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. 2026 Sep 12;12(10):3373–3384. doi: 10.1021/acsinfecdis.6c00350

Development of GuaB Inhibitors to Treat Mycobacterium tuberculosis Infection

Eric M Kofoed *, Terry D Crawford ‡, Christine M Bowman ∥, Jessica Sims ○, Tahnee J Dening ◆, Shumei Wang ‡, Yuebiao Zhou ‡, Seth F Harris ⊥, Min Xu #, Ping Wu ⊥, Ying Yang §, Manda K Wong ⊥, Racquel Corpuz ⊥, Yiming Xu ∇, Anjani Ganti ∥, Savita Ubhayakar ∥, Chloe Hu ○, Kevin R Clark ∇, Zhijuan Cai ⧅, Kwong Wah Lai ⧅, Xiaojing Wang ‡, Renwei Zhang ⧅, Michael FT Koehler ‡, Emily J Hanan ‡, Ignacio Aliagas ‡, Julie Ng , Jared Silverman ⊞, Khisimuzi Mdluli ⊞, Sharookh Kapadia †, Man-Wah Tan †,*
PMCID: PMC13660411  PMID: 42917107

Abstract

Mycobacterium tuberculosis (Mtb) is responsible for more deaths per year than any other infectious disease. Resistance of circulating Mtb to the standard of care treatment regimen is an urgent global health crisis for which novel antibiotic treatments are needed. We have developed extremely potent, orally bioavailable, small-molecule inhibitors of inosine 5′-monophosphate dehydrogenase (GuaB) that catalyze the rate-limiting step in de novo guanine biosynthesis. The inhibitors exhibited bactericidal activity against Mtb in extracellular, intracellular, and hypoxic environments that capture features of advanced disease pathology in patients. Mtb GuaB2 inhibitors (GuaBi) were efficacious in three different in vivo mouse models of Mtb lung infection: the C57BL/6 super acute, C57BL/6 chronic, and C3HeB/FeJ (Kramnik) models. In the chronic BALB/c infection model of primarily intracellular Mtb, no benefit was observed, whereas in the C3HeB/FeJ model, a bedaquiline, pretomanid, and moxifloxacin treatment regimen that included GuaBi led to a rapid elimination of culturable Mtb infection in half of the mice. Notably, the C3HeB/FeJ model recapitulates advanced pathophysiology and heterogeneous disease that includes caseous necrotic granulomas, intracellular and extracellular reservoirs of Mtb, and lethal progressive infection. These results support further evaluation of a GuaBi-containing therapeutic regimen in relapse-capable preclinical models.

Keywords: Mycobacterium, Tuberculosis, GuaB, Antibiotics, Kramnik, Antimicrobial

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Introduction

Mycobacterium tuberculosis (Mtb) is the causative agent of the disease tuberculosis and is the leading cause of death from an infectious disease, leading to 1.3 million deaths in 2022. − Over 1.7 billion people are infected with Mtb, and 10.6 million people become sick from Mtb each year. Compounding the enormous impact of Mtb on global health, the emergence of resistance to frontline antibiotics reduces treatment options available for patients. , New antibiotic regimens are needed for the treatment of tuberculosis and improving global public health.

Mtb is an obligate human pathogen that is difficult to treat due to the complex pathology of disease and adaptation to survive in different host environments. Especially pernicious is the ability of Mtb to replicate within lung macrophages that are the frontline defense of the host innate immune system against bacteria. , Mtb can persist for decades as a chronic latent infection encased within granulomas that are in a stalemate with the host. , Over time, cellular granulomas harboring intracellular bacteria develop into necrotic caseating lesions that are hypoxic and contain large numbers of extracellular bacteria. , The ability of Mtb to thrive in unique host environments (intracellular, extracellular, normoxic, hypoxic, lipid-rich caseum) and in various states (replicating, dormant, nonreplicating) makes it difficult to eradicate. , The ability of Mtb to adapt to diverse environments presents a challenge to achieving pharmacodynamic concentration of antibiotics across different sites of infection in patients. , Taken together, the complex pathophysiology of the disease helps explain why multiple antibiotics, each with a distinct mechanism of action, are required to cure Mtb infections. The World Health Organization recommends treatment of drug-susceptible, pulmonary Mtb infections with a 4–6 month regimen that includes multiple antibiotics, whereas treatment for drug-resistant Mtb may be much longer and carry greater safety risks. , Each antibiotic in each regimen must contribute activity against Mtb in one or more of the many host environments and bacterial reservoirs.

Pharmacologic inhibition of the de novo purine biosynthesis enzyme PurF was active in vivo against Mtb in mouse lung infection models and showed that purine salvage was not sufficient to rescue inhibition of de novo purine biosynthesis in mice. Targeting inosine-5′-monophosphate (IMP) dehydrogenase that is encoded by the GuaB2 gene in Mtb − (Figure A) is a novel and attractive candidate antibiotic target, because it is the rate-limiting step in de novo guanine biosynthesis and was shown to be effective in mouse models of Acinetobacter baumannii infection. Consequences of inhibiting Mtb GuaB2 are that the pool of available guanosine 5′-triphophate (GTP) plummets, resulting in prevention of RNA and DNA synthesis, translation elongation, and cell division. The Mtb GuaB2 enzyme converts IMP to xanthosine-5′-monophosphate (XMP) through an oxidation reaction that requires the cofactor NAD and transitions through a covalent enzyme-substrate intermediate. The Mtb genome encodes three separate genes that have been denoted as GuaB1, GuaB2, and GuaB3, but only GuaB2 exhibits catalytic activity of a functional inosine 5′-monophosphate dehydrogenase (IMPDH), , and only GuaB2 is essential for bacterial viability of Mtb both in vitro and in vivo mouse lung infection. , GuaB1 is a guanosine 5′-monophosphate reductase, whereas GuaB3 has an unknown function and lacks catalytic activity in Mtb. , Therefore, GuaB2 is the only functional ortholog with IMPDH activity that is expressed in Mtb, which explains why only Mtb GuaB2 is essential. , However, attempts at pharmacologic inhibition of Mtb GuaB2 in BALB/c mice were initially disappointing, and it was hypothesized that the lack of activity was due to purine salvage that circumvented the essentiality of de novo purine biosynthesis.

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Role of Mtb GuaB2 in de novo guanine biosynthesis and characterization of antibacterial activity of a GuaB inhibitor against Mtb in extracellular, intracellular, and hypoxic environments. A) Schematic of the de novo purine biosynthesis and nucleobase salvage pathways in Mtb. B) Chemical structure of GuaBi (G1), Mtb GuaB2 inhibitory concentration (IC50), and Mtb H37Ra and Mtb H37Rv minimal inhibitory concentration (MIC). C) Kinetics of bactericidal activity across a dose-response (0, 0.04, 0.09, 0.35 μM) of G1 against Mtb H37Ra enumerated by plating for colony-forming units (CFUs) over time. D) Bactericidal efficacy of G1 against Mtb H37Ra in the presence of 0, 25, 50, and 100 μM exogenous guanine across a dose-response with viable bacteria enumerated after 21 days. E) Intracellular activity of G1 against Mtb H37Rv-GFP-infected human THP-1 macrophages after 4 days of treatment measured by quantitative fluorescence microscopy. F) C57BL/6 Bone marrow-derived macrophages (BMDMs) infected with Mtb H37Rv overlaid with different concentrations (vehicle, 1.6 μM, 3.1 μM, and 12.5 μM) of G1 for 4 days followed by enumeration of viable intracellular bacteria. G) BMDM infection with Mtb H37Rv overlaid with different doses (vehicle, 0.25 μM, 0.5 μM, and 2 μM) of rifampicin. H) Mtb H37Ra grown under aerobic (21% O2) and non-replicating hypoxic (1% O2) conditions treated with a dose-response of G1 (left panel) and isoniazid (right panel) for 19 days followed by CFU enumeration. Data are the mean and standard deviation of N ≥ 3 independent biological replicates; statistical significance (*p < 0.05, **p < 0.005, ***p < 0.001) was determined using a Welch’s t-test or Holm–Sidak (GraphPad Prism).

Various GuaB inhibitor scaffolds have been described to date that exhibit antibacterial activity in vitro, ,− but poor pharmacokinetic and safety profiles have prevented their use in vivo. We previously reported the efficacy of GuaBi G1 against A. baumannii in a neutropenic mouse model of infection that required a PK booster to achieve effective concentrations in vivo. Recently, we described a distinct chemical scaffold that had exquisite activity against Mtb GuaB2 and improved ADME properties that support safe long-term oral dosing required for mouse Mtb efficacy studies. Here we show that a novel inhibitor, G9, targeting Mtb GuaB2 is effective in multiple mouse Mtb infection models, including acute, chronic, and the high-bar C3HeB/FeJ model. The antibiotic combination of GuaBi with bedaquiline (B), pretomanid (P), and moxifloxacin (M), and B, P, and sutezolid (U) improved bactericidal activity in C3HeB/FeJ mice. These data suggest that GuaBi could play a role in a pan-TB treatment regimen that may shorten treatment duration and overcome antibiotic resistance.

Results

Characterization of Mtb GuaB2 Inhibitor G1 Activity in Different Environments

The GuaB inhibitor (G1) traps the covalent enzyme (Mtb GuaB2)-substrate (IMP) intermediate with an inhibitory concentration (IC50) of 0.3 nM and a minimal inhibitory concentration (MIC) against the Mtb strain H37Rv of 0.3 μM (Figure B). Dose-dependent bactericidal activity (0, 0.04, 0.08, 0.4 μM G1) against extracellular Mtb H37Ra grown in broth culture was observed with durable sterilization of the culture at five-fold the MIC (Figures C and S1). Nucleobase salvage is an alternative method for bacteria to obtain guanine required for viability in addition to de novo biosynthesis. We determined the concentration of exogenous nucleobases required for protection of Mtb H37Ra from G1-mediated bactericidal activity by performing G1 dose-responses in the presence of 0, 25, 50, and 100 μM exogenous guanine, guanosine, or xanthine (Figures D and S1). Confirming previous results, the concentration of exogenous guanine or guanosine required to prevent bactericidal activity was 100 μM, , whereas xanthine addition did not prevent killing of Mtb H37Ra by G1 ,, (Figure S1). Guanine and guanosine were undetectable in human and mouse blood, indicating that exogenous levels are insufficient for salvage pathways to rescue extracellular Mtb from GuaB inhibition in vivo.

Mtb is highly adapted to survival and replication within macrophages, , and restriction of Mtb replication within macrophages is a desirable feature for new Mtb antibiotics. , However, intermediate levels of free guanine (40 μM) were found inside human macrophages, raising the possibility that intracellular Mtb in macrophages could overcome G1 activity. Intracellular activity of antibiotics against Mtb H37Rv-GFP in PMA-activated THP-1 human macrophages was measured using quantitative fluorescent microscopy and automated cell segmentation analysis. G1 exhibited dose-dependent inhibition against replicating intracellular Mtb H37Rv in human THP-1 macrophages with an IC50 value of 0.058 μM (Figure E).

To determine if this inhibitory effect resulted in bacterial killing, Mtb H37Rv-infected C57BL/6 mouse bone marrow-derived macrophages were treated with G1, and viable bacteria were enumerated by CFU plating (Figure F). G1 exhibited bactericidal activity against intracellular Mtb H37Rv measured after 4 days of exposure at 10× MIC (3.1 μM) and 40× MIC (12.5 μM) but was not bactericidal at 5× MIC (1.6 μM). In contrast, the positive control rifampicin exhibited a one-log bactericidal activity against intracellular Mtb H37Rv at all concentrations greater than 2× MIC (0.25 μM) (Figure G). Another key requirement for Mtb antibiotics is the ability to kill the bacteria under hypoxic conditions, a feature of advanced pathology where some bacteria are in a dormant, non-replicating state. , Using a control antibiotic, isoniazid, we observed a 16-fold shift in the minimal bactericidal concentration (MBC) for non-replicating bacteria compared to replicating Mtb (Figure H). In contrast, G1 had an equipotent MBC against both replicating and non-replicating Mtb (Figures H and S2). Taken together, these data demonstrate that G1 is bactericidal against Mtb under a suite of in vitro conditions that mimic important host environments and physiological states observed during infection.

Antibiotic Combinations Using GuaBi In Vitro

Treatment of tuberculosis requires antibiotic combinations; therefore, we evaluated in vitro drug–drug interactions between G1 and standard of care Mtb antibiotics against Mtb H37Ra. Fractional inhibitory concentration values in 2-dimensional combinations of G1, linezolid, bedaquiline, rifampicin, moxifloxacin, and clofazimine were performed to identify antagonistic, synergistic, additive, and indifferent combinations. Synergy was observed between G1 and rifampicin, linezolid, and clofazimine in vitro (Table and Supporting Information) and highlighted the potential for GuaB inhibitors to add value to therapeutic regimens. No antagonism was found with any of these combinations. Combination with antibiotics that impact cell processes that require a stable purine nucleotide pool was a logical candidate for a novel regimen based on the expected reduction of intracellular GTP by G1, but rifampicin was deprioritized due to preexisting resistance.

1. Fractional Inhibitory Concentration (F.I.C.) of G1 in Combination with Standard-of-Care Antibiotics against Mtb H37Raa .

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Fractional inhibitory concentration [F.I.C. = (MICA+B/MICA) + (MICA+B/MICB)] values in 2-dimensional combinations of G1, linezolid, bedaquiline, rifampicin, moxifloxacin, and clofazimine using an 8-point checkerboard assay and Alamar blue readout.

Discovery of Mtb GuaB2 Inhibitor Active against Mtb H37Rv In Vitro and In Vivo

Mtb GuaB2 is an essential and vulnerable target in Mtb, ,, and we hypothesized that pharmacologic inhibition of Mtb GuaB2 during infection will lead to bactericidal activity in vivo. Medicinal chemistry progress on the G1 scaffold has led to the discovery of a new lead compound G9, with better PK properties and amenable to oral dosing, which is a practical requirement for new TB drugs (Figure A). Our focus shifted from G1 to G9 due to its reduced hepatic clearance and plasma protein binding and superior PK (Table ). G9 exhibited potent biochemical inhibitory activity (Mtb GuaB2 IC50 = 0.94 nM), selectivity for the bacterial GuaB over the human homology hIMPDH2 (hIMPDH2 IC50 > 50 μM), and potent MIC against virulent Mtb H37Rv (MIC = 0.058 μM). G9 also displayed a favorable pharmacokinetic profile, low P-gp efflux, no observed drug–drug interaction risk, an excellent in vitro safety profile (Supplemental Table 1), and a predicted human half-life amenable to oral QD dosing (Table ).

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Discovery of potent inhibitors of Mtb GuaB2 active against Mtb H37Rv in vitro and in an acute C57BL/6 mouse lung infection model. A) Chemical structure of GuaBi (G9), Mtb GuaB2 inhibitory concentration (IC50), and Mtb H37Rv minimal inhibitory concentration (MIC). B) Crystal structure of G9 (teal) bound to M. tuberculosis GuaB2ΔCBS protein with IMP (magenta) bound determined at 1.35 Å resolution (PDB 12LG). The binding region of interest is situated at the junction between two GuaB protomers of the tetrameric arrangement, seen here in a side view of the tetramer relative to its broader plane. C) The binding site is located at an interface between two GuaB protomers of the tetramer, packing against the IMP enzymatic substrate. D) The IMP molecule (magenta) and the G9 inhibitor (“LIG”) with salient protein pocket amino acids labeled via the 3-letter codes and residue position numbers. GuaB protomers are in different shading, and residues in the neighboring partner display a tick mark on the residue numbering. Ordered water molecules are shown as small gray detached spheres to mark their dominant positions. E) Activity against Mtb H37Ra was determined over a dose-response to G9 (0, 0.09, 0.19, 0.39 μM) where viable bacteria were enumerated over time by plating for colony-forming units (CFUs). F) Activity against Mtb H37Rv was determined over a dose-response to G9 (0, 0.06, 0.6, 6 μM), and isoniazid (4 μM) where viable bacteria were enumerated over time by CFU plating. G) Acute C57BL/6 mouse Mtb H37Rv lung infection model testing the effect of escalating doses (5, 10, 25, 50, 100 mg/kg b.i.d.) of G9 on bacterial load in the lungs of mice after 8 days of treatment, compared to untreated and isoniazid (10 mg/kg q.d.) controls. Pretreatment refers to bacterial load in the lung 1 day post infection, prior to treatment. H) Pharmacokinetics of G9 measured in C57BL/6 mouse blood 2 and 24 h after escalating b.i.d. doses during acute lung infection with Mtb H37Rv. Data represent the mean and standard deviation of independent biological replicates (E,F: N ≥ 3; G,H: N ≥ 5) and statistical significance (*p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001) was determined using the Holm–Sidak (E,F) or Mann–Whitney (G) test (GraphPad Prism).

2. Chemical, Biochemical, Antibiotic, and Pharmacokinetic Properties of G9 .

Measurement G1 G9
LogD 7.4/kinetic solubility 2.7/140 μM 1.9/146 μM
IC50 Mtb GuaB2/MIC Mtb H37Rv (virulent) 0.3 nM/0.3 μM 0.94 nM/0.058 μM
Hep CLhep (H/R/M/D/C) (mL/min/kg) 15/36/70/22/35 <6.2/20/22/<7.9/18
% PPB (H/R/M/D/C) 94/98/95/ND/ND 79.5/94.3/68.7/74/82.6
gMDCK Papp [A to B] (MDR1 efflux), (10–6 cm/sec) 1.9 (4.7) 19 (4.3)
Cyp inhibition/TDI 3A4(T) 8.8 μM others >10 μM/ND All >10 μM/>10 μM
PXR activation ND >10 μM
Mouse PK CLb (mL/min/kg)/Vss (L/kg)/%F 132/1.6/ND 8/0.8/75
Predicted human PK CLb (mL/min/kg)/Vss (L/kg)/T 1/2 (hr) ND 2.45/1.3/12.6
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Clearance in hepatocytes, scaled up using the well-stirred model, assuming binding constants (fu,plasma and fu,inc) equal to 1. Not detected (ND).

High-Resolution Co-Crystal Structure of G9 with Mtb GuaB2ΔCBS

Structure-based drug development enables medicinal chemistry to improve the pharmacokinetic properties of small molecule inhibitors while retaining the biochemical potency. The co-crystal structure of G9 bound to Mtb GuaB2 protein-IMP was determined to 1.35 Å resolution (Figures B–D and S3, and Table S2) (PDB 12LG). The G9 binding site was identified at an interface between two Mtb GuaB2 protomers of the tetramer, packing against the IMP enzymatic substrate and occupying a cavity that is otherwise observed to accommodate the NADH cofactor. G9 bound to Mtb GuaB2-IMP showed a similar binding mode and selectivity as reported for other GuaB inhibitors in this scaffold family. ,, The aromatic head group of G9 forms π–π stacking interactions against IMP bound in the central pocket, occluding NADH co-factor, and is oriented by an H-bond at the saturated tetrahydropyran moiety to Gly336 of Mtb GuaB2. The tail of the molecule is bent down around VanderWaals contacts to Ala285, a non-canonical H-bond to the side chain of Glu458, and a bulkier substituent that mediates contacts with Tyr487 and other features in the neighboring Mtb GuaB2 protomer. Several of these residues are notable for affecting species selectivity in GuaB. ,

Characterization of Mtb GuaB2 Inhibitor G9 Activity In Vitro

Bactericidal activity of G9 was shown by a dose-response of 0, 0.09, 0.19, and 0.39 μM against extracellular Mtb H37Ra in broth cultures, where durable sterilization of culture was achieved after 15 days (Figure E). We confirmed activity in the virulent strain Mtb H37Rv using a time-kill study of G9 at 0.06 (1× MIC), 0.6 (10× MIC), and 6 μM (100× MIC), or isoniazid (INH) at 4 μM (10× MIC), and viable bacteria were enumerated over time (Figure F). G9 showed dose-dependent, bacterial killing (4-log reduction) over 21 days. For INH, we observed rapid bacterial killing over the first 7 days followed by rapid growth (Figure F). We hypothesize the observed regrowth is driven by the emergence of resistant mutants and is consistent with the high frequency of spontaneous resistance to INH (∼3.5 × 10–06). We confirmed this by selecting for spontaneous mutants of Mtb H37Ra resistant to INH and calculated a frequency of ∼1.4 × 10–06 at 4× MIC (Table S3). Sequencing of resistant isolates revealed mutations primarily in the gene encoding catalase peroxidase (katG: D487N, G269C, W300R, W49R, P422 frameshift), which is required to activate the INH prodrug. In contrast, selections with Mtb H37Ra for spontaneous resistance to G9 resulted in a frequency of resistance <1 × 10–09 and 5.2 × 10–09 at 10× MIC and 4× MIC G9, respectively (Table S3). All G9-resistant mutants possessed only on-target mutations in Mtb GuaB2 (E458Q).

Single-Agent Activity of G9 in Acute and Chronic Mtb In Vivo Infection Models

The in vitro activity, pharmacokinetic profile, and ADME properties of G9 allowed us to test whether pharmacologic inhibition of Mtb GuaB2 was efficacious in vivo. First, we tested the efficacy of G9 in an acute C57BL/6 mouse Mtb H37Rv infection model. In this model, a high intratracheal dose of Mtb H37Rv (1E5 CFU/mouse) was administered, and treatments (vehicle control, isoniazid, G9 at 5, 10, 25, 50, and 100 mg/kg; b.i.d.) were dosed orally beginning 1 day post-infection (pre-treatment) for 8 consecutive days. Viable Mtb from mouse lungs at the end of the study were enumerated by CFU plating. After 8 days, isoniazid treatment resulted in a >3-log reduction in the number of Mtb compared to mice treated with the vehicle control (Figure G). Treatment with G9 led to a dose-proportional reduction in Mtb of ∼1–2 log compared to the vehicle control, but only the 100 mg/kg dose significantly reduced bacterial burden relative to the pre-treatment (Figure G). Serum levels of G9, total and unbound, were assessed for all doses at 24 h to assess the pharmacokinetics (Figure H). For the 50 mg/kg and 100 mg/kg doses, the unbound G9 concentrations in the serum at 24 h were sufficient to ensure enough drug was present to cover the 0.24 μM MBC target for Mtb H37Rv. Specifically, we detected 2.17 μM of G9 at 24 h for the 50 mg/kg dose and 6.84 μM at the 100 mg/kg b.i.d. doses (Figure H and Table S4). For the 100 mg/kg dose group, we observe a significant, ∼0.5-log reduction in Mtb load compared to the pre-treatment group (P = 0.0127*; Figure G), suggesting that 24 h coverage at 10× the broth MBC (0.6 μM; Figure F) is necessary for in vivo bactericidal activity. The higher concentration of drug required to observe bactericidal activity in vivo suggests that the intramacrophage H37Rv MBC (e.g., Figure F; 10× above broth MIC) more accurately predicted bactericidal activity in the superacute lung infection that models intracellular bacterial infection.

We next tested G9 in a more challenging Mtb infection model. Chronic infection in C57BL/6 mice was established by using a low-dose aerosol intranasal infection (100 CFU/mouse) of Mtb H37Rv. Following infection, 21 days of bacterial outgrowth led to a stable level of 5E6 CFU of Mtb H37Rv per mouse lung (Figure A). Mice were treated orally with either vehicle control, isoniazid (10 mg/kg q.d.), G9 (100 mg/kg b.i.d.), or G9 (200 mg/kg q.d.) for 28 or 56 days prior to bacterial enumeration. After 28 days of dosing, only isoniazid reduced the number of CFU/lung (Figure A). After 56 days of treatment, in addition to isoniazid, the high-dose G9 (200 mg/kg q.d.) group showed a significant reduction of Mtb CFU per lung, and the low-dose G9 group was dose-proportionally less effective (Figure A). Pharmacokinetics of G9 in the chronic model predicted 24 h coverage of the broth MBC of Mtb H37Rv in both the 100 mg/kg b.i.d. and 200 mg/kg q.d. dosing groups (Figure S4C and D).

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In vivo activity of GuaB inhibitor G9 against Mycobacterium tuberculosis as a single agent and in combination with other antibiotics in the chronic C57BL/6, BALB/c, and C3HeB/FeJ mouse models of lung infection. A) Chronic C57BL/6 mouse Mtb H37Rv lung infection model testing the effect of different doses of G9 (100 mg/kg b.i.d. and 200 mg/kg q.d.) on bacterial load in the lungs of mice after 49 and 77 days post infection, compared to untreated and isoniazid as controls. B) Chronic BALB/c Mtb Erdman lung infection model with oral treatments begun on day 14, followed by enumeration of bacterial burden after two months of treatment (day 70). Treatment combinations were: untreated, G9 (100 mg/kg q.d.), bedaquiline (B) (25 mg/kg q.d.)/pretomanid (P) (50 mg/kg q.d.), B/P/G9, B/P/sutezolid (U) (50 mg/kg q.d.), B/P/U/G9, B/P/moxifloxacin (M) (100 mg/kg q.d.), B/P/M/G9, and B/P/M/pyrazinamide (Z) (150 mg/kg q.d.). C) Chronic C3HeB/FeJ Mtb Erdman lung infection model using a low-dose aerosol infection allowed to progress for 56 days, after which oral treatment was begun once/day (q.d.) with G9 alone or in combination with other antibiotics for one or two months as described in B above. Significant changes in bacterial load were compared pairwise across treatment groups that included G9 after one and two months. (Mann–Whitney test, GraphPad Prism; N = 5 mice per group; *p < 0.05, **p < 0.005, ****p < 0.0001). The dotted line indicates the limit of detection, where no viable bacteria were detected.

Antibiotic Combinations Including G9 in Chronic BALB/c and C3HeB/FeJ Lung Infection Models

Antibiotics with novel mechanisms of action are needed to overcome antibiotic resistance and provide a pan-TB treatment regimen that can cure patients on an all-comer basis. Encouraged by the activity of G9 as a single agent in vivo, we tested it in combination with standard-of-care antibiotics to determine whether it could contribute to a regimen that more rapidly clears Mtb. BALB/c mice were infected with a high-dose aerosol of Mtb Erdman and allowed to establish infection for 14 days prior to oral administration of antibiotic treatments (Figure B). Treatment combinations (untreated, G9 (100 mg/kg q.d.), bedaquiline (B) (25 mg/kg q.d.)/pretomanid (P) (50 mg/kg q.d.), B/P/G9, B/P/sutezolid (U) (50 mg/kg q.d.), B/P/U/G9, B/P/moxifloxacin (M) (100 mg/kg q.d.), B/P/M/G9, and B/P/M/pyrazinamide (Z) (150 mg/kg q.d.)) were administered for 2 months, followed by enumeration of bacteria burden in lungs by plating for CFUs. Bacterial expansion of Mtb Erdman in BALB/c mice was significant between day 1 and day 14 (P = 0.016*; Figure B). No significant reductions in bacterial burden were observed by the addition of G9 to any combination in this chronic BALB/c mouse infection model, and the addition of G9 to the combination of B+P led to a slight increase in CFUs (P = 0.016*; Figure B). Direct comparison of plating conditions of lung homogenates with or without 0.1 mM guanine had no effect on recovery of viable Mtb Erdman bacteria in these studies (Figure S5).

An alternative Mtb chronic infection model using C3HeB/FeJ mice was also assessed. Mice were administered Mtb Erdman using low-dose aerosol intranasal infection and allowed to establish an infection. After 56 days, untreated lungs harbored 5 × 108 CFU (Figure C, untreated) and exhibited advanced pathology. Treatments with G9 as a single agent or in combination with approved antibiotics were dosed orally for one and two months prior to measuring the bacterial burden in the lungs. When comparing treatment effects over time, all treatment combinations led to a significant reduction in viable Mtb in the lungs of mice when compared to the pre-treatment group, and most groups exhibited a significant reduction after two months of treatment compared to one month (B/P*, B/P/M*, and B/P/M/G9*, B/P/Uns, B/P/U/G9ns, and B/P/M/Z*) (Figure S6). After one month of treatment as a single agent, G9 (100 mg/kg q.d.) led to a significant reduction in the number of Mtb bacteria (Figure C; P = 0.016*) but did not significantly improve depth of bacterial clearance when combined with other antibiotics (Figure C). All mice treated with G9 as a single agent survived, in contrast to untreated mice that do not survive 4 months postinfection. Notably, after two months of treatment, we observed a significant improvement in bacterial clearance from the lungs of mice treated with the B/P/M/G9 combination, including several mice with no bacteria in their lungs after treatment (Figure C; P = 0.028*). Two months of treatment with a combination of B/P/U/G9 did not lead to a statistically significant decrease in CFUs compared to B/P/U; however, two of the mice in this group had possessed no culturable Mtb in the lungs (Figure C).

Discussion

We have identified a novel class of antibiotics targeting Mtb GuaB2 that demonstrates efficacy as a single agent across acute C57BL/6, chronic C57BL/6, and C3HeB/FeJ Mtb mouse infection models but that lacked efficacy in a chronic BALB/c infection model. Combination of G9 with bedaquiline, pretomanid, and moxifloxacin led to a significant improvement in bactericidal activity in the C3HeB/FeJ Mtb Erdman infection model. Historically, the potential for the de novo purine biosynthesis pathway as an antibiotic target has been questioned because the presence of bypass mechanisms via nucleobase salvage could render these enzyme targets non-essential in vivo. ,, Our findings, alongside recent studies on PurF inhibition, , provide compelling evidence that guanine salvage is insufficient to sustain bacterial proliferation during infection. Although human macrophages reportedly contain ∼40 μM free guanine, and TB patient lung samples possess ∼5 μM free guanine, these concentrations fall below the threshold required to rescue Mtb from GuaB2 inhibition (Figures D and S1). The BALB/c chronic Mtb infection model is a static, primarily intracellular, latent infection model that does not reflect advanced pathology of human disease. However, it does model intracellular Mtb, and higher than predicted concentrations of G9 may be required to cover the target bactericidal concentration in macrophages and in the chronic BALB/c model. Alternatively, Mtb lesions could differ between chronic BALB/c and C57BL/6 mouse infection models, leading to different drug exposure, target attainment, and pharmacodynamics. ,

A key finding in this study is the contrasting performance of an antibiotic regimen containing the GuaB2 inhibitor G9 in the BALB/c versus C3HeB/FeJ mouse chronic infection models. Different effects of G9 combinations in the BALB/c versus C3HeB/FeJ infection models suggested that bacterial reservoirs modeled by these two mouse strains were differentially susceptible to GuaB inhibition. For example, primarily intracellular bacteria typical of BALB/c infection may respond differently from more heterogeneous bacterial reservoirs in a C3HeB/FeJ infection. The added benefit of G9 in the C3HeB/FeJ model suggested an improved activity against extracellular bacteria in necrotic granulomas and warrants further evaluation in relapse-capable preclinical models. The possibility that these mice may be cured of Mtb infection after only 2 months of combination treatment must be rigorously tested using a nonrelapsing cure study design. The mechanistic basis for failure in the chronic BALB/c model remains speculative but may include altered drug penetration into sites of infection, or populations of bacteria may possess altered sensitivity to GuaB2 inhibition. Phenotypic resistance of Mtb in macrophages and in vivo could be driven by several factors, including metabolic flux and enhanced reliance on purine salvage within the intracellular niche, target upregulation by adaptive increases in GuaB2 expression within the phagosome, pharmacodynamic alterations in Mtb cell wall permeability in the dormant state, and potential efflux of inhibitors from activated macrophages. Alternatively, penetration of G9 into cellular granulomas in BALB/c mice may limit drug exposure, or bacteria in these lesions could exhibit extreme drug tolerance. Measurement of macrophage-, lesion-, and caseum-specific drug exposure may help differentiate among these possibilities. Whether dosing higher could overcome these differences and improve activity in vivo is an open question. In contrast, the C3HeB/FeJ model, which better reflects the heterogeneous pathophysiology of human TB, showed a marked improvement in bactericidal activity when G9 was added to the B/P/M (bedaquiline, pretomanid, moxifloxacin) regime backbone. This suggests that GuaB2 inhibition is particularly effective against the large, extracellular bacterial populations found within hypoxic, necrotic, caseous granulomas previously described for this model. These results independently confirmed the vulnerability of non-replicating Mtb to inhibition of de novo purine biosynthesis shown by the bactericidal activity of PurF inhibition.

Conclusion

The dramatic improvement in the ADME and pharmacokinetic profiles of G9 allowed for the first rigorous test of long-term GuaB2 inhibition in vivo. The ability of G9 to accelerate bactericidal activity in necrotic lesions addresses one of the greatest hurdles in TB therapy: eradication of non-replicating persister populations that lead to relapse. The synergy observed between GuaB inhibitors and standard-of-care agents such as rifampicin and linezolid in vitro suggests that G9 could be a versatile component for future regimens. Improved bactericidal activity observed after treatment with B/P/M/G9 in the two-month C3HeB/FeJ study supports further evaluation of GuaBi-containing regimens in relapse-capable preclinical models, which, if successful, could fundamentally transform the global TB landscape by reducing treatment duration and improving patient adherence.

Methods

Bacterial Cultures, MIC, F.O.R., and Time-Kill Assays

Mycobacterium tuberculosis (Mtb) H37Ra (ATCC 25177), Mtb H37Rv (ATCC 27294), and Mtb Erdman (ATCC 35801) were grown on Middlebrook 7H10 agar plates (Hardy Diagnostics W30) or in Middlebrook 7H9 broth (Sigma M0178) + 10% OADC supplement (Sigma M0678) incubated at 37 °C for the indicated times. Bacterial minimal inhibitory concentration (MIC) and bactericidal concentration (MBC) were determined using a 12-day Alamar blue (Thermo DAL1100) microtiter assay. Bactericidal activity and time kill studies (0–28 days) were determined by sampling 1 mL cultures grown static at 37 °C in 2 mL polypropylene assay blocks treated with dose-responses of G1 or G9 over time, and viable bacteria were enumerated by plating for colony-forming units (CFUs) by serial dilution onto 7H10 agar plates. MICs and MBCs for M. tuberculosis H37Rv were performed by TICRO Biosciences. The MICs are reported as the average of at least three independent experiments.

Intracellular activity of G1 was performed at Evotec Biosciences by infecting human THP-1 macrophages in the presence of PMA with Mtb H37Rv-GFP (MOI = 0.2) in a roller bottle for 2 h at 37 °C, washed, and seeded in 384-well plates containing compounds in a dose-response. After 4 days of incubation at 37 °C, 5% CO2, cells were fixed with 4% paraformaldehyde, stained with DRAQ5, and high-content imaging was performed. Growth was measured by using GFP fluorescence and automated cell segmentation software. Bactericidal activity against intracellular Mtb H37Rv grown in C57BL/6 mouse bone marrow-derived macrophages was performed by TICRO Biosciences. In brief, differentiated BMDMs were infected with Mtb H37Rv (MOI = 0.2) for 4 h at 37 °C and incubated for 4 days with compound G1 or rifampicin. Intracellular bacteria were enumerated by lysis of macrophages and plated for viable CFUs. Normoxic (21% O2) and hypoxic (1% O2) activity of G1, G9, and isoniazid against Mtb H37Ra was determined after 20 days of incubation at 37 °C in 1 mL static cultures by CFU plating and Alamar blue assay.

Frequency of resistance studies was performed using Mtb H37Ra grown at 37 °C for 21 days, and the number of emergent drug-resistant mutants was counted. Mutants we restreaked on selective agar and known resistance-conferring genes (GuaB, KatG, InhA, and OxyR) were then amplified by colony PCR, Sanger sequenced, and analyzed for mutations.

Biochemical Characterization of Mtb GuaB2 and hIMPDH2 in the Presence of G1 and G9

M. tuberculosis GuaB2 and hIMPDH2 Nad­(P)­H-Glo Assays

Mtb GuaB2 inhibitor G1 and G9 biochemical IC50 values were determined using enzyme reaction reagents (Mtb GuaB2, hIMPDH2, IMP, and NAD+) diluted in assay buffer (50 mM Tris, pH 8.0, 100 mM KCl, 3 mM EDTA, 0.002% Tween-20, 1 mM DTT) and detected with the addition of the NAD­(P)­H-Glo detection system (Promega, G9062). See the Extended Methods for details.

Animal Usage Declaration

All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at Genentech with identification numbers 22-1184, 22-1520, 22-1186, and 23-1187. Genentech is an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited institution in accordance with the Guide for the Care and Use of Laboratory Animals and applicable laws and regulations.

In Vitro Metabolism Studies in Mouse Hepatocytes

The incubation of G9 with cryopreserved mouse hepatocytes was conducted according to the standard in-house protocol and analyzed by an LC–MS/MS system. See the Extended Methods for details.

In Vitro Plasma Protein Binding Study

Plasma protein binding experiments were performed in triplicate (n = 3) using a single-use rapid equilibrium dialysis (RED) plate from Thermo Fisher Scientific Inc. (Rockford, IL) by following the standard protocol from the supplier. See the Extended Methods for details.

In Vivo Pharmacokinetic Study in Mouse: Bioanalysis

G9 blood concentrations in mouse PK studies were determined by LC–MS/MS. See the Extended Methods for details.

Expression and Purification

The M. tuberculosis GuaB2 with CBS domain deletion (S2-R529_ΔE126-R252) was cloned into a modified pET52b vector to produce a fusion protein with an N-terminal His6 tag, purified using affinity chromatography on a 5 mL HisTrap FF column (GE Healthcare), and further purified by size-exclusion chromatography using a Superdex 200 26/60 column (GE Healthcare). Fractions containing the protein were pooled and concentrated to 21 mg/mL for crystallization.

Crystallization

M. tuberculosis GuaB2ΔCBS was crystallized by room temperature vapor diffusion setups in the presence of 5 mM IMP under the following conditions: 0.1 M TRIS pH 7.5, 0.2 M LiSO4, and 5% PEG 4000. Complex structures were obtained by soaking the crystals overnight in artificial mother liquor with 5 mM compound and 5 mM IMP. Crystals were cryo-protected with 30% ethylene glycol, 2 mM IMP, and 2 mM compound in mother liquor and flash frozen in liquid nitrogen. Data sets were collected at SSRL 12-1.

Structure Determination

The structure of Mtb GuaB2 bound to G9 was determined by molecular replacement (MR) using our prior structures, those originally derived from an MR search with published GuaB CBS-deletion coordinates (e.g., V. cholera PDB 4IX2). Mtb GuaB2 crystallized with one monomer per asymmetric unit (asu), while the four-fold operator of the I4 symmetry spacegroup reproduced the frequently observed GuaB tetramer arrangement in which the NAD binding region (and where our inhibitors are located) occurs at the interface between adjacent protomers. Structures were manually rebuilt using COOT and refined with Phenix , and Buster. Molecular graphics were prepared with the PyMOL Molecular Graphics System, Version 2.5.5, Schrödinger, LLC (2010). Structural models have been refined to good statistics (Supplemental Table 2) and deposited with structure factors in the PDB under code 12LG.

Synthesis of G9

graphic file with name id6c00350_0006.webp

General Chemistry Methods

All commercial solvents and reagents were used as obtained. NMR analysis was performed in a deuterated solvent with a Bruker Avance 400- or 500-MHz NMR spectrometer, referenced to trimethylsilane (TMS). See the Extended Methods for details.

Supplementary Material

id6c00350_si_001.xlsx (67.2KB, xlsx)
id6c00350_si_002.pdf (3.6MB, pdf)

Acknowledgments

We would like to thank William Reilley from TICRO Biosciences, Evotec, and WuXi for experimental design, planning, and execution of in vitro, ex vivo, and in vivo infection studies. Special thanks to Steven T. Rutherford for critical review of the manuscript. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (P30GM133894). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsinfecdis.6c00350.

  • 2D MIC data Mtb H37Ra and F.I.C. summary (XLSX)

  • Bactericidal dose-response curves of G1 against Mycobacterium tuberculosis H37Ra (Mtb) measured after 21-day broth culture in 7H9 + 10% OADC supplemented with varying concentrations of vehicle or exogenous nucleobase xanthine, guanine, and guanosine (Figure S1); activity of GuaB inhibitors G1 and G9 against Mtb H37Ra grown for 20 days in aerobic (21% O2) and hypoxic (1% O2) conditions (Figure S2); an omit map (ligand omitted from model) contoured at 3 sigma shows unambiguous density for the ligand pose; Mtb GuaB2ΔCBS co-crystallized with G9 (PDB 12LG) shows unbiased density that indicates the high quality of starting information graphically (Figure S3); pharmacokinetics of G9 in C57BL/6 mice using different routes of administration, formulation, and simulated PK in chronic Mtb H37Rv infection (Figure S4); effect of guanine on recovery of viable Mtb Erdman from chronic BALB/c and C3HeB/FeJ lung infections (Figure S5); effect of antibiotic combinations over time in the chronic C3HeB/FeJ Mtb Erdman lung infection model (Figure S6); in vitro safety profile of G9 (Table S1); data collection and refinement statistics (PDB 12LG) (Table S2); frequency of spontaneous resistance to G1 and isoniazid in Mtb H37Ra and the genetic nature of the resistance alleles (Table S3); PK data summary for super acute C57BL/6 Mtb H37Rv lung infection model (Table S4); Extended Methods: bacterial cultures, MIC, FOR, and time-kill assays; biochemical characterization of Mtb GuaB2 and hIMPDH2 in the presence of G1 and G9; M. tuberculosis GuaB2 NAD­(P)­H-Glo assay; hIMPDH2 NAD­(P)­H-Glo assay; animal usage declaration; pharmacokinetics and metabolism of G9; in vitro metabolism studies in mouse hepatocytes; in vivo pharmacokinetic study in mouse: bioanalysis; expression and purification; crystallization; structure determination; synthesis of G9; general chemistry methods (PDF)

⟠.

E.M.K. and T.D.C. contributed equally.

The authors declare the following competing financial interest(s): Most authors were employed by Genentech, Inc. a member of the Roche Group when the research was conducted.

Published as part of ACS Infectious Diseases special issue “Bridging Gaps in Global Health with Medicinal Chemistry”.

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