WHAT IS CLAIMED IS: 1. A compound of the formula (IA) or (IIA) or a pharmaceutically acceptable salt thereof, wherein X is O or S; R1 is halo, C1-C8 alkyl, C6-C10 aryl, 5- to 12-membered heteroaryl, phenoxy, benzyl, - C(O)RA, -ORA,-SRA or -NHRA, or one or more R1 in combination with the atoms to which each is attached combine to form a C9-C14 aryl or C9-C12 bicyclic heteroaryl, wherein each hydrogen atom in C6-C10 aryl, 5- to 12-membered heteroaryl, phenoxy, benzyl, C1-C8 alkyl, -C(O)C2-C6 alkenylene-phenyl, C9-C12 aryl and C9-C12 bicyclic heteroaryl is optionally substituted by halo, cyano, or -NO2; R2 is C6-C10 aryl or 5- to 12-membered heteroaryl, wherein each hydrogen atom in C6- C10 aryl and 5- to 12-membered heteroaryl is optionally substituted by halo, hydroxy, or -NO2; R11 is nitro or N(H)COR3; R12 is H or C(O)C1-C6 alkyl; R13 is H or C1-C6 alkyl; R14 is H, C6-C10 aryl, or 5 to 12-membered heteroaryl, wherein each hydrogen atom in C6-C10 aryl and 5 to 12-membered heteroaryl is optionally substituted by halo, 5 to 12- membered heteroaryl, -NHC(O)C1-C6 alkyl, C1-C6 alkoxy, -C(O)C1-C6 alkoxy, -RA, -ORA, - SRA, -C(O)RA or -NHRA; each RA is individually C1-C6 alkyl, C6-C10 aryl, C2-C6 alkenyl-C6-C10 aryl, -C(O)C1-C6 alkyl, C1-C6 alkoxy, or 5 to 12-membered heteroaryl, wherein each hydrogen atom in C1-C6 alkyl, C6-C10 aryl, C2-C6 alkenyl-C6-C10 aryl, -C(O)C1-C6 alkyl, C1-C6 alkoxy, or 5 to 12- membered heteroaryl is optionally substituted by nitro or halo; or two RA combine to form C4- C6 cycloalkyl optionally substituted by phenyl; R3 is C6-C10 aryl, 5 to 12-membered heteroaryl, or C9-C12 bicyclic aryl, wherein each hydrogen atom in C6-C10 aryl, 5 to 12-membered heteroaryl, and C9-C12 bicyclic aryl is optionally substituted by halo, C1-C6 alkyl, or nitro; and n is 0, 1, 2, or 3. 2. A compound of the formula (I) (II) wherein R1 is H, halo, C6-C10 aryl, C5-C10 heteroaryl, substituted C6-C10 aryl, substituted C5-C10 heteroaryl, phenoxy, C1-C8 alkyl, or R1 in combination with the atom to which it is attached forms a C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl, wherein said substituted C6-C10 aryl and substituted C5-C10 heteroaryl comprise 1 to 5 substituents selected from halo and -NO2; R2 is C6-C8 aryl, C5-C8 heteroaryl, substituted C6-C10 aryl, substituted C5-C10 heteroaryl, wherein said substituted C6-C10 aryl and substituted C5-C10 heteroaryl comprise 1 to 5 substituents selected from halo and -NO2; R3 is C6-C8 aryl, C5-C8 heteroaryl, C9-C12 bicyclic aryl, C9-C12 bicyclic heteroaryl, substituted C6-C8 aryl, substituted C5-C8 heteroaryl, substituted C9-C12 bicyclic aryl or substituted C9-C12 bicyclic heteroaryl, wherein said substituted C6-C10 aryl, substituted C5-C10 heteroaryl, substituted C9-C12 bicyclic aryl or substituted C9-C12 bicyclic heteroaryl comprise 1 to 5 substituents selected from halo and -NO2; and R15 and R16 are each independently hydrogen or C1-C6 alkyl; or a pharmaceutically acceptable salt thereof. 3. The compound of claim 1 or 2 having the general structure of formula I: wherein R3 is C5-C8 aryl, C5-C8 heteroaryl, substituted C5-C8 aryl, substituted C5-C8 heteroaryl, wherein said substituted C5-C10 aryl and substituted C5-C10 heteroaryl comprise 1 to 5 substituents selected from halo and -NO2; or a pharmaceutically acceptable salt thereof. 4. The compound of any one of claims 1-3, having the general structure of formula I: or a pharmaceutically acceptable salt thereof, wherein R11 is halo; R12 is -NO2; R16 is C1-C6 alkyl; and R15 is H. 5. The compound of claim 3, wherein R11 is F; and R16 is -CH2CH3. 6. A compound or a pharmaceutically acceptable salt thereof, having the formula of wherein R10 is C5-C8 aryl, C5-C8 heteroaryl, C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl, substituted C5-C8 aryl, substituted C5-C8 heteroaryl, substituted C9-C12 bicyclic aryl or substituted C9-C12 bicyclic heteroaryl, wherein said substituted C5-C10 aryl, substituted C5-C10 heteroaryl, substituted C9-C12 bicyclic aryl or substituted C9-C12 bicyclic heteroaryl comprise 1 to 5 substituents selected from halo, phenoxy, -NR5R6, -OR5, -S(O)2NR5R6, or –NHSO2R7; R5 and R6 are each independently hydrogen, C1-C6 alkyl, C6-C10 aryl, bicyclic C6-C10 heteroaryl, substituted C1-C6 alkyl, substituted C6-C10 aryl, or substituted bicyclic C6-C10 heteroaryl, wherein said substituted C5-C10 aryl, substituted C5-C10 heteroaryl and or substituted bicyclic C6-C10 heteroaryl comprise 1 to 5 substituents selected from halo, -NO2 and –OC1-C6 alkyl; and R7 is C5-C8 aryl, C5-C8 heteroaryl, substituted C5-C8 aryl, substituted C5-C8 heteroaryl, wherein said substituted C5-C10 aryl and substituted C5-C10 heteroaryl comprise 1 to 5 substituents selected from halo and -NO2; or a pharmaceutically acceptable salt thereof 7. The compound of claim 6, wherein R10 is C5-C8 aryl, C5-C8 heteroaryl, substituted C5-C8 aryl, substituted C5-C8 heteroaryl, wherein said substituted C5-C10 aryl, substituted C5-C10 heteroaryl, comprise 1 to 5 substituents selected from halo, phenoxy, -NR5R6, and -OR5; and R5 and R6 are each independently hydrogen or -CH3. 8 The compound of claim 1 or 2, wherein said compound is Compound 49 Compound 50. 9. A pharmaceutical comprising a compound of any one of claims 1-8 and a pharmaceutically acceptable carrier. 10. A method of inhibiting the GroEL/ES and HSP60/10 chaperonin system in a microorganism said method comprising contacting said microorganism with the pharmaceutical composition of claim 8. 11. A method of inhibiting the growth of Trypanosoma brucei and/or treating a Trypanosoma brucei infection, said method comprising administering compound of Compound 49 Compound 50. to a subject in need of treatment. 12. A method of inhibiting the growth of Trypanosoma brucei and/or treating a Trypanosoma brucei infection, said method comprising administering a therapeutically effective amount of a compound of any one of claims 1-8 to a subject in need of treatment. |
or a pharmaceutically acceptable salt thereof. In certain embodiments, X is O or S. In some embodiments X is O. In some embodiments X is S. In some embodiments, n is 0, 1, 2, or 3. In some embodiments, n is 0 and the phenyl ring is unsubstituted. In some embodiments, n is 1. In certain embodiments when n is 1, R 1 is optionally substituted phenoxy. In some embodiments, n is 2. In some embodiments, n is 2 and the two R 1 in combination with the atoms to which each is attached combine to form a C 9 - C 14 aryl (e.g., ) or C 9 -C 12 bicyclic heteroaryl, each of which may be optionally substituted, for example by a heteroaryl. In some embodiments, R 1 is halo, C 1 -C 8 alkyl, C 6 -C 10 aryl, 5- to 12-membered heteroaryl, phenoxy, benzyl, -C(O)R A , -OR A ,-SR A or -NHR A . In some embodiments, one or more R 1 in combination with the atoms to which each is attached combine to form a C 9 -C 14 aryl or C9-C12 bicyclic heteroaryl. In certain embodiments, each hydrogen atom in C6-C10 aryl, 5- to 12-membered heteroaryl, phenoxy, benzyl, C 1 -C 8 alkyl, -C(O)C 2 -C 6 alkenylene-phenyl, C 9 -C 12 aryl and C9-C12 bicyclic heteroaryl is optionally substituted by halo, cyano, or -NO2. In some embodiments, R 2 is C 6 -C 10 aryl or 5- to 12-membered heteroaryl, wherein each hydrogen atom in C6-C10 aryl and 5- to 12-membered heteroaryl is optionally substituted by halo, hydroxy, or -NO 2 . For example R 2 may be phenyl optionally substituted by alkoxy, ester, halo such as chloro, etc. In some embodiments, R 2 is 5- to 12-membered heteroaryl, for example , each of which may be optionally substituted. In some embodiments, R 11 is nitro or -N(H)COR 3 . In some embodiments, R 12 is H or -C(O)C1-C6 alkyl (e.g, -C(O)ethyl). In some embodiments, R 13 is H or C 1 -C 6 alkyl (e.g., methyl). In some embodiments, R 14 is H, C6-C10 aryl (e.g., phenyl), or 5 to 12-membered heteroaryl, wherein each hydrogen atom in C 6 -C 10 aryl and 5 to 12-membered heteroaryl is optionally substituted by halo, 5 to 12-membered heteroaryl, -NHC(O)C1-C6 alkyl, C1-C6 alkoxy, -C(O)C 1 -C 6 alkoxy, -R A , -OR A , -SR A , -C(O)R A or -NHR A . In some embodiments, each R A is C1-C6 alkyl (e.g., methyl), C6-C10 aryl, C2-C6 alkenyl- C 6 -C 10 aryl, -C(O)C 1 -C 6 alkyl, C 1 -C 6 alkoxy (e.g., methoxy), or 5 to 12-membered heteroaryl, wherein each hydrogen atom in C1-C6 alkyl, C6-C10 aryl, C2-C6 alkenyl-C6-C10 aryl, -C(O)C1-C6 alkyl, C 1 -C 6 alkoxy, or 5 to 12-membered heteroaryl is optionally substituted by nitro or halo; or two R A combine to form C4-C6 cycloalkyl optionally substituted by phenyl, for example if R 14 is phenyl, two R A can combine with the phenyl to form . In some embodiments, R 3 is C 6 -C 10 aryl, 5 to 12-membered heteroaryl, or C 9 -C 12 bicyclic aryl, wherein each hydrogen atom in C6-C10 aryl, 5 to 12-membered heteroaryl, and C9- C 12 bicyclic aryl is optionally substituted by halo, C 1 -C 6 alkyl, or nitro. Initial screening of a small library of 960 compounds received from the Medicines for Malaria Venture (MMV) foundation, identified 37 initial GroEL inhibitor hits. These initial 37 compounds served as a basis for the development 14 analogs of compound 23, having the structure: . Compounds were screened for killing of E. coli, the ESKAPE bacteria, M. tuberculosis, and T. brucei parasites in liquid culture. In accordance with one embodiment the compounds of the present disclosure have utility as inhibitors of M. tuberculosis and T. brucei parasites. In accordance with one embodiment a composition comprising any of analogs 39-52 of Table 1, and in one embodiment compounds 49 and 50, are used as potent and selective inhibitors against T. brucei. Compounds 49 and 50 and functional derivatives thereof have more potent and selective activity against T. brucei than Suramin and Nifurtimox - the first line treatments used for treating HAT. These compounds are anticipated to have additional applications against other infectious organisms. Developing analogs of compound 23 Compound 23 was chosen as the model for analogs because of its potency inhibiting actively-replicating M. tuberculosis and GroEL folding functions. Additionally, in 2013, Wilson et al. found this compound to have whole cell antituberculosis activity, with evidence supporting inhibition of Pks13 – an essential step in the process of synthesizing mycolic acids, a part of the M. tuberculosis cell wall that is imperative in the pathogenesis of the bacteria. Analogs were prepared in which the left-hand side of the molecule was retained along with the amide linker and the aryl on the right side of the compound was varied in an effort to modulate the reactivity – with the exception of 38 (the parent amine starting material) and 51 and 52 (both contained unsubstituted phenyl rings). Analog 49 included a para-nitrofluoro ring in an effort to increase the efficiency of the nucleophilic aromatic substitution reaction with GroEL cysteines. Analog 50 included a nitrofuran group as we previously reported this to be efficacious in GroEL-targeting antibacterials due to its ability to act as a prodrug and become activated via nitroreductases. The general synthesis of these analogs is presented in Figure 1, which resulted in the generation of 15 additional analogs for testing (see Table 1 for the structures of the different analogs).
Table 1: Dose-response results for the compound 23 analogs tested in the GroEL/ES-mediated refolding assays and native reporter enzyme counter-screens. Biochemical Assay IC 50 (µM) Overall, the compound 23 analogs were less potent at inhibiting E. coli GroEL refolding functions (Table 1), with the exception of 49, which was ~2-fold more potent than 23. Of particular significance was IC 50 values for these analogs exhibited high correlations between the two GroEL-mediated client protein refolding assays and were inactive in the native MDH and Rho reporter counter-screens (with the exception of compound 50 in the native Rho counter- screen), supporting that inhibitors were functioning against the GroEL/ES-mediated refolding cycle and were not false-positives of the MDH and Rho reporter enzymes. Compound 50 was also tested with the E. coli NfsB nitroreductase, resulting in a GroEL/ES-dMDH refolding IC50 value of 13 uM – slightly more potent than when tested without the NfsB nitroreductase (17 uM IC 50 ), suggesting it may act through a pro-drug mechanism. The compound 23 analogs were all tested in the T. brucei, ESKAPE and Mtb bacterial proliferation assays and human cell viability counter-screens. Most analogs were less toxic against human cells, though compounds 49 and 50 had comparable cytotoxicity to compound 23 (Table 2). Compounds 39 and 50 were exceptionally potent at killing T. brucei parasites with high selectivity compared to cytotoxicity to human colon and intestine cells. While most compounds were inactive against the ESKAPE bacteria, parent compound 23 and nitrofuran analog 50 were particularly potent against M. tuberculosis (EC90 = 4.1 μM and 0.20 μM, respectively), with weak-moderate activity against E. coli, S. aureus, and E. faecium. Further study is needed to determine the mechanism of this inhibition, as compound 50 was largely inactive against the other Gram-negative KAPE bacteria. As nitrofuran analog 50 was potent and selective for killing both T. brucei and M. tuberculosis, a panel of 14 additional nitrofuran-containing amide analogs based on formula II were synthesized as described in Figure 1 and evaluated for their ability to inhibit GroEL/ES- mediated folding functions (in the absence and presence of E. coli NfsB nitroreductase to examine their pro-drug potential via metabolizing the nitrofurans to active metabolites), their efficacy at killing T. brucei parasites, and cytotoxicity to human colon and intestine cells – results are presented in Table 3. All compounds were stronger GroEL/ES inhibitors in the presence of NfsB, suggesting they can function as pro-drugs activated by nitroreductases. All compounds were more potent than Nifurtimox at inhibiting GroEL/ES folding functions and killing T. brucei parasites, with four proving more potent at killing T. brucei parasites than Suramin (54.60, 63, and 65).
Table 2: Dose-response testing of the compound 23 analogs in cell viability assays. Presented are antibiotic efficacy against actively replicating T. brucei (EC 50 ) parasites and M. tuberculosis, E. coli, and the ESKAPE bacteria (EC90) and cytotoxicity (CC50) to human colon (FHC) and intestine (FHs 74Int) cell lines. Table 3: Dose-response results for the nitrofuran analogs tested in the GroEL/ES- mediated refolding assays, native MDH reporter enzyme counter-screen, and T. brucei and human intestine and colon cell viability assays. Results for HAT therapeutics Nifurtimox and Suramin are shown for comparison. GroEL/ES-dMDH refolding results are shown from testing in the absence and presence of E. coli NfsB nitroreductase. In accordance with one embodiment, a compound of the formula (I) (II) is provided wherein R 1 is H, halo, C 6 -C 10 aryl, C 6 -C 10 heteroaryl, phenoxy, C 1 -C 8 alkyl, or R 1 in combination with the atom to which it is attached forms a C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl, wherein one or more of the hydrogen atoms of the R 1 substituents is optionally substituted with halogen, -OR 5 , -NR 5 R 6 , -S(O)2NR 5 R 6 , or –NHSO2R 7 ; R 2 is C 5 -C 8 aryl, C 5 -C 8 heteroaryl, substituted C 5 -C 10 aryl, substituted C 5 -C 10 heteroaryl, wherein said substituted C5-C10 aryl and substituted C5-C10 heteroaryl comprise 1 to 5 substituents selected from halo and -NO 2 ; R 3 is C5-C8 aryl, C5-C8 heteroaryl, a C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl, wherein each of the C 5 -C 8 aryl, C 5 -C 8 heteroaryl, a C 9 -C 12 bicyclic aryl or C 9 -C 12 bicyclic heteroaryl groups further comprises at least one -NO2 and one halo substituent; R 5 and R 6 are each independently hydrogen, C1-C6 alkyl, C6-C10 aryl, or bicyclic C6-C10 heteroaryl, wherein each hydrogen atom in C1-C6 alkyl, aryl, and heteroaryl is optionally substituted with halogen or –OC1-C6 alkyl; R 7 is C5-C6 aryl, or C5-C16 heteroaryl wherein one or more of the hydrogen atoms of the C5-C6 aryl, or C5-C16 heteroaryl is optionally substituted with halo, -OR 5 or -NO2; and R 15 and R 16 are each independently hydrogen or -CH 3 ; or a pharmaceutically acceptable salt thereof, optionally wherein R 5 and R 6 are each independently hydrogen or -CH 3 . In accordance with one embodiment, a compound of the formula (I) (II) is provided wherein R 1 is H, halo, C6-C10 aryl, C6-C10 heteroaryl, phenoxy, C1-C8 alkyl, or R 1 in combination with the atom to which it is attached forms a C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl; R 3 is C5-C6 aryl, C5-C6 heteroaryl, wherein the C5-C6 aryl, and C5-C6 heteroaryl further comprises at least one -NO 2 and one halo substituent; and R 15 and R 16 are each independently hydrogen or -CH3; or a pharmaceutically acceptable salt thereof. In accordance with one embodiment, a compound of the formula (I) (II) is provided wherein R 1 is H; R 3 is R 11 is halo; R 12 is NO2; and R 15 and R 16 are each independently hydrogen or C 1 -C 6 alkyl; or a pharmaceutically acceptable salt thereof, optionally wherein R 15 is F and R 16 is each independently hydrogen or C 1 -C 3 alkyl. In another embodiment, the disclosure relates to a compound or a pharmaceutically acceptable salt thereof, having the formula II wherein R 10 is C5-C8 aryl, C5-C8 heteroaryl, C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl, wherein one or more of the hydrogen atoms of the R 10 substituent is optionally substituted with halo, phenoxy, -OR 5 , -NR 5 R 6 , -S(O)2NR 5 R 6 , CO2(C1-C4 alkyl), -C(O)NH2 or –NHSO2R 7 ; R 5 and R 6 are each independently hydrogen, C 1 -C 6 alkyl, C 6 -C 10 aryl, or bicyclic C 6 -C 10 heteroaryl, wherein each hydrogen atom in C1-C6 alkyl, aryl, and heteroaryl is optionally substituted with halogen or –OC 1 -C 6 alkyl; and R 7 is C5-C6 aryl, or C5-C16 heteroaryl wherein one or more of the hydrogen atoms of the C 5 -C 6 aryl, or C 5 -C 16 heteroaryl is optionally substituted with halo, -OR 5 or nitro; or a pharmaceutically acceptable salt thereof, optionally wherein R 5 and R 6 are each independently hydrogen or -CH 3 . In one embodiment a compound having the general structure of is provided wherein R 1 is halo or -OH; R 15 is H or C 1 -C 6 alkyl; X is S or O, or a pharmaceutically acceptable salt thereof. In accordance with one embodiment a compound having the formula is provided wherein X is S or O, and R 1 is H, halo, C6-C10 aryl, C6-C10 heteroaryl, phenoxy, C1- C 8 alkyl, or R 1 in combination with the atom to which it is attached forms a C 9 -C 12 bicyclic aryl or C9-C12 bicyclic heteroaryl; or a pharmaceutically acceptable salt thereof. In accordance with one embodiment a compound having the formula is provided wherein R 1 is H, halo, C6-C10 aryl, C6-C10 heteroaryl, phenoxy, C1-C8 alkyl, or R 1 in combination with the atom to which it is attached forms a C 9 -C 12 bicyclic aryl or C 9 -C 12 bicyclic heteroaryl; or a pharmaceutically acceptable salt thereof. The compound or a pharmaceutically acceptable salt thereof, selected from the group consisting
A pharmaceutical composition comprising any of the compounds disclosed herein, or a pharmaceutically acceptable salt thereof, and at least one diluent, carrier or excipient. A method of treating a microbial infection comprising administering to a subject in need of such treatment an effective amount of at least one compound as disclosed herein, or a pharmaceutically acceptable salt thereof. In embodiment 1 a compound having the formula: (I) (II) wherein R 1 is H, halo, C5-C10 aryl, C5-C10 heteroaryl, substituted C5-C10 aryl, substituted C5-C10 heteroaryl, phenoxy, C 1 -C 8 alkyl, or R 1 in combination with the atom to which it is attached forms a C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl, wherein said substituted C5-C10 aryl and substituted C 5 -C 10 heteroaryl comprise 1 to 5 substituents selected from halo and -NO 2 ; R 2 is C5-C8 aryl, C5-C8 heteroaryl, substituted C5-C10 aryl, substituted C5-C10 heteroaryl, wherein said substituted C 5 -C 10 aryl and substituted C 5 -C 10 heteroaryl comprise 1 to 5 substituents selected from halo and -NO2; R 3 is C 5 -C 8 aryl, C 5 -C 8 heteroaryl, C 9 -C 12 bicyclic aryl, C 9 -C 12 bicyclic heteroaryl, substituted C5-C8 aryl, substituted C5-C8 heteroaryl, substituted C9-C12 bicyclic aryl or substituted C 9 -C 12 bicyclic heteroaryl, wherein said substituted C 5 -C 10 aryl, substituted C 5 -C 10 heteroaryl, substituted C9-C12 bicyclic aryl or substituted C9-C12 bicyclic heteroaryl comprise 1 to 5 substituents selected from halo and -NO 2 ; R 15 and R 16 are each independently hydrogen or C1-C6 alkyl; or a pharmaceutically acceptable salt thereof, optionally wherein R 5 and R 6 are each independently hydrogen or -CH3 is used to treating a microbial infection, including inhibiting the growth of Mycobacterium tuberculosis or T. brucei and/or treating Mycobacterium tuberculosis T. brucei infection. In embodiment 2, the compound used in embodiment 1 has the general structure of formula I: wherein R 3 is C5-C8 aryl, C5-C8 heteroaryl, substituted C5-C8 aryl, substituted C5-C8 heteroaryl, wherein said substituted C 5 -C 10 aryl and substituted C 5 -C 10 heteroaryl comprise 1 to 5 substituents selected from halo and -NO2; or a pharmaceutically acceptable salt thereof, optionally wherein R 15 and R 16 are each independently hydrogen or -CH3. In embodiment 3, the compound used in embodiment 1 or 2 has the general structure of formula I: wherein R 11 is halo; R 12 is -NO2; R 16 is C1-C6 alkyl; and R 15 is H. In embodiment 4, the compound used in any one of embodiments 1-3 is provided wherein R 16 is -CH2CH3. In embodiment 5, the compound used in embodiment 1 has the structure of formula II (II) wherein R 10 is C5-C8 aryl, C5-C8 heteroaryl, C9-C12 bicyclic aryl or C9-C12 bicyclic heteroaryl, substituted C 5 -C 8 aryl, substituted C 5 -C 8 heteroaryl, substituted C 9 -C 12 bicyclic aryl or substituted C9-C12 bicyclic heteroaryl, wherein said substituted C5-C10 aryl, substituted C5-C10 heteroaryl, substituted C 9 -C 12 bicyclic aryl or substituted C 9 -C 12 bicyclic heteroaryl comprise 1 to 5 substituents selected from halo, phenoxy, -NR 5 R 6 , -OR 5 , -S(O) 2 NR 5 R 6 , or –NHSO 2 R 7 ; R 5 and R 6 are each independently hydrogen, C 1 -C 6 alkyl, C 6 -C 10 aryl, bicyclic C 6 -C 10 heteroaryl, substituted C1-C6 alkyl, substituted C6-C10 aryl, or substituted bicyclic C6-C10 heteroaryl, wherein said substituted C 5 -C 10 aryl, substituted C 5 -C 10 heteroaryl and or substituted bicyclic C6-C10 heteroaryl comprise 1 to 5 substituents selected from halo, -NO2 and –OC1-C6 alkyl; and R 7 is C5-C8 aryl, C5-C8 heteroaryl, substituted C5-C8 aryl, substituted C5-C8 heteroaryl, wherein said substituted C 5 -C 10 aryl and substituted C 5 -C 10 heteroaryl comprise 1 to 5 substituents selected from halo and -NO2; or a pharmaceutically acceptable salt thereof In embodiment 6, the compound used in embodiment 5 is provided wherein R 10 is C5-C8 aryl, C5-C8 heteroaryl, substituted C5-C8 aryl, substituted C5-C8 heteroaryl, wherein said substituted C 5 -C 10 aryl, substituted C 5 -C 10 heteroaryl, comprise 1 to 5 substituents selected from halo, phenoxy, -NR 5 R 6 , and -OR 5 ; and R 5 and R 6 are each independently hydrogen or -CH 3 . In embodiment 7, the compound used in embodiment 5 or 6 is provided wherein said compound is Compound 49 Compound 50. In embodiment 8, a pharmaceutical composition is provided comprising a compound of any one of embodiments 1-7 and a pharmaceutically acceptable carrier. In embodiment 9, a method of inhibiting the GroEL/ES and HSP60/10 chaperonin system in a microorganism is provided wherein said method comprises contacting said microorganism with the pharmaceutical composition of embodiment 8. In embodiment 10, a method of inhibiting the growth of Trypanosoma brucei and/or treating a Trypanosoma brucei infection is provided, said method comprising administering compound of Compound 49 Compound 50. to a subject in need of treatment. EXAMPLE 1 Abbreviations: The examples described herein use materials, including but not limited to, those described by the following abbreviations known to those skilled in the art:
General synthetic methods. Unless otherwise stated, all chemicals were purchased from commercial suppliers and used without further purification. Reaction progress was monitored by thin-layer chromatography on silica gel 60 F254 coated glass plates (EM Sciences). Flash chromatography was performed using a Biotage Isolera One flash chromatography system and eluting through Biotage KP-Sil Zip or Snap silica gel columns for normal-phase separations (hexanes:EtOAc gradients), or Snap KP-C18-HS columns for reverse-phase separations (H2O:MeOH gradients). Reverse-phase high-performance liquid chromatography (RP-HPLC) was performed using a Waters 1525 binary pump, 2489 tunable UV/Vis detector (254 and 280 nm detection), and 2707 autosampler. For preparatory HPLC purification, samples were chromatographically separated using a Waters XSelect CSH C18 OBD prep column (part number 186005422, 130 Å pore size, 5 μm particle size, 19x150 mm), eluting with a H2O:CH3CN gradient solvent system. Linear gradients were run from either 100:0, 80:20, or 60:40 A:B to 0:100 A:B (A = 95:5 H2O:CH3CN, 0.05% TFA; B = 5:95 H2O:CH3CN, 0.05% TFA. Products from normal-phase separations were concentrated directly, and reverse-phase separations were concentrated, diluted with H2O, frozen, and lyophilized. For primary compound purity analyses (HPLC-1), samples were chromatographically separated using a Waters XSelect CSH C18 column (part number 186005282, 130 Å pore size, 5 μm particle size, 3x150 mm), eluting with the above H2O:CH3CN gradient solvent systems. For secondary purity analyses of final test compounds (HPLC-2), samples were chromatographically separated using a Waters XBridge C18 column (either part number 186003027, 130 Å pore size, 3.5 μm particle size, 3x100 mm, or part number 186003132, 130 Å pore size, 5.0 μm particle size, 3x100 mm), eluting with a H2O:MeOH gradient solvent system. Linear gradients were run from either 100:0, 80:20, 60:40, or 20:80 A:B to 0:100 A:B (A = 95:5 H2O:MeOH, 0.05% TFA; B = 5:95 H 2 O:MeOH, 0.05% TFA). All test compounds were found to be >95% in purity from both RP-HPLC analyses, with the exceptions of 3 (90%), 10 (93%), 12 (N/A as the compound has no chromophore), 17 (89%), 22 (86%), 32 (93%), and 37 (94%). Mass spectrometry data were collected using an Agilent analytical LC-MS at the IU Chemical Genomics Core Facility (CGCF). 1 H-NMR and 13 C-NMR spectra were recorded on either Bruker 600 MHz or 300 MHz spectrometers. Chemical shifts are reported in parts per million and calibrated to the d6-DMSO solvent peaks at 2.50 ppm ( 1 H) and 39.51 ppm ( 13 C). The general amide coupling procedure to give parent inhibitor 23 and analogs is presented below, followed by characterization data for each analog. General procedure for the amide coupling to give inhibitor 23 and analogs. To stirring mixtures of either aniline or ethyl 2-amino-5-carbamoyl-4-methylthiophene- 3-carboxylate (1 eq.) in anhydrous acetonitrile were added the respective R-COCl reagents (1 eq.) and Cs 2 CO 3 (1.3 eq.). Note that for any analogs where the R-CO 2 H starting materials were only commercially available, the acids were first converted to the acid chlorides by stirring in thionyl chloride at 60°C for 1 h, then concentrating. The reactions were allowed to stir at room temperature overnight, then diluted with water/DMSO to solubilize, and flash chromatographic purification (reverse-phase with a water:MeOH gradients) afforded the products as solids after lyophilization. If necessary, products were further purified by preparatory RP-HPLC (water:CH 3 CN and/or water:MeOH gradients), concentrated, and lyophilized. Analog characterization data. (38) Ethyl 2-amino-5-carbamoyl-4-methylthiophene-3-carboxylate. Commercially obtained starting material. 1 H NMR (600 MHz, DMSO-d6) δ ppm 1.27 (t, J=7.2 Hz, 3 H) 2.47 (s, 3 H) 4.20 (q, J=7.2 Hz, 2 H) 7.07 (br. s., 2 H) 7.64 (s, 2 H); 13 C-NMR (150 MHz, d 6 -DMSO) δ 14.29, 16.05, 59.18, 105.46, 113.06, 139.82, 164.38, 164.83, 164.99; MS (ESI) C9H11N2O3S [M-H]- m/z expected = 227.1, observed = 226.9. HPLC-1 = 98%, HPLC-2 = >99%. (39) Ethyl 5-carbamoyl-2-(2-fluorobenzamido)-4-methylthiophene-3-carbox ylate. Yield = 22%. 1 H NMR (600 MHz, DMSO-d6) δ ppm 1.34 (t, J=7.2 Hz, 3 H), 2.56 (s, 3 H), 4.36 (q, J=7.0 Hz, 2 H), 7.41-7.60 (m, 4 H), 7.70-7.79 (m, 1 H), 8.06 (t, J=7.5 Hz, 1 H), 12.28 (d, J=10.6 Hz, 1 H); 13 C-NMR (150 MHz, d6-DMSO) δ 14.00, 15.47, 61.01, 114.41, 116.69, 116.85, 118.96, 119.03, 124.05, 125.52, 131.64, 135.44, 135.49, 136.94, 148.05, 159.18, 159.94, 160.84, 164.22, 165.01; MS (ESI) C16H14FN2O4S [M-H]- m/z expected = 349.1, observed = 349.0. HPLC-1 = >99%, HPLC-2 = >99%. (40) Ethyl 5-carbamoyl-2-(4-fluorobenzamido)-4-methylthiophene-3-carbox ylate. Yield = 11%. 1 H NMR (600 MHz, DMSO-d 6 ) δ ppm 1.36 (t, J=7.0 Hz, 3 H), 2.56 (s, 3 H), 4.37 (q, J=7.0 Hz, 2 H), 7.41-7.61 (m, 4 H), 8.00 (dd, J=7.7, 5.5 Hz, 2 H), 12.07 (br. s., 1 H); 13 C-NMR (150 MHz, d6-DMSO) δ 14.01, 15.41, 61.14, 114.36, 116.30, 116.44, 123.72, 128.26, 130.13, 130.20, 136.94, 148.62, 162.36, 164.02, 164.22, 165.48, 165.69; MS (ESI) C 16 H 14 FN 2 O 4 S [M- H]- m/z expected = 349.1, observed = 349.0. HPLC-1 = >99%, HPLC-2 = >99%. (41) Ethyl 5-carbamoyl-2-(2-chlorobenzamido)-4-methylthiophene-3-carbox ylate. Yield = 6%. 1 H NMR (600 MHz, DMSO-d6) δ ppm 1.30 (t, J=7.2 Hz, 3 H), 2.55 (s, 3 H), 4.30 (q, J=7.2 Hz, 2 H), 7.47-7.59 (m, 3 H), 7.59-7.69 (m, 2 H), 7.79 (d, J=7.3 Hz, 1 H), 11.71 (s, 1 H); 13 C-NMR (150 MHz, d 6 -DMSO) δ 13.92, 15.24, 61.02, 115.20, 123.81, 127.82, 130.04, 130.29, 130.51, 132.94, 133.05, 137.05, 146.99, 163.16, 164.13, 164.71; MS (ESI) C16H14ClN2O4S [M- H]- m/z expected = 365.0, observed = 365.0. HPLC-1 = >99%, HPLC-2 = >99%. (42) Ethyl 5-carbamoyl-2-(3-chlorobenzamido)-4-methylthiophene-3-carbox ylate. Yield = 29%. 1 H NMR (600 MHz, DMSO-d 6 ) δ ppm 1.37 (t, J=7.2 Hz, 3 H), 2.56 (s, 3 H), 4.37 (q, J=7.1 Hz, 2 H), 7.52 (br. s., 2 H), 7.64-7.69 (m, 1 H), 7.76-7.80 (m, 1 H), 7.86-7.89 (m, 1 H), 7.93 (t, J=1.8 Hz, 1 H), 12.00 (s, 1 H); 13 C-NMR (150 MHz, d 6 -DMSO) δ 13.98, 15.35, 61.16, 114.86, 123.95, 125.91, 127.21, 131.23, 132.83, 133.82, 133.93, 137.04, 147.99, 162.14, 164.16, 165.17; MS (ESI) C 16 H 14 ClN 2 O 4 S [M-H]- m/z expected = 365.0, observed = 365.0. HPLC-1 = >99%, HPLC-2 = >99%. (43) Ethyl 5-carbamoyl-2-(4-chlorobenzamido)-4-methylthiophene-3-carbox ylate. Yield = 11%. 1 H NMR (600 MHz, DMSO-d 6 ) δ ppm 1.36 (t, J=7.2 Hz, 3 H), 2.56 (s, 3 H), 4.37 (q, J=7.0 Hz, 2 H), 7.52 (br. s., 2 H), 7.71 (d, J=8.1 Hz, 2 H), 7.94 (d, J=8.1 Hz, 2 H), 12.08 (s, 1 H); 13 C-NMR (150 MHz, d 6 -DMSO) δ 14.01, 15.41, 61.16, 114.50, 123.84, 129.19, 129.37, 130.45, 136.96, 137.98, 148.45, 162.40, 164.19, 165.45; MS (ESI) C16H14ClN2O4S [M-H]- m/z expected = 365.0, observed = 365.0. HPLC-1 = >99%, HPLC-2 = >99%. (44) Ethyl 5-carbamoyl-2-(3,4-dichlorobenzamido)-4-methylthiophene-3-ca rboxylate. Yield = 9%. 1 H NMR (600 MHz, DMSO-d6) δ ppm 1.37 (t, J=7.0 Hz, 3 H), 2.56 (s, 3 H), 4.38 (q, J=7.0 Hz, 2 H), 7.53 (br. s., 2 H), 7.88 (dd, J=8.4, 2.2 Hz, 1H), 7.93 (d, J=8.4 Hz, 1 H), 8.13 (d, J=1.8 Hz, 1 H), 11.97 (s, 1 H); 13 C-NMR (150 MHz, d6-DMSO) δ 14.46, 15.82, 61.86, 115.65, 124.61, 127.88, 129.95, 132.02, 132.54, 132.80, 136.32, 137.51, 148.18, 161.97, 164.60, 165.52; MS (ESI) C16H13Cl2N2O4S [M-H]- m/z expected = 399.0, observed = 398.9. HPLC-1 = 97%, HPLC-2 = 96%. (45) Ethyl 5-carbamoyl-4-methyl-2-(3-methylbenzamido)thiophene-3-carbox ylate. Yield = 6%. 1 H NMR (600 MHz, DMSO-d6) δ ppm 1.37 (t, J=67.0 Hz, 3 H), 2.42 (s, 3 H), 2.56 (s, 3 H), 4.37 (q, J=7.1 Hz, 2 H), 7.45-7.57 (m, 4 H), 7.72 (br. s., 1 H), 7.75 (s, 1 H), 12.04 (s, 1 H); 13 C-NMR (150 MHz, d6-DMSO) δ 14.00, 15.42, 20.93, 61.12, 114.18, 123.82, 124.29, 127.76, 129.16, 131.66, 133.75, 137.00, 138.75, 148.67, 163.45, 164.25, 165.39; MS (ESI) C 17 H 17 N 2 O 4 S [M-H]- m/z expected = 345.1, observed = 345.0. HPLC-1 = >99%, HPLC-2 = >99%. (46) Ethyl 5-carbamoyl-4-methyl-2-(4-methylbenzamido)thiophene-3-carbox ylate. Yield = 6%. 1 H NMR (600 MHz, DMSO-d6) δ ppm 1.36 (t, J=7.2 Hz, 3 H), 2.41 (s, 3 H), 2.56 (s, 3 H), 4.37 (q, J=7.2 Hz, 2 H), 7.43 (m, J=8.1 Hz, 2 H), 7.50 (br. s., 2 H), 7.83 (m, J=8.1 Hz, 2 H), 12.07 (s, 1 H); 13 C-NMR (150 MHz, d 6 -DMSO) δ 14.01, 15.44, 21.12, 61.11, 113.9, 123.54, 127.25, 128.80, 129.79, 136.96, 143.55, 148.92, 163.19, 164.26, 165.55; MS (ESI) C 17 H 17 N 2 O 4 S [M-H]- m/z expected = 345.1, observed = 345.0. HPLC-1 = >99%, HPLC-2 = >99%. (47) Ethyl 5-carbamoyl-4-methyl-2-(3-nitrobenzamido)thiophene-3-carboxy late. Yield = 4%. 1 H NMR (600 MHz, DMSO-d6) δ ppm 1.38 (t, J=7.2 Hz, 3 H), 2.56 (s, 3 H), 4.39 (q, J=7.1 Hz, 2 H), 7.54 (br. s., 2 H), 7.94 (t, J=8.1 Hz, 1 H), 8.34 (d, J=7.7 Hz, 1 H), 8.53 (d, J=8.4 Hz, 1 H), 8.68 (s, 1 H), 12.11 (s, 1 H); 13 C-NMR (150 MHz, d6-DMSO) δ 13.97, 15.33, 61.20, 115.35, 122.23, 124.25, 127.37, 131.03, 133.30, 133.53, 137.05, 147.80, 148.03, 161.85, 164.12, 165.04; MS (ESI) C16H14N3O6S [M-H]- m/z expected = 376.1, observed = 375.9. HPLC-1 = >99%, HPLC-2 = >99%. (48) Ethyl 5-carbamoyl-4-methyl-2-(4-nitrobenzamido)thiophene-3-carboxy late. Yield = 3%. 1 H NMR (600 MHz, DMSO-d6) δ ppm 1.37 (t, J=7.0 Hz, 3 H), 2.57 (s, 3 H), 4.39 (q, J=7.2 Hz, 2 H), 7.55 (br. s., 2 H), 8.18 (d, J=8.8 Hz, 2 H), 8.46 (d, J=8.8 Hz, 2 H), 12.16 (s, 1 H); 13 C-NMR (150 MHz, d6-DMSO) δ 14.02, 15.38, 61.22, 115.21, 124.23, 124.32, 128.97, 137.00, 137.24, 147.82, 149.92, 161.96, 164.11, 165.25; MS (ESI) C 16 H 14 N 3 O 6 S [M-H]- m/z expected = 376.1, observed = 376.0. HPLC-1 = >99%, HPLC-2 = 99%. (49) Ethyl 5-carbamoyl-2-(2-fluoro-5-nitrobenzamido)-4-methylthiophene- 3-carboxylate. Yield = 68%. 1 H NMR (600 MHz, DMSO-d 6 ) δ ppm 1.35 (t, J=7.2 Hz, 3 H), 2.55 (s, 3 H), 4.36 (q, J=7.0 Hz, 2 H), 7.54 (br. s., 2 H), 7.77 (t, J=9.9 Hz, 1 H), 8.51-8.60 (m, 1 H), 8.74 (dd, J=6.1, 2.8 Hz, 1 H), 12.33 (d, J=9.9 Hz, 1 H); 13 C-NMR (150 MHz, d 6 -DMSO) δ 13.98, 15.42, 61.14, 115.02, 118.75, 118.92, 120.53, 120.44, 124.54, 127.06, 130.15, 130.22, 136.97, 14.36, 147.44, 158.18, 162.18, 163.91, 164.07, 164.95; MS (ESI) C 16 H 13 FN 3 O 6 S [M-H]- m/z expected = 394.1, observed = 393.9. HPLC-1 = >99%, HPLC-2 = 97%. (50) Ethyl 5-carbamoyl-4-methyl-2-(5-nitrofuran-2-carboxamido)thiophene -3- carboxylate. Yield = 22%. 1 H NMR (600 MHz, DMSO-d 6 ) δ ppm 1.39 (t, J=7.0 Hz, 3 H), 2.56 (s, 3 H), 4.40 (q, J=7.0 Hz, 2 H), 7.57 (br. s., 2 H), 7.68 (d, J=3.7 Hz, 1 H), 7.87 (d, J=4.0 Hz, 1 H), 12.08 (s, 1 H); 13 C-NMR (150 MHz, d6-DMSO) δ 13.96, 15.38, 61.33, 113.84, 115.24, 118.65, 124.82, 137.00, 145.66, 146.79, 151.71, 153.17, 163.97, 164.85; MS (ESI) C14H12N3O7S [M-H]- m/z expected = 366.0, observed = 366.0. HPLC-1 = 97%, HPLC-2 = 98%. (51) 2-fluoro-5-nitro-N-phenylbenzamide. Yield = 89%. 1 H NMR (300 MHz, DMSO-d6) δ ppm 7.08-7.21 (m, 1 H), 7.38 (t, J=7.9 Hz, 2 H), 7.63-7.78 (m, 3 H), 8.46 (ddd, J=9.1, 4.3, 3.0 Hz, 1 H), 8.54 (dd, J=5.9, 2.9 Hz, 1 H), 10.68 (s, 1 H); 13 C-NMR (75 MHz, d 6 -DMSO) δ 117.89, 118.22, 119.89, 124.30, 125.75, 125.82, 126.03, 127.94, 128.08, 138.45, 143.78, 143.81, 160.56, 160.65, 184.09; MS (ESI) C 13 H 8 FN 2 O 3 [M-H]- m/z expected = 259.1, observed = 259.0. HPLC-1 = >99%, HPLC-2 = 97%. (52) 2,3,4,5,6-pentafluoro-N-phenylbenzamide. Yield = 79%. 1 H NMR (300 MHz, DMSO- d6) δ ppm 7.12-7.25 (m, 1 H), 7.40 (t, J=7.9 Hz, 2 H), 7.60-7.74 (m, 2 H), 11.00 (s, 1 H); 13 C- NMR (75 MHz, d 6 -DMSO) δ 119.62, 124.79, 129.12, 137.90, 154.87; MS (ESI) C 13 H 5 F 5 NO [M-H]- m/z expected = 286.0, observed = 286.0. HPLC-1 = >99%, HPLC-2 = >99%. General procedure for the amide coupling to give nitrofuran inhibitor analogs 53-66. To stirring mixture of the respective arylamine (1 eq.) in dichloromethane was added 5- nitro2-furoyl chloride (1.1 eq.) followed by pyridine (1.2 eq.), then the reactions were left to stir at RT overnight. The following day, the reactions were concentrated and flash chromatographic purification over silica (hexanes:EtOAc gradient) afforded the products as solids. If necessary, products were further purified by preparatory RP-HPLC (water:CH3CN gradient), concentrated, and lyophilized. (53) N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl )-5-nitrofuran-2- carboxamide. Yield = 86%. 1 H NMR (600 MHz, DMSO-d6) δ ppm 2.27 (s, 3 H), 6.04 (s, 1 H), 7.41 (d, J=8.1 Hz, 2 H), 7.51 (d, J=7.7 Hz, 2 H), 7.56 (d, J=9.5 Hz, 2 H), 7.61 (d, J=3.7 Hz, 1 H), 7.83 (d, J=3.3 Hz, 1 H), 10.45 (s, 1 H); 13 C-NMR (150 MHz, d6-DMSO) δ 17.48, 38.09, 113.43, 116.94, 119.00, 127.67, 129.18, 129.61, 130.83, 131.73, 133.00, 133.63, 133.93, 136.45, 147.48, 151.77, 154.94; MS (ESI) C20H13Cl2N3O4 [M-H]- m/z expected = 428.0210, observed = 428.0172. HPLC-1 = 98%, HPLC-2 = 97%. (54) (E)-5-nitro-N-(4-(3-(3-nitrophenyl)acryloyl)phenyl)furan-2-c arboxamide. Yield = 10%. 1 H NMR (600 MHz, DMSO-d6) δ ppm 7.71 (d, J=2.9 Hz, 1 H), 7.76 (t, J=7.9 Hz, 1 H), 7.82 - 7.89 (m, 2 H), 7.98 (d, J=8.1 Hz, 2 H), 8.19 (d, J=15.4 Hz, 1 H), 8.28 (d, J=8.1 Hz, 3 H), 8.35 (d, J=7.7 Hz, 1 H), 8.80 (br. s., 1 H), 10.95 (s, 1 H); 13 C-NMR (150 MHz, d6-DMSO) δ 113.45, 117.18, 119.98, 122.95, 124.67, 130.04, 130.37, 133.03, 135.13, 136.67, 141.11, 142.57, 147.45, 148.45, 151.92, 154.92, 187.52; MS (ESI) C20H13N3O7 [M-H]- m/z expected = 406.0681, observed = 406.0649. HPLC-1 = 98%, HPLC-2 = 97%. (55) 5-nitro-N-(4-phenoxyphenyl)furan-2-carboxamide. Yield = 92%. 1 H NMR (600 MHz, DMSO-d6) δ ppm 7.01 (d, J=8.1 Hz, 2 H), 7.06 (m, J=8.4 Hz, 2 H), 7.13 (t, J=7.3 Hz, 1 H), 7.39 (t, J=7.5 Hz, 2 H), 7.63 (d, J=3.3 Hz, 1 H), 7.75 (m, J=8.4 Hz, 2 H), 7.82 (d, J=3.3 Hz, 1 H), 10.67 (s, 1 H); 13 C-NMR (150 MHz, d6-DMSO) δ 113.49, 116.45, 118.25, 119.16, 122.47, 123.29, 130.04, 133.46, 147.96, 151.74, 152.98, 154.44, 156.95; MS (ESI) C 17 H 12 N 2 O 5 [M-H]- m/z expected = 323.0673, observed = 323.0652. HPLC-1 = >99%, HPLC-2 = >99%. (56) 5-nitro-N-(4-(phenylamino)phenyl)furan-2-carboxamide. Yield = 83%. 1 H NMR (600 MHz, DMSO-d6) δ ppm 6.81 (t, J=7.2 Hz, 1 H), 7.06 (d, J=7.7 Hz, 2 H), 7.09 (d, J=8.4 Hz, 2 H), 7.22 (t, J=7.5 Hz, 2 H), 7.60 (d, J=6.2 Hz, 3 H), 7.77 - 7.85 (m, 1 H), 8.18 (s, 1 H), 10.50 (s, 1 H); 13 C-NMR (150 MHz, d6-DMSO) δ 113.54 (s, 1 C) 116.03, 116.38, 117.03, 119.49, 122.06, 129.18, 130.11, 140.20, 143.51, 148.32, 151.66, 154.08; MS (ESI) C 17 H 13 N 3 O 4 [MH] + m/z expected = 324.0979, observed = 324.0914. HPLC-1 = 98%, HPLC-2 = >99%. (57) N-(4-acetamidophenyl)-5-nitrofuran-2-carboxamide. Yield = 48%. 1 H NMR (600 MHz, DMSO-d 6 ) δ ppm 2.03 (s, 3 H), 7.55 - 7.60 (m, 2 H), 7.60 - 7.63 (m, 1 H), 7.64 (d, J=8.1 Hz, 2 H), 7.76 - 7.85 (m, 1 H), 9.96 (s, 1 H), 10.57 (s, 1 H); 13 C-NMR (150 MHz, d6-DMSO) δ 23.92, 113.49, 116.28, 119.24, 121.18, 132.83, 136.01, 148.08, 151.71, 154.31, 168.11; MS (ESI) C13H11N3O5 [M-H]- m/z expected = 288.0626, observed = 288.0609. HPLC-1 = >99%, HPLC-2 = >99%. (58) N-(4-methoxyphenyl)-5-nitrofuran-2-carboxamide. Yield = 78%. 1 H NMR (600 MHz, DMSO-d6) δ ppm 3.75 (s, 3 H), 6.96 (d, J=8.4 Hz, 2 H), 7.59 (d, J=3.7 Hz, 1 H), 7.64 (d, J=8.4 Hz, 2 H), 7.81 (d, J=3.7 Hz, 1 H), 10.52 (s, 1 H); 13 C-NMR (150 MHz, d 6 -DMSO) δ 55.21, 113.51, 113.94, 116.17, 122.32, 130.73, 148.18, 151.66, 154.25, 156.15; MS (ESI) C12H10N2O5 [M-H]- m/z expected = 261.0517, observed = 261.0496. HPLC-1 = 98%, HPLC-2 = >99%. (59) methyl 4-(5-nitrofuran-2-carboxamido)benzoate. Yield = 21%. 1 H NMR (600 MHz, DMSO-d6) δ ppm 3.86 (s, 3 H), 7.66 - 7.72 (m, 1 H), 7.81 - 7.85 (m, 1 H), 7.92 (d, J=8.1 Hz, 2 H), 8.00 (d, J=8.1 Hz, 2 H), 10.91 (s, 1 H); 13 C-NMR (150 MHz, d 6 -DMSO) δ 52.01, 113.41, 117.10, 119.99, 125.14, 130.23, 142.31, 147.44, 151.91, 154.87, 165.72; MS (ESI) C13H10N2O6 [M-H]- m/z expected = 289.0466, observed = 289.0460. HPLC-1 = 97%, HPLC-2 = >99%. (60) N-(3-chloro-4-(4-chlorophenoxy)phenyl)-5-nitrofuran-2-carbox amide. Yield = 93%. 1 H NMR (600 MHz, DMSO-d6) δ ppm 6.97 (m, J=8.4 Hz, 2 H), 7.24 (d, J=8.8 Hz, 1 H), 7.42 (m, J=8.4 Hz, 2 H), 7.64 (d, J=3.7 Hz, 1 H), 7.74 (d, J=9.2 Hz, 1 H), 7.83 (d, J=2.9 Hz, 1 H), 8.06 (s, 1 H), 10.83 (s, 1 H); 13 C-NMR (150 MHz, d 6 -DMSO) δ 113.47, 116.94, 118.58, 120.97, 122.20, 122.29, 124.89, 126.97, 129.88, 135.48, 147.16, 147.47, 151.83, 154.67, 155.77; MS (ESI) C 17 H 10 Cl 2 N 2 O 5 [M-H]- m/z expected = 390.9894, observed = 390.9882. HPLC-1 =97%, HPLC-2 = >99%. (61) 5-nitro-N-(quinolin-6-yl)furan-2-carboxamide. Yield = 41%. 1 H NMR (600 MHz, DMSO-d 6 ) δ ppm 7.52 (dd, J=8.1, 4.0 Hz, 1 H), 7.71 (d, J=3.3 Hz, 1 H), 7.85 (d, J=3.3 Hz, 1 H), 7.99 - 8.09 (m, 2 H), 8.36 (d, J=8.4 Hz, 1 H), 8.46 (s, 1 H), 8.79 - 8.87 (m, 1 H), 10.94 (s, 1 H); 13 C-NMR (150 MHz, d 6 -DMSO) δ 113.49, 116.85, 117.14, 121.94, 124.13, 128.09, 129.60, 135.77, 145.15, 147.72, 149.73, 151.88, 154.91; MS (ESI) C14H9N3O4 [M-H]- m/z expected = 282.0520, observed = 282.0514. HPLC-1 = >99%, HPLC-2 = 99%. (62) N-(4-(benzo[d]thiazol-2-ylthio)-3-chlorophenyl)-5-nitrofuran -2-carboxamide. Yield = 74%. 1 H NMR (600 MHz, DMSO-d 6 ) δ ppm 7.52 (dd, J=8.1, 4.0 Hz, 1 H), 7.71 (d, J=3.3 Hz, 1 H), 7.85 (d, J=3.3 Hz, 1 H), 8.00 - 8.08 (m, 2 H), 8.36 (d, J=8.4 Hz, 1 H), 8.46 (s, 1 H), 8.80 - 8.89 (m, 1 H), 10.94 (s, 1 H); 13 C-NMR (150 MHz, d 6 -DMSO) δ 113.46, 117.49, 120.19, 121.49, 121.73, 121.87, 122.30, 124.62, 126.53, 134.89, 138.75, 138.88, 141.94, 147.12, 151.96, 153.39, 155.06, 167.61; MS (ESI) C18H10ClN3O4S2 [M-H]- m/z expected = 429.9728, observed = 429.9709. HPLC-1 = >99%, HPLC-2 = >99%. (63) N-(9H-fluoren-3-yl)-5-nitrofuran-2-carboxamide. Yield = 46%. 1 H NMR (600 MHz, DMSO-d 6 ) δ ppm 3.96 (s, 2 H), 7.30 (t, J=7.3 Hz, 1 H), 7.38 (t, J=7.3 Hz, 1 H), 7.57 (d, J=7.3 Hz, 1 H), 7.67 (d, J=3.3 Hz, 1 H), 7.73 (d, J=8.4 Hz, 1 H), 7.83 (d, J=3.3 Hz, 1 H), 7.86 (d, J=7.3 Hz, 1 H), 7.90 (d, J=8.1 Hz, 1 H), 8.05 (s, 1 H), 10.70 (s, 1 H); 13 C-NMR (150 MHz, d 6 - DMSO) δ 36.54, 113.51, 116.45, 117.48, 119.48, 119.73, 120.19, 125.08, 126.45, 126.80, 136.76, 137.62, 140.75, 143.01, 143.73, 148.03, 151.79, 154.50; MS (ESI) C 18 H 12 N 2 O 4 [M-H]- m/z expected = 319.0724, observed = 319.0718. HPLC-1 = 99%, HPLC-2 = 98%. (64) N-(4-(benzo[d]thiazol-2-ylthio)phenyl)-5-nitrofuran-2-carbox amide. Yield = 69%. 1 H NMR (600 MHz, DMSO-d 6 ) δ ppm 7.34 (t, J=7.5 Hz, 1 H), 7.45 (t, J=7.7 Hz, 1 H), 7.70 (d, J=4.0 Hz, 1 H), 7.84 (d, J=8.4 Hz, 4 H), 7.93 (d, J=7.7 Hz, 1 H), 7.97 (d, J=7.7 Hz, 2 H), 10.92 (s, 1 H); 13 C-NMR (150 MHz, d 6 -DMSO) δ 113.46, 117.10, 121.34, 121.77, 121.82, 123.45, 124.45, 126.45, 134.78, 136.42, 140.47, 147.49, 151.90, 153.49, 154.89, 169.75; MS (ESI) C 18 H 11 N 3 O 4 S 2 [M-H]- m/z expected = 396.0118, observed = 396.0095. HPLC-1 = >99%, HPLC-2 = >99%. (65) N-(2,3-di(furan-2-yl)quinoxalin-6-yl)-5-nitrofuran-2-carboxa mide. Yield = 60%. 1 H NMR (600 MHz, DMSO-d 6 ) δ ppm 6.69 - 6.77 (m, 4 H), 7.74 (d, J=3.7 Hz, 1 H), 7.86 (d, J=3.7 Hz, 2 H), 7.90 (d, J=8.4 Hz, 1 H), 8.06 - 8.17 (m, 2 H), 8.63 (s, 1 H), 11.12 (s, 1 H); 13 C-NMR (150 MHz, d 6 -DMSO) δ 112.15, 112.23, 112.54, 113.02, 113.49, 116.88, 117.29, 125.12, 129.25, 137.30, 139.80, 140.49, 141.03, 142.52, 144.75, 145.01, 147.45, 150.38, 151.94, 155.14; MS (ESI) C 21 H 12 N 4 O 6 [M-H]- m/z expected = 415.0684, observed = 415.0672. HPLC-1 = >99%, HPLC-2 = 96%. (66) 5-nitro-N-phenylfuran-2-carboxamide. Yield = 86%. 1 H NMR (600 MHz, DMSO-d6) δ ppm 7.16 (t, J=7.3 Hz, 1 H), 7.39 (t, J=7.7 Hz, 2 H), 7.64 (d, J=3.7 Hz, 1 H), 7.73 (d, J=7.7 Hz, 2 H), 7.82 (d, J=3.7 Hz, 1 H), 10.62 (s, 1 H); 13 C-NMR (7150 MHz, d6-DMSO) δ 113.46, 116.50, 120.71, 124.57, 128.82, 137.79, 147.93, 151.76, 154.59; MS (ESI) C 11 H 8 N 2 O 4 [M-H]- m/z expected = 231.0411, observed = 231.0395. HPLC-1 = >99%, HPLC-2 = >99%. General materials for biochemical & cell-based experiments. DH5α and BL21 (DE3) E. coli cells were purchased from New England Biolabs. The Mycobacterium tuberculosis proliferation assay used an H37Rv strain that researchers at the Seattle Children’s Research Institute (Seattle, WA, USA) previously engineered to express a codon-optimized mCherry fluorescent protein (TOPred). Trypanosoma brucei Plimmer and Bradford parasites (Lister 427 VSG 221 [TetR T7RNAP] transgenic bloodstream form) were obtained from the ATCC (PRA-383). FHs 74 Int (CCL-241) cells and FHC (CRL-1831) cells were obtained from the ATCC. E. coli GroEL and GroES Purification E. coli GroEL was expressed from a trc-promoted and Amp(+) resistance marker plasmid in DH5α E. coli cells. GroES was expressed from a T7-promoted and Amp(+) resistance plasmid in E. coli BL21 (DE3) cells. Transformed colonies were plated onto Ampicillin-treated LB agar and incubated for 24 h at 37°C. Cells were then grown at 37°C in Ampicillin-treated LB medium until an OD600 of ~0.5 was reached, then were induced with 0.8 mM IPTG and continued to grow for 2-2.5 h at 37°C. The cultures were centrifuged at 8,000 rpm and the cell pellets were collected and re-suspended in Buffer A (50 mM Tris-HCl, pH 7.4, and 20 mM NaCl) supplemented with half a Pierce TM protease inhibitor tablet, EDTA-free (Thermo Scientific). The combined suspension was lysed by sonication, the lysate was centrifuged at 14,000 rpm, and the clarified lysate was passed through a 0.45 μm filter (Millipore). Anion exchange purification: The filtered lysate was loaded onto a GE HiScale Anion exchange column (Q Sepharose fast flow anion exchange resin) that was equilibrated with 2 column volumes of Buffer A. The loaded column was washed with 10% Buffer B (50 mM Tris.HCl, pH 7.4, 1 M NaCl) for 3 CVs when purifying GroES and 28% Buffer B for 3 CVs when purifying GroEL, then bound protein was eluted with 3 CVs of 10-50% gradient flow of Buffer B when purifying GroES and 3 CVs of 28-60% gradient flow of Buffer B when collecting GroEL. Protein-containing fractions, as identified by SDS-PAGE, were collected, spin concentrated using a 10 kDa Amicon Ultra-15 centrifugal filter (EMD Millipore), and dialyzed overnight with 10 kDa SnakeSkin™ dialysis tubing (Thermo Scientific) at 4°C in 2 L of 50 mM Tris-HCl, pH 7.4, and 150 mM NaCl solution (Dialysis Buffer). Size exclusion chromatography: The dialyzed protein was loaded onto a Superdex 200 column (HiLoad 26/600, GE) column that was equilibrated with 2 column volumes of SEC Buffer (50 mM Tris-HCl, pH 7.4 and 150 mM NaCl). The loaded column was eluted with 1 column volume of 100% SEC Buffer and the column was washed with 2 CVs of the same solution. Protein-containing fractions, as identified by SDS-PAGE, were collected, spin concentrated using a 10 kDa Amicon Ultra-15 centrifugal filter (EMD Millipore). The final protein concentration was determined by Coomassie Protein Assay Kit (Thermo Scientific). Batches of protein for testing were stored at 4°C for up to six weeks and then discarded. Determination of compounds’ refolding inhibition in dMDH reporter assay Reagent preparation: For these assays, four primary reagent stocks were prepared: 1) GroEL/ES-dMDH or HSP60/10-dMDH binary complex stock; 2) ATP initiation stock; 3) EDTA quench stock; 4) MDH enzymatic assay stock. Denatured MDH (dMDH) was prepared by 2-fold dilution of MDH (5 mg/ml, soluble pig heart MDH from Roche, product #10127248001) with denaturant buffer (7 M guanidine-HCl, 200 mM Tris, pH 7.4, and 50 mM DTT). MDH was completely denatured by incubating at room temperature for 60 min. The binary complex solutions were prepared by adding the dMDH stock to GroEL (or HSP60) in folding buffer (50 mM Tris-HCl, pH 7.4, 50 mM KCl, 10 mM MgCl 2 , and 1 mM DTT), followed by addition of GroES (or HSP10). The binary complex stocks were prepared immediately prior to dispensing into the assay plates and had final protein concentrations of 83.3 nM GroEL (Mr 800 kDa) or HSP60 (Mr 400 kDa), 100 nM GroES or HSP10 (Mr 70 kDa), and 20 nM dMDH in folding buffer. For the ATP initiation stock, ATP solid was diluted into folding buffer to a final concentration of 2.5 mM. Quench solution contained 600 mM EDTA (pH 8.0). The MDH enzymatic assay stock consisted of 20 mM sodium mesoxalate and 2.4 mM NADH in reaction buffer (50 mM Tris-HCl, pH 7.4, 50 mM KCl, and 1 mM DTT). Assay protocol: First, 30 µL aliquots of the GroEL/ES-dMDH or HSP60/10-dMDH binary complex stocks were dispensed into clear, 384-well polystyrene plates. Next, 0.5 µL of the compound stocks (10 mM to 4.6 µM, 3-fold dilutions series in DMSO) were added by pin- transfer (V&P Scientific). The chaperonin-mediated refolding cycles were initiated by addition of 20 µL of ATP stock (reagent concentrations during refolding cycle: 50 nM GroEL or HSP60, 60 nM GroES or HSP10, 12 nM dMDH, 1 mM ATP, and compounds of 100 μM to 46 nM, 3- fold dilution series), and the refolding reactions incubated at 37°C. The incubation times were determined from refolding time-course control experiments until they reached ~90% completion of refolding of the denatured MDH – generally ~15-40 min for GroEL/ES, and ~40- 60 min for HSP60/10. Next, the assays were quenched by addition of 10 µL of the EDTA stock, to final concentration of 100 mM. Enzymatic activity of the refolded MDH was initiated by addition of 20 µL MDH enzymatic assay stock (20 mM sodium mesoxalate and 2.4 mM NADH in reaction buffer, 50 mM Tris pH 7.4, 50 mM KCl, 1 mM DTT), and followed by measuring the NADH absorbance in each well at 340 nm using a Molecular Devices, SpectraMax Plus384 microplate reader (NADH absorbs at 340 nm, while NAD + does not). A 340 nm measurements were recorded at 0.5 minutes (start point) and at successive time points until the amount of NADH consumed reached ~90% (end point, generally between 20-35 minutes). The differences between the start and end point A 340 values were used to calculate the % inhibition of the GroEL/ES or HSP60/10 machinery by the compounds. IC50 values for the test compounds were obtained by plotting the % inhibition results in GraphPad Prism and analyzing by non-linear regression using the log (inhibitor) vs. response (variable slope) equation. Results presented represent the averages of IC 50 values obtained from at least four replicates. Counter-screening compounds for inhibition of native MDH enzymatic activity. Reagent preparations and assay protocol: This assay was performed as described above for the GroEL/ES-dMDH refolding assay; however, the assay protocol differed in the sequence of compound addition to the assay plates. The refolding reactions were allowed to proceed for 45- 50 min at 37°C in the absence of test compounds (nearly complete refolding of MDH occurs), then quenched with the EDTA stock. Compounds were then pin-transferred into the plates after the EDTA quenching step; thus, compounds’ effects are only possible by inhibiting the fully refolded MDH reporter substrate. Next, enzymatic activity of the refolded MDH was initiated by addition of 20 µL MDH enzymatic assay stock (20 mM sodium mesoxalate and 2.4 mM NADH in reaction buffer(50 mM Tris pH 7.4, 50 mM KCl, 1 mM DTT), and followed by measuring the NADH absorbance in each well at 340 nm using a Molecular Devices SpectraMax Plus384 microplate reader (NADH absorbs at 340 nm, while NAD+ does not). A340 nm measurements were recorded at 0.5 minutes (start point) and at successive time points until the amount of NADH consumed reached ~90% (end point, generally between 15-30 minutes). Compounds were tested in 8-point, 3-fold dilution series (62.5 μM to 29 nM during the reporter reaction) in clear, flat-bottom 384-well microtiter plates. DMSO was used as negative control, and previously discovered native MDH inhibitors were used as positive controls. IC 50 values for the test compounds were obtained by plotting the % inhibition results in GraphPad Prism and analyzing by non-linear regression using the log (inhibitor) vs. response (variable slope) equation. Results presented represent the averages of IC50 values obtained from at least four replicates. Evaluating compounds for inhibition in the GroEL/ES-dRho refolding assay. Reagent preparation: For this assay, five primary reagent stocks were prepared: 1) GroEL/ES- dRho binary complex stock; 2) ATP initiation stock; 3) Enzyme solution; 4) Formaldehyde quench solution; 5) Fe(NO 3 ) 3 assay stock. Denatured Rho (dRho) was prepared by 3-fold dilution of Rho (Roche product #R1756, stock diluted to 10 mg/ml with H2O) with denaturant buffer (12 M Urea, 50 mM Tris, pH 7.4, and 10 mM DTT). Rho was completely denatured by incubating at room temperature for 40 min. The binary complex solution was prepared by slowly adding the dRho stock to a stirring solution of concentrated GroEL in modified folding buffer (50 mM Tris-HCl, pH 7.4, 50 mM KCl, 10 mM MgCl2, 5 mM Na2S2O3 and 1 mM DTT) and then incubated at room temperature for 10 min. The solution was centrifuged at 16,000 x g for 5 minutes, and the supernatant was collected and added to a solution of GroES in modified folding buffer to give final protein concentrations of 100 nM GroEL, 120 nM GroES, and 80 nM dRho in modified folding buffer. The binary complex stock was prepared immediately prior to use. For the ATP initiation stock, ATP solid was diluted into modified folding buffer to a final concentration of 2.0 mM. The thiocyanate enzymatic assay stock was prepared to contain 70 mM KH2PO4, 80 mM KCN, and 80 mM Na2S2O3 in water. The formaldehyde quench solution contained 30% formaldehyde in water. The ferric nitrate reporter stock contained 8.5% w/v Fe(NO3)3 and 11.3% v/v HNO3 in water. Assay protocol: First, 10 µL aliquots of the GroEL/ES-dRho complex stock was dispensed into clear, 384-well polystyrene plates. Next, 0.5 µL of the compound stocks (10 mM to 4.6 µM, 3- fold dilutions in DMSO) were added by pin-transfer. The chaperonin-mediated refolding cycle was initiated by addition of 10 µL of ATP stock (reagent concentrations during refolding cycle: 50 nM GroEL, 60 nM GroES, 40 nM dRho, 1 mM ATP, and compounds of 250 µM to 114 nM, 3-fold dilution series). After incubating for 45 minutes at 37°C for the refolding cycle, 30 µL of the thiocyanate enzymatic assay stock was added and incubated for 60 min at room temperature for the refolded rhodanese enzymatic reporter reaction. The rhodanese-catalyzed thiosulfate- cyanide reaction was quenched by adding 10 µL of the formaldehyde quench solution, and then 40 µL of the ferric nitrate reporter stock was added to quantify the amount of thiocyanate produced during the enzymatic reporter reaction, which is proportional to the amount of dRho refolded by GroEL/ES. After incubating at room temperature for 15 min, the absorbance by Fe(SCN) 3 was measured at 460 nm using a Molecular Devices, SpectraMax Plus384 microplate reader. A second set of baseline control plates were prepared analogously, but without binary solution, to correct for possible interference from compound absorbance or turbidity. IC 50 values for the test compounds were obtained by plotting the A460 results in GraphPad Prism and analyzing by non-linear regression using the log(inhibitor) vs. response (variable slope) equation. Results presented represent the averages of IC50 values obtained from at least four replicates. Counter-screening compounds for inhibition of native Rho enzymatic activity. Reagent preparations and assay protocol: Reagents were identical to those used in the GroEL/ES-dRho refolding assay described above; however, the assay protocol differed in the sequence of compound addition to the wells. Compounds were pin-transferred after the 60-minute incubation for the refolding cycle, but prior to the addition of the thiocyanate enzymatic assay stock. Thus, the refolding reactions were allowed to proceed for 60 min at 37°C in the absence of test compounds, but the enzymatic activity of the refolded rhodanese reporter enzyme was monitored in the presence of test compounds (inhibitor concentration range during the enzymatic reporter reaction is 100 µM to 46 nM – 3-fold dilutions). IC50 values for the rhodanese reporter enzyme were determined as described above. Results presented represent the averages of IC 50 values obtained from at least three replicates. Bacterial Proliferation Assays. Bacterial Strains: NEB 5-alpha Escherichia coli (a derivative of DH5α E. coli, New England Biolabs #C2987H); Enterococcus faecium - (Orla-Jensen) Schleifer and Kilpper-Balz strain NCTC 7171 (ATCC #19434); Staphylococcus aureus - Rosenbranch strain Seattle 1945 (ATCC #25923); Klebsiella pneumoniae - (Schroeter) Trevisan strain NCTC 9633 (ATCC #13883); Acinetobacter baumannii - Bouvet and Grimont strain 2208 (ATCC 19606); Pseudomonas aeruginosa - (Schroeter) Migula strain NCTC 10332 (ATCC #10145); Enterobacter cloacae - E. cloacae, subsp. cloacae (Jordan) Hormaeche and Edwards strain CDC 442-68 (ATCC #13047). Growth Media: E. coli were grown with LB medium and all ESKAPE bacteria were grown in Mueller-Hinton Broth 2, Cation-Adjusted (CA-MHB) medium (Millipore), with all liquid cultures supplemented with 20-25 mg/L Ca2+ and 10-12.5 mg/L Mg 2+ to mimic physiological free concentrations of these cations. General Assay Protocol: Stock bacterial cultures were streaked onto LB or CA-MHB agar plates and grown overnight at 37°C. Fresh aliquots of cation supplemented media were inoculated with single bacterial colonies and the cultures were grown overnight at 37°C with shaking (240 rpm). The following morning, cultures were diluted 10-fold into fresh media and grown at 37°C until bacteria had reached mid-log phase growth (OD600 ~ 0.4-0.6). The cultures were then diluted into fresh media to achieve final CFU/mL of 8.33 x10 5 CFU/mL. Aliquots of these diluted cultures (30 μL) were added to clear, flat-bottom 384-well polystyrene plates that were previously stamped with 0.5 μL of test compounds in 20 μL of media (yielding initial density of 5 x 10 5 CFU/mL). All compounds were tested in dose-response with concentration ranges during the proliferation assays from 100 μM to 46 nM (3-fold dilution series). A second set of baseline control plates were prepared analogously, but without any bacteria added, to correct for possible compound absorbance and/or precipitation. Plates were sealed with "Breathe-Easier" oxygen permeable membranes (Diversified Biotech) and left to incubate at 37°C without shaking (stagnant assay) for 20-24 hours. Plates were then read at 600 nm using a Molecular Devices SpectraMax Plus384 microplate reader. EC90 values for the test compounds were obtained by plotting the OD 600 results in GraphPad Prism and analyzing by non-linear regression using the [agonist] vs. response - Find ECanything equation. Results presented represent the averages of EC90 values obtained from at least four replicates. Evaluating compounds for inhibition of M. tuberculosis proliferation. M. tuberculosis (strain H37Rv) was grown in Middlebrook 7H9 medium supplemented with 0.05% Tween 80, 10% v/v oleic acid, and albumin dextrose catalase (OADC) supplement (Becton Dickinson) (7H9-Tw-OADC). Stock bacterial cultures were inoculated in a startup culture and grown to a logarithmic phase of OD 590 ~0.7. This was sub-cultured in fresh media (1:10 dilution) and grown to an OD5901.0, then diluted again into fresh media to achieve a final OD 590 reading of 0.04 (just prior to dispensing into plates). Compound plates were prepared by adding 4 µL of compound stocks to 96 µL of fresh medium in 96-well plates. Aliquots of the diluted Mtb cultures (100 µL) were then added to the compound plates, which were incubated in sealed bags at 37ºC for 5 days. All compounds were first evaluated in singlicate at a single concentration of 200 µM, with compounds showing >50% inhibition re-evaluated in dose- response format (inhibitor concentration range of 200 μM to 390 nM – 2-fold dilutions) to determine EC 50 values. After 5 days, OD 590 values were read and % growth inhibition for each well was calculated. EC90 values for the test compounds were obtained by plotting the OD590 results in GraphPad Prism and analyzing by non-linear regression using the [agonist] vs. response - Find ECanything equation. Results presented represent the averages of EC90 values obtained from at least duplicate experiments. T. brucei cell viability assay protocol. Test compounds were evaluated using a T. brucei cell viability assay in 384-well plate format. Briefly, 55 μL of 2000 parasites/mL (110 parasites/well) of Trypanosoma brucei brucei (strain BF427) in HMI-9 medium were dispensed in to clear, 384-well polystyrene plates (BRAND cell culture grade plates, 781980). Plates were sealed with "Breathe Easy" oxygen permeable membranes (Diversified Biotech) and incubated at 37°C, 5% CO2 for 24 h. Next, 1 µL of the compound stocks (10 mM to 4.6 µM, 3-fold dilutions in DMSO) were pre-diluted by pin-transfer into 20 µL HMI-9 medium, then 5 µL of these diluted compounds were added to the parasite assay plates to give an inhibitor concentration range of 42 µM to 19 nM during the assay (the final DMSO concentration of 0.42% was maintained during the assay). Parasites were incubated for an additional 48 h at 37°C and 5% CO 2 . Cell viability was then measured by adding 10 µL of Alamar Blue reagent to give 10% v/v in the assay. Plates were incubated at 37°C and 5% CO 2 and sample fluorescence (535 nm excitation, 590 nm emission) monitored over time using a Molecular Devices FlexStation II 384-well plate reader, and cell viability was calculated as per vendor instructions. EC50 and EC90 values for the test compounds were obtained by plotting the % Alamar Blue reduction results in GraphPad Prism and analyzing by non-linear regression using the [agonist] vs. response - Find ECanything equation. DMSO was used as negative control, and pentamidine, suramin, and nifurtimox (drugs used to treat HAT) were used as positive controls. Results presented represent the averages of EC 50 and EC 90 values obtained from at least four replicates. Evaluating compounds for effects on human intestine and colon cell viability. Evaluation of compound cytotoxicity to FHs 74 Int intestine and FHC colon cells was performed using Alamar Blue-based viability assays. FHs 74 Int cells were maintained in Hybri-care Medium ATCC 46-X supplemented with 30ng/ml epidermal growth factor (EGF) and 10% FBS. FHC cells were maintained in DMEM: F-12 media, supplemented with 10mM HEPES (final concentration 25 mM), 10 ng/ml cholera toxin, 0.005 mg/ml insulin, 0.005mg/ml transferrin, 100 ng/ml hydrocortisone, and 10% FBS (Sigma, F2242). All assays were carried out in 384-well plates (BRAND cell culture grade plates, 781980). Cells at 80% confluence were harvested and diluted in growth medium, then 45 µL of the FHs 74 Int cells (1,500 cells/well) or FHC cells (1,500 cells/well) were dispensed per well, and plates were sealed with "Breathe Easy" oxygen permeable membranes (Diversified Biotech) and incubated at 37°C, 5% CO 2 , for 24 h. The following day, 1 µL of the compound stocks (10 mM to 4.6 µM, 3-fold dilutions in DMSO) were pre-diluted by pin-transfer into 25 µL of the relevant growth mediums. Then, 15 µL aliquots of the diluted compounds were added to the cell assay plates to give inhibitor concentration ranges of 100 μM to 46 nM during the assay (final DMSO concentration of 0.1% was maintained during the assay). Plates were sealed with "Breathe Easy" oxygen permeable membranes and incubated for an additional 48 h at 37°C and 5% CO2. The Alamar Blue reporter reagents were then added to a final concentration of 10%, the plates incubated at 37°C and 5% CO2, and sample fluorescence (535 nm excitation, 590 nm emission) was read using a Molecular Devices FlexStation II 384-well plate reader (readings taken between 4-24 h of incubation so as to achieve signals in the 30-60% range for conversion of resazurin to resorufin). Cell viability was calculated as per vendor instructions (Thermo Fisher - Alamar Blue cell viability assay manual). Cytotoxicity CC50 values for the test compounds were obtained by plotting the % resazurin reduction results in GraphPad Prism and analyzing by non- linear regression using the log(inhibitor) vs. response (variable slope) equation. Results presented represent the averages of CC 50 values obtained from at least four replicates for FHs 74 Int small intestine cells and four replicates for FHC colon cells. Control compounds, calculation of IC 50 values, and statistical considerations. For the chaperonin-mediated biochemical assays, DMSO was used as a negative control, and a panel of our previously discovered and reported chaperonin inhibitors were used as positive controls: e.g., compounds 8, 9, and 18 from Johnson et. al 2014 and Abdeen et. al 2016; suramin and compound 2h-p from Abdeen et. al 2016; compounds 20R, 20L, and 28R from Abdeen et. al 23. All IC 50 results reported are averages of values determined from individual dose-response curves in assay replicates as follows: 1) Individual IC50 values from assay replicates were first log-transformed and the average log(IC 50 ) values and standard deviations (SD) calculated; 2) Replicate log(IC50) values were evaluated for outliers using the ROUT method in GraphPad Prism (Q of 10%); and 3) Average IC 50 values were then back- calculated from the average log(IC50) values. For compounds where log(IC50) values were greater than the maximum compound concentrations tested (i.e. >1.6, >1.8, >2.0, and >2.4 – or >42, >63, >100, and >250 μM, respectively), results were represented as 0.1 log units higher than the maximum concentrations tested (i.e.1.7, 1.9, 2.1, and 2.5 – or 53, 79, 126, and 316 μM, respectively) so as not to overly bias comparisons because of the unavailability of definitive values for these inactive compounds.