhibits the bacteria's ability to cope with thiol oxidation.

[069] Antimicrobial activity of paraquat, which generates intracellular reactive oxygen species, was compared the antimicrobial activity of paraquat with auranofin to assess the effect of auranofin on the ability of S. aureus to cope with oxidative stress. S. aureus cultures were treated for 3 hr with the indicated concentrations of paraquat, 700 nM auranofin, alone or in combination. In the absence of auranofin, paraquat showed little antimicrobial activity (0.8 log decrease in CFU/mL at 5 mM), but when coupled with auranofin, the two compounds had synergistically (1.6 log decrease in CFU/mL over dose-wise additivity) in their bacterial killing (FIG. 3D). These results demonstrate that auranofin severely impairs the bacterial defense against oxidative stress.

Example 5. Defense against auranofin in gram-negatives is mediated by glutathione

[070] To identify whether gram-negative E. coli, which possesses possess both Trx and GSH systems, would be susceptible to auranofin, we evaluated the ability of strains of E . coli absent in either glutathione (AgshA) or thioredoxin reductase (AtrxB) to grow in the presence of auranofin. Wild type or E. coli lacking glutathione (AgshA), thioredoxin reductase (AtrxB), or glutathione reductase ( gor) were treated with indicated concentrations of auranofin for 6 hr and growth was assayed by absorbance. While E. coli lacking TrxB showed increased resistance to auranofin compared to the wild-type, E . coli lacking GSH showed 4-fold increase in sensitivity to killing by auranofin (FIG. 3E). These results suggest that the GSH-GR system helps defend against loss of TrxR reducing capability in auranofin-treated E . coli and further establishes that TrxR is the relevant in vivo target in bacteria.

Example 6. Auranofin is effective in a murine systemic infection model of S. aureus

[071] To ascertain potential in vivo efficacy of auranofin, we tested its ability to protect mice from MRSA-induced mortality in a peritonitis model.

[072] Briefly, MRSA strain Sanger 252 was grown to mid-logarithmic phase in Todd-Hewitt broth, washed into phosphate-buffered saline, and ~ 109 colony-forming units were injected intraperitoneally (IP) into 8 week old female CD1 mice (Charles River Laboratories, Wilmington MA). One hour after infection, the mice were treated with a single i.p. injection of two different dosages of auranofin (0. 12 or 0.012 mg/ kg, which are equivalent to standard human doses of 6 mg or 3 mg per day or equivalent vehicle control. Treatment was continued IP once daily (with 0.12 mg/kg or 0 .12mg/kg) throughout the study, and mortality was monitored for 7 days. Auranofin at these doses appeared to be well tolerated by the mice and was sufficient to provide significant protection against mortality (FIG. 4), (P < 0.03, Mantel-Cox test.). Mice receiving 0.12 mg/kg auranofin 50% of the mice survived out to 7 days, compared with 32.5% of mice receiving 0.012 mg/kg and none of the mice in the vehicle control group. These results suggest that the potent in vitro activity of auranofin translates into anti-MRSA activity in a mouse model at well-tolerated doses. Finally, efforts by our group to determine the maximum tolerated dose in mice have found limited toxicity up to 70 mg/kg daily dosing for 7 days, suggesting doses higher than 6 mg/day would likely be tolerated in humans.

Example 7. Auranofin inhibits M. tuberculosis mycothione reductase in vitro.

[073] Mtb mycothiol reductase from H37Ra (100% identity to Rv2855c from H37Rv) was cloned into the pET28a expression vector (EMD Millipore) and electroporated into E . coli BL21(DE3)pLysS cells. Cultures were grown at 37°C in LB medium supplemented with 100 mg/ml kanamycin. Protein expression was induced with 500 mM isopropyl-b-D-thiogalactopyranoside (IPTG) and cells were grown overnight at 16°C. Cultures were then harvested by centrifugation (4000 g, 4°C, 10 min). The bacterial pellet was resuspended in buffer (50 mM Tris-HCl, 150

mM NaCl, 1 mM dithiotreitol (DTT), 10 mM imidazole, 0 .1% Triton X-100 v/v, pH 7.5) and lysed by passing through a M-l 10P microfluidizer at 4°C (Microfluidics) and then centrifuged at 8000 g,

4°C, 45 min. The cleared lysate was incubated with washed Ni-NTA agarose beads (500 µΐ per 10 ml of lysate) for one hour while shaking at 4°C. After applying the beads to a polypropylene column, non-specifically bound proteins were removed by washing (50 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 20 mM imidazole, 0.1% Triton X-100 v/v, pH 7.5). The protein was eluted in 50 mM

Tris-HCl, 150 mM NaCl, 1 mM DTT, 200 mM imidazole, 0 .1% Triton X-100 v/v, pH 7.5. Fractions with the highest protein content were pooled and the His-tag and was removed by incubation with PreScission protease (GE Healthcare). The enzyme was further purified by Ni-NTA chromatography and concentrated using an Amicon Ultra 15, 30 kDa (EMD Millipore).

[074] Mycothione reductase activity assays were performed in a total volume of 50 µΐ in black clear-bottom 384-well plates at 30°C. Standard reaction mixes contain 500 µΜ MSSM and 500 µΜ NADPH unless otherwise noted, in 50 mM HEPES, pH 8.0, with 2 mM EDTA. Enzyme activity was monitored in kinetic mode on a Spectramax M5 plate reader by measuring loss of absorbance at 340 nm. [075] Mycothione reductase (Mtr) was cloned from M. tuberculosis strain H37Ra and heterologously expressed in E. coli BL21(DE3)pLysS (Promega). Mtr was preincubated with NADPH and varying concentrations auranofin. Reactions were initiated by the addition of mycothione and activity was monitored by measuring a decrease in absorbance at 340 nm.

[076] Results show that auranofin inhibits inhibits M. tuberculosis mycothione reductase in vitro (see FIG. 6)

Example 8. Cloning, expression and purification of thioredoxin reductase (TrxR) and thioredoxin (Trx).

[077] TrxB2 and TrxC were cloned from M. tuberculosis strain H37Ra and TrxB was cloned from S. aureus NCTC8325. All three were cloned into the pET28a expression vector and purified via Ni- NTA chromatography.

[078] Mycobacterium tuberculosis thioredoxin reductase (Rv3913) was cloned into the pET28a expression vector (EMD Millipore, Billerica, MA) and electroporated into E. coli BL21(DE3)pLysS cells. Cultures were grown at 37°C in LB medium supplemented with 100 mg/ml kanamycin. Protein expression was induced with 500 mM isopropyl-b-D-thiogalactopyranoside (IPTG) and cells were grown overnight at 16°C. Cultures were then harvested by centrifugation (4000 g, 4°C, 10 min). The bacterial pellet was resuspended in lysis buffer (50 mM Tris-HCl, 150 mMNaCl, 1 mM dithiotreitol (DTT), 10 mM Imidazole, 0 .1% Triton X-100 v/v, pH 7.5) and lysed by passing through a M-l 10P microfluidizer at 4°C (Microfluidics, Westwood, MA) and then spun down at 8000 g, 4°C, 45 min.

The cleared lysate was incubated with washed Ni-NTA agarose beads (500 µΐ per 10 ml of lysate) for one hour while shaking at 4°C. After applying the beads to a polypropylene column, non-specifically bound proteins were removed by washing (50 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 20 mM Imidazole, 0.1% Triton X-100 v/v, pH 7.5). The protein was eluted in 50 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 200 mM Imidazole, 0.1% Triton X-100 v/v, pH 7.5. Fractions with the highest protein content were pooled and the His-tag and was removed by incubation with PreScission protease (GE Healthcare, Pittsburgh, PA). The enzyme was further purified by Ni-NTA chromatography and concentrated using an Amicon Ultra 15, 30 kDa (EMD Millipore).

[079] Thioredoxin reductase activity assays were performed in a total volume of 50 µΐ in black clear-bottom 384-well plates at 30°C. Standard reaction mixes contain 100 µΜ 5,5'-dithiobis- (2nitrobenzoic acid) (DTNB), 20 µΜ NADPH, and 10 µΜ thioredoxin unless otherwise noted, in 50 mM HEPES pH 7.5 with 2 mM EDTA. Absorbance at 412 nm was read on a Spectramax M5 plate reader as an indication of enzyme activity.

Example 9. Auranofin toxicity on HepG2 cells. [080] Auranofin cytotoxicity was assayed against HepG2 cells for 48 hr at varying concentrations. Viability was measured using CellTiter-Glo. The CC50 for auranofin was 4.3 µΜ . FIG. 7 shows auranofin can inhibit M . tuberculosis mycothione reductase in vitro. CLAIMS

What is claimed is:

1. A method of treating a bacterial infection in a subject, comprising administering a metal thiolate complex, wherein the metal thiolate complex is cytotoxic to a bacteria.

2. The method of claim 1, wherein the bacteria is a gram-positive bacteria. 3. The method of claim 2, wherein the gram-positive bacteria is of a Mycobacterium genus. 4. The method of claim 2, wherein the gram-positive bacteria is of a Bacillus genus. 5. The method of claim 2, wherein the gram-positive bacteria is of an Enterococcus genus. 6. The method of claim 2, wherein the gram-positive bacteria is Mycobacterium tuberculosis. 7. The method of claim 2, wherein the gram-positive bacteria is Bacillus subtilis. 8. The method of claim 2, wherein the gram-positive bacteria is selected from Enterococcus faecium and Enterococcusfaecalis. 9. The method of claim 2, wherein the gram-positive bacteria is Staphylococcus aureus. 10. The method of claim 9, wherein the Staphylococcus aureus is resistant to a drug. 11. The method of claim 10, wherein the drug is an antibiotic agent selected from vancomycin and linezolid. 12. The method of claim 10, wherein the drug is methicillin.

13. The method of claim 1, wherein the bacteria expresses a thioredoxin and a thioredoxin reductase. 14. The method of claim 13, wherein the bacteria is Staphylococcus aureus and the thioredoxin is TrxC. 15. The method of claim 13, wherein the bacteria is Mycobacterium tuberculosis and the thioredoxin reductase is TrxB2.

16. The method of claim 1, wherein the bacteria does not express a glutathione reductase.

17. The method of claim 1, wherein the bacteria is a dormant bacteria.

18. The method of claim 1, wherein the metal thiolate complex is a gold salt.

19. The method of claim 1, wherein the metal thiolate complex is a gold complex.

20. The method of claim 1, wherein the metal thiolate complex is an organogold compound.

21. The method of claim 1, wherein the metal thiolate complex is selected from auranofin, aurothioglucose, disodium aurothiomalate, sodium aurothiosulfate, sodium aurothiomalate and pharmaceutically acceptable derivatives thereof.

22. The method of claim 1, wherein the metal thiolate complex is selected from auranofin and pharmaceutically acceptable derivatives thereof.

23. The method of claim 1, further comprising administering an agent selected from paraquat, diamide, rifampicin, and a combination thereof. 24. A method of inhibiting a thioredoxin reductase or substrate thereof in a bacteria comprising contacting the bacteria with a metal thiolate complex. 25. The method of claim 24, wherein the contacting comprises administering the metal thiolate complex to a subject infected with the bacteria. 26. The method of claim 24, wherein the bacteria does not express a glutathione reductase. 27. The method of claim 24, wherein the metal thiolate complex comprises a compound selected from auranofin and pharmaceutically acceptable derivatives thereof. 28. The method of claim 24, wherein the thioredoxin reductase is a first enzyme and the method further comprises administering a therapeutic agent that inhibits at least one additional enzyme. 29. The method of claim 28, wherein the at least one additional enzyme comprises an enzyme bearing a reactive cysteine residue. 30. The method of claim 28, wherein the at least one additional enzyme is selected from a cysteine protease and a sulfurtransferase. 31. The method of claim 28, wherein the at least one additional enzyme is an oxidoreductase. 32. The method of claim 31, wherein the oxidoreductase is an NADPH-dependent oxidoreductase. 33. The method of claim 31, wherein the oxidoreductase is a mycothione reductase. 34. The method of claim 24, wherein the bacteria is a gram-positive bacteria. 35. The method of claim 34, wherein the gram-positive bacteria is selected from Mycobacterium tuberculosis and Bacillus subtilis. 36. The method of claim 34, wherein the gram-positive bacteria is a drug-resistant strain of bacteria. 37. The method of claim 36, wherein the drug-resistant strain of bacteria is a strain of bacteria selected from Enterococcusfaecium and Enterococcusfaecalis. 38. The method of claim 36, wherein the drug-resistant strain of bacteria is a strain of Staphylococcus aureus. 39. The method of claim 38, wherein the strain of Staphylococcus aureus is resistant to an antibiotic agent selected from methicillin, vancomycin, and linezolid. 40. The method of claim 24, wherein the metal thiolate complex is a gold salt. 41. The method of claim 24, wherein the metal thiolate complex is a gold complex. 42. The method of claim 24, wherein the metal thiolate complex is an organogold compound. 43. The method of claim 24, wherein the metal thiolate complex is selected from auranofin, aurothioglucose, disodium aurothiomalate, sodium aurothiosulfate, sodium aurothiomalate and pharmaceutically acceptable derivatives thereof. 44. The method of claim 24, wherein the metal thiolate complex is selected from auranofin and pharmaceutically acceptable derivatives thereof. 45. A method for preventing a bacterial infection in a subject, comprising administering a metal thiolate complex. 46. The method of claim 45, wherein the bacterial infection is an infection with a gram-positive bacterium. 47. The method of claim 45, wherein the bacterial infection is an infection with a bacterium that expresses thioredoxin reductase and does not express glutathione reductase. 48. The method of claim 45, wherein the metal thiolate complex is a gold salt. 49. The method of claim 45, wherein the metal thiolate complex is a gold complex. 50. The method of claim 45, wherein the metal thiolate complex is an organogold compound. 51. The method of claim 45, wherein the metal thiolate complex is selected from auranofin, aurothioglucose, disodium aurothiomalate, sodium aurothiosulfate, sodium aurothiomalate and pharmaceutically acceptable derivatives thereof. 52. The method of claim 45, wherein the metal thiolate complex is selected from auranofin and pharmaceutically acceptable derivatives thereof. 53. The method of claim 45, further comprising administering an agent selected from paraquat, diamide, rifampicin, and a combination thereof. 54. A method of treating tuberculosis in a subject comprising administering a therapeutic agent to the subject, wherein the therapeutic agent modulates an activity of a thioredoxin reductase or a substrate thereof. 55. The method of claim 54, wherein the thioredoxin reductase is TrxB2. 56. The method of claim 54, wherein the therapeutic agent inhibits the activity of the thioredoxin reductase. 57. A method of treating a Staphylococcus aureus infection in a subject comprising administering a therapeutic agent to the subject, wherein the therapeutic agent modulates a thioredoxin reductase or substrate thereof. 58. The method of claim 57, wherein the thioredoxin reductase is TrxC. 59. A composition comprising auranofin and a therapeutic agent. 60. The composition of claim 59, wherein the therapeutic agent is selected from an antibiotic agent and an anti-inflammatory agent. 61. The composition of claim 59, wherein the therapeutic agent is a thiol oxidizing agent. 62. The composition of claim 61, wherein the thiol oxidizing agent is diamide. 63. The composition of claim 59, wherein the therapeutic agent is paraquat. 64. The composition of claim 60, wherein the antibiotic agent is rifampicin.

INTERNATIONAL SEARCH REPORT International application No.

PCT/US2016/015409

A . CLASSIFICATION O F SUBJECT MATTER IPC(8) - A61K 31/28; A61K 31/70; A61K 31/7135; A61K 39/04; A61 P 31/04; A61 P 31/06 (2016.01) CPC - A61K 31/70; A61K 31/715; A61K 31/7004; C07H 1 1/04; C07H 23/00 (2016.05) According to International Patent Classification (IPC) o r to both national classification and IPC

B . FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols) IPC(8) - A61K 31/28; A61K 31/70; A61K 31/7135; A61K 39/04; A61 P 31/04; A61 P 31/06 (2016.01 ) CPC - A61K 31/70; A61K 31/715; A61K 31/7004; C07H 1 1/04; C07H 23/00 (2016.05)

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched IPC(8) - A61K 31/28; A61K 31/70; A61K 31/7135; A61K 39/04; A61 P 31/04; A61P 31/06 (2016.01 ); CPC - A61K 31/70; A61K 31/715; A61K 31/7004; C07H 11/04; C07H 23/00 (2016.05) ;USPC - 514/4.1 , 17.1, 2 3 (keyword delimited)

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)

PatBase, STN, Google Patents, Google Scholar Search terms used: thioredoxin reductase anti-inflammatory combinations auranofin tuberculosis

C . DOCUMENTS CONSIDERED T O B E RELEVANT

Category* Citation o f document, with indication, where appropriate, of the relevant passages Relevant to claim No.

U S 201 1/0104308 A 1 (STAMLER e t al) 0 5 May 201 1 (05.05.201 1) entire document 5 9

CASSETTA et al. Drug repositioning: auranofin a s a prospective antimicrobial agent for the 1-3, 6-13, 15, 18-22, 24, treatment of severe staphylococcal infections. Biometals 27(4): 787-791, 2014. [retrieved o n 02 25, 27-35, 40-56, 59-64 March 2016]. Retrieved from the Internet. entire document

U S 2009/0005422 A 1 (HOLMGREN e t al) 0 1 January 2009 (01 .01.2009) entire document 1-3, 6-13, 15, 18-22, 24, 25, 27-35, 40-56, 59-64

P, X HARBUT et al. Auranofin exerts broad-spectrum bactericidal activities by targeting thiol-redox 1-3, 6-13, 15, 18-22, 24, homeostasis. PNAS 112(14): 4453-4458, 2015. [retrieved on 0 6 May 2016]. Retrieved from the 25, 27-35, 40-56, 59-64 Internet. entire document

□ Further documents are listed in the continuation of Box C . □ See patent family annex.

Special categories of cited documents: "T" later document published after the international filing date or priority A " document defining the general state of the art which is not considered date and not in conflict with the application but cited to understand to be of particular relevance the principle or theory underlying the invention E" earlier application or patent but published on or after the international "X" document of particular relevance; the claimed invention cannot be filing date considered novel or cannot be considered to involve an inventive L" document which may throw doubts on priority claim(s) or which is step when the document is taken alone cited to establish the publication date of another citation or other "Y" document of particular relevance; the claimed invention cannot be special reason (as specified) considered to involve an inventive step when the document is O" document referring to an oral disclosure, use, exhibition or other combined with one or more other such documents, such combination means being obvious to a person skilled in the art

P" document published prior to the international filing date but later than "&" document member of the same patent family the priority date claimed

Date of the actual completion o f the international search Date of mailing of the international search report

06 May 2016 0 JU 2016

Name and mailing address o f the ISA/ Authorized officer Mail Stop PCT, Attn: ISA/US, Commissioner for Patents Blaine R . Copenheaver P.O. Box 1450, Alexandria, V A 22313-1450 Facsimile No. 571-273-8300

Form PCT/ISA/210 (second sheet) (January 201 5) INTERNATIONAL SEARCH REPORT International application No. PCT/US2016/015409

Box No. II Observations where certain claims were found unsearchable (Continuation of item 2 of first sheet)

This international search report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons:

Claims Nos.: because they relate to subject matter not required to be searched by this Authority, namely:

□ Claims Nos,; because they relate to parts of the international application that do not comply with the prescribed requirements to such an extent that no meaningful international search can be carried out, specifically:

Claims Nos.: because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a).

Box No. I Observations where unity of invention is lacking (Continuation of item 3 of first sheet)

This International Searching Authority found multiple inventions in this international application, as follows:

See Extra Sheet

As all required additional search fees were timely paid by the applicant, this international search report covers all searchable claims.

As all searchable claims could be searched without effort justifying additional fees, this Authority did not invite payment of additional fees.

□ As only some of the required additional search fees were timely paid by the applicant, this international search report covers only those claims for which fees were paid, specifically claims Nos..

4. No required additional search fees were timely paid by the applicant. Consequently, this international search report is restricted to the invention first mentioned in the claims; it is covered by claims Nos.: 1-3, 6, 13, 15, 18-22, 24, 25, 27-35, 40-56, 59-64

The additional search fees were accompanied by the applicant's protest and, where applicable, the payment of a protest fee. The additional search fees were accompanied by the applicant's protest but the applicable protest fee was not paid within the time limit specified in the invitation. No protest accompanied the payment of additional search fees.

Form PCT/lSA/2 10 (continuation of first sheet (2)) (January 201 5) INTERNATIONALSEARCH REPORT International application No.

PCT/US2016/015409

Continued from Box No. Ill Observations where unity of invention is lacking

Claims 1-3, 6, 13, 15, 18-22, 24, 25, 27-35, 40-56, and 59-64 have been analyzed subject to the restriction that the claims read on the methods and compositions as described in the Lack of Unity of Invention (See Box IV). The claims are restricted to a method of treating a bacterial infection in a subject, comprising administering a metal thiolate complex, wherein the metal thiolate complex is cytotoxic to a bacteria, wherein the bacteria is a gram-positive bacteria, wherein the gram-positive bacteria is of a Mycobacterium genus, where the gram-positive bacteria is Mycobacterium tuberculosis; wherein the metal thiolate is a gold salt, wherein the gold salt is a gold complex, wherein metal thiolate complex is an organogold compound, wherein the metal thiolate complex is auranofin.

This application contains the following inventions or groups of inventions which are not so linked as to form a single general inventive concept under PCT Rule 13.1 . In order for all inventions to be examined, the appropriate additional examination fees need to be paid.

Group l+: claims 1-64 are drawn to methods and compositions.

The first invention of Group l+ is restricted to a method of treating a bacterial infection in a subject, comprising administering a metal thiolate complex, wherein the metal thiolate complex is cytotoxic to a bacteria, wherein the bacteria is a gram-positive bacteria, wherein the gram-positive bacteria is of a Mycobacterium genus, where the gram-positive bacteria is Mycobacterium tuberculosis; wherein the metal thiolate is a gold salt, wherein the gold salt is a gold complex, wherein metal thiolate complex is an organogold compound, wherein the metal thiolate complex is auranofin; and compositions thereof. It is believed that claims 1-3, 6, 13, 15, 18-22, 24, 25, 27-35, 40-56, and 59-64 read on this first named invention and thus these claims will be searched without fee to the extent that they read on the above embodiment.

Applicant is invited to elect additional formula(e) for each additional compound to be searched in a specific combination by paying an additional fee for each set of election. Each additional elected formula(e) requires the selection of a single definition for each compound variable. An exemplary election would be a method of treating a bacterial infection in a subject, comprising administering a metal thiolate complex, wherein the metal thiolate complex is cytotoxic to a bacteria, wherein the bacteria a gram-positive bacteria, wherein the gram-positive bacteria is of a Bacillus genus, where the gram-positive bacteria is Bacillus subtilis; wherein the metal thiolate is a gold salt, wherein the gold salt is a gold complex, wherein metal thiolate complex is an organogold compound, wherein the metal thiolate complex is auranofin; and compositions thereof. Additional formula(e) will be searched upon the payment of additional fees. Applicants must specify the claims that read on any additional elected inventions. Applicants must further indicate, if applicable, the claims which read on the first named invention if different than what was indicated above for this group. Failure to clearly identify how any paid additional invention fees are to be applied to the "+" group(s) will result in only the first claimed invention to be searched/examined.

The inventions listed in Groups l+ do not relate to a single general inventive concept under PCT Rule 13.1, because under PCT Rule 13.2 they lack the same or corresponding special technical features for the following reasons:

The Groups l+ formulae do not share a significant structural element requiring the selection of alternatives for the metal thiolate complex and bacteria.

The Groups l+ share the technical features of a method of treating a bacterial infection in a subject, comprising administering a metal thiolate complex, wherein the metal thiolate complex is cytotoxic to a bacteria; a method of inhibiting a thioredoxin reductase or substrate thereof in a bacteria comprising contacting the bacteria with a metal thiolate complex; a method for preventing a bacterial infection in a subject, comprising administering a metal thiolate complex; a method of treating tuberculosis in a subject comprising administering a therapeutic agent to the subject, wherein the therapeutic agent modulates an activity of a thioredoxin reductase or a substrate thereof; a method of treating a Staphylococcus aureus infection in a subject comprising administering a therapeutic agent to the subject, wherein the therapeutic agent modulates a thioredoxin reductase or substrate thereof; and a composition comprising auranofin and a therapeutic agent. However, these shared technical features do not represent a contribution over the prior art.

Specifically, "Drug repositioning: auranofin as a prospective antimicrobial agent for the treatment of severe staphylococcal infections" to Cassetta et al. teach a method of treating a bacterial infection in a subject, comprising administering a metal thiolate complex, wherein the metal thiolate complex is cytotoxic to a bacteria (See Pg. 1, Abstract, Auranofin, (AF), a gold(l) complex in clinical use for the therapy of rheumatoid arthritis, is reported here to produce remarkable bactericidal effects in vitro against Staphylococcus sp. Noticeably, a similar antimicrobial action and potency are also noticed toward a few methicillin-resistant Staphylococcus aureus strains).

Additionally, US 201 1/0104308 A 1 to Stamler et al. teach a method of inhibiting a thioredoxin reductase or substrate thereof in a bacteria (See Para. [001 1], Examples of diseases treatable in the first embodiment herein are... infection, e.g. a bacterial...) comprising contacting the bacteria with a metal thiolate complex (See Para. [0014], A therapeutically effective amount of Trx reductase inhibitor is an amount that causes amelioration of symptoms and/or pathology of the disease being treated in the first embodiment herein. For auranofin, an oral dose is 6 mg once a day or 3 mg twice a day and an injection dose is 20-55 mg once a week.);a method for preventing a bacterial infection in a subject (See Para. [001 1], Examples of diseases treatable in the first embodiment herein are... infection, e.g. a bacterial...), comprising administering a metal thiolate complex (See Para. [0014], A therapeutically effective amount of Trx reductase inhibitor is an amount that causes amelioration of symptoms and/or pathology of the disease being treated in the first embodiment herein. For auranofin, an oral dose is 6 mg once a day or 3 mg twice a day and an injection dose is 20-55 mg once a week.); and a composition comprising auranofin and a therapeutic agent (See Para. [0038]).

Form PCT/ISA/2 10 (extra sheet) (January 2015) INTERNATIONAL SEARCH REPORT International application No. PCT/US2016/015409

Additionally, US 2009/0005422 A 1 to Holmgren et al. teach a method of treating tuberculosis in a subject (See Abstract; Claim 4) comprising administering a therapeutic agent to the subject (Claim 10), wherein the therapeutic agent modulates an activity of a thioredoxin reductase or a substrate thereof (See Paras. [0045] and [0046], Paras. [0070] and [0071]; Claim 7, A method of prophylaxing an animal or human against infection with a bacteria lacking glutathione... administering... ebeselen...; Claim 0, A method of treating a mammal infected with a prokaryote, comprising administering at least one prokaryotic thioredoxin reductase inhibitor...); and a method of treating a Staphylococcus aureus infection in a subject (See Abstract; Claim 4; Para. [0008]) comprising administering a therapeutic agent to the subject (Claim 10), wherein the therapeutic agent modulates a thioredoxin reductase or substrate thereof (See Para. [0008], The growth of methicillin resistant Staphylococcus aureus was shown to be inhibited by 0.20 ug per ml of ebselen. ..;Paras. [0070] and [0071]; Claim 7, A method of prophylaxing an animal or human against infection with a bacteria lacking glutathione. ..administering... ebeselen...; Claim 10, A method of treating a mammal infected with a prokaryote, comprising administering at least one prokaryotic thioredoxin reductase inhibitor...).

The inventions listed in Groups l+ therefore lack unity under Rule 13 because they do not share a same or corresponding special technical feature.

Form PCT/ISA/210 (extra sheet) (January 2015)