Human fungal pathogens resistant to conventional therapeutics pose a major threat to global human health. Thus, there is an urgent need to discover new antifungal drugs that act via novel mechanisms of action. Here, we show that commercial herbicides that inhibit acetohydroxyacid synthase (AHAS) have potent and broad-spectrum antifungal activity in vitro and that chlorimuron ethyl, a member of the sulfonylurea herbicide family, has antifungal activity in a mouse model. Thus, this study shows that AHAS inhibitors have strong potential to be developed into potent antifungal therapeutic agents.

The increased prevalence of drug-resistant human pathogenic fungal diseases poses a major threat to global human health. Thus, new drugs are urgently required to combat these infections. Here, we demonstrate that acetohydroxyacid synthase (AHAS), the first enzyme in the branched-chain amino acid biosynthesis pathway, is a promising new target for antifungal drug discovery. First, we show that several AHAS inhibitors developed as commercial herbicides are powerful accumulative inhibitors of Candida albicans AHAS (K i values as low as 800 pM) and have determined high-resolution crystal structures of this enzyme in complex with several of these herbicides. In addition, we have demonstrated that chlorimuron ethyl (CE), a member of the sulfonylurea herbicide family, has potent antifungal activity against five different Candida species and Cryptococcus neoformans (with minimum inhibitory concentration, 50% values as low as 7 nM). Furthermore, in these assays, we have shown CE and itraconazole (a P450 inhibitor) can act synergistically to further improve potency. Finally, we show in Candida albicans-infected mice that CE is highly effective in clearing pathogenic fungal burden in the lungs, liver, and spleen, thus reducing overall mortality rates. Therefore, in view of their low toxicity to human cells, AHAS inhibitors represent a new class of antifungal drug candidates.

The number of diagnosed pathogenic fungal infections continues to rise significantly each year, with an estimated 1 billion people worldwide now at risk (1) and ∼2 million people per annum succumbing to these infections (2), a number that exceeds the annual death toll for tuberculosis or malaria (3). Despite this, major advances in the search for novel antifungal treatments have been a rarity (4). Individuals commonly affected by fungal infections (e.g., candidiasis, cryptococcosis, aspergillosis, or Pneumocystis pneumonia) are transplant recipients, the immunocompromised suffering HIV, or patients receiving immunosuppressant therapy. However, existing antifungal therapeutics are limited to just a few classes of drugs (e.g., polyenes, azoles, echinocandins, and fluorinated pyrimidine analogs), and these are costly to produce. Furthermore, treatment using these medications requires high doses to be taken over a prolonged time, which can result in serious side effects and lack of compliance (5, 6). In addition to these factors, several clinically relevant pathogenic fungi, including Aspergillus fumigatus, Candida glabrata, Candida krusei, and Cryptococcus spp., are intrinsically resistant to some first-line antifungals (4, 5, 7). Moreover, acquired resistance to all these classes of antifungal drugs has been extensively reported (8⇓⇓–11), and the prevalence of multidrug-resistant strains of pathogenic fungi is continuing to rise (4, 12). Thus, it is clear that new drugs are urgently needed to combat this major threat to human health.

A potential target for the development of new antifungal drugs is acetohydroxyacid synthase (AHAS; EC 2.2.1.6), the first enzyme in the branched-chain amino acid (BCAA) biosynthesis pathway. BCAAs are synthesized by a common pathway in plants and microorganisms; however, this pathway is absent in animals, making it an especially attractive drug target (13). It has previously been shown that AHAS activity is crucial for survival of Candida albicans and Cryptococcus neoformans in vitro, and BCAA auxotrophic strains of these two fungal pathogens are avirulent in vivo (14, 15). These findings suggest AHAS inhibitors could be useful as antifungal agents.

It has been 30 y since the first AHAS inhibitors were introduced as commercial herbicides, with five families of compounds [i.e., sulfonylureas (SUs), imidazolinones (IMIs), pyrimidinyl-benzoates (PBs), triazolopyrimidines (TPs), and sulfonylamino-carbonyl-triazolinones (SCTs)] still in use today (16), which is a testament to their robustness as biocidal agents. Further enhancing their credentials as drug candidates, these compounds display low toxicity in mammals (LD 50 in rat >5 g/kg), which is expected since animals do not possess the BCAA pathway (13, 17). Studies on AHAS inhibitors as antifungals have been sporadic and inconclusive. Chlorimuron ethyl (CE) (Fig. 1A), an SU herbicide, has previously been shown to inhibit the growth of C. albicans in culture with a minimum inhibitory concentration, 50% (MIC 50 ) of 2 μM (18), and several noncommercial TPs have activity against C. albicans, C. neoformans, and A. fumigatus with MIC 50 values in the range of 1–4 μg/mL (19). However, beyond these reports, a comprehensive study into the suitability of the AHAS-inhibiting herbicides as antifungal agents has yet to be undertaken.

Results

CaAHAS and CnAHAS Inhibition by Commercial Herbicides. Representatives of the five families of herbicides that target AHAS (SI Appendix, Fig. S1) were tested as inhibitors of C. albicans (Ca) AHAS and C. neoformans (Cn) AHAS. Members of the SU and TP families are the most potent inhibitors (Table 1), with CE having a K i of 24.26 nM for CaAHAS and 119.7 nM for CnAHAS (Fig. 1B), metosulam (MT) a K i of 800 pM for CaAHAS, and cloransulam methyl a K i of 35.3 nM for CnAHAS. Members of the SCT, PB, and IMI families inhibit CaAHAS, but with K i values only in the micromolar range, while the SCTs and PBs inhibit CnAHAS, with K i values in the millimolar range (Table 1). CnAHAS is partially resistant to SU herbicides, including bensulfuron methyl (BSM), ethoxysulfuron (ES), and sulfometuron methyl (SM). This lower inhibitory activity appears to be due to the P188A and A191L substitutions in the CnAHAS herbicide binding pocket compared with CaAHAS (discussed below) (SI Appendix, Fig. S2A). Two noncommercial SUs derived from the chemical structure of CE, iodomuron ethyl (IE) and iodomuron methyl (IM) (18) (SI Appendix, Fig. S1), were also tested as inhibitors of the fungal enzymes. Compared with CE, these compounds show a two- to fourfold reduction in the K i values against CaAHAS; however, this improvement in binding affinity is not extended to CnAHAS (Table 1). Table 1. Inhibition constants and kinetic rate constants of accumulative inhibition of commercial herbicides for fungal AHASs Recently, we demonstrated AHAS inhibition by commercial herbicides fits a model of time-dependent accumulative inhibition from which first-order rate constants of enzyme inactivation (k iapp ) and enzyme recovery (k 3 ) can be determined (20). Here, we found that the five families of herbicides show different levels of reversible accumulative inhibition for CaAHAS and CnAHAS (Table 1 and SI Appendix, Fig. S3). The k iapp and k 3 values were determined by curve fitting to Eq. 3 (20) and show that three families of herbicides (i.e., SUs, TPs, and SCTs) are highly effective in promoting the inactivation of CaAHAS (k iapp values from 0.33 to 21.26 min−1). Despite the high level of conservation observed in the herbicide-binding site of CnAHAS and CaAHAS (SI Appendix, Fig. S2A), the former enzyme is less prone to undergo inactivation by commercial herbicides with k iapp values in the range of 0.13–3.99 min−1. k 3 values for CaAHAS in the presence of IE, and for CnAHAS in the presence of pyroxsulam (PYS), flumetsulam (FT), or propoxycarbazone (PC) are so low that the inactivation evoked by these herbicides is virtually irreversible (Table 1). This implies that, when k iapp >> k 3 , the enzyme’s ability to recover from herbicide-induced inactivation determines the potency of accumulative inhibition (k iapp /k 3 ).

CaAHAS Structures in Complex with Herbicide. To understand the molecular basis of herbicide inhibition of CaAHAS, we cocrystallized this enzyme in complex with five SUs, CE, BSM, SM, IE, and IM; two TPs, MT and PS; and the SCT, PC (SI Appendix, Table S1). The overall fold of these structures is similar to that of the uninhibited enzyme, with rmsd values between 0.145 and 0.271 Å when all Cα atoms are superposed, but in these structures the I192–Q198 region, which is involved in herbicide binding (SI Appendix, Fig. S5), is now fully resolved.

Conformational Changes in CaAHAS upon Herbicide Binding. A comparison of uninhibited CaAHAS and the CaAHAS-inhibitor complexes shows that, in the uninhibited enzyme, R376 and W582 protrude toward the herbicide-binding cavity and occupy the location taken by the heterocyclic ring of all three families of herbicides (SI Appendix, Fig. S9). This implies that these residues would block herbicide binding. However, the side chains of R376 and W582 change their conformation so as to make numerous contacts with the herbicides. The adjustments of W582 are the most important for herbicide binding because these allow the π stacking interaction that locks the herbicides into the binding pocket. Other residues in the herbicide-binding site, including P188, A191, K247, and D375, undergo less pronounced side-chain and backbone adjustments to accommodate the herbicides (SI Appendix, Fig. S9). Compared with AtAHAS (22, 23) the herbicide-binding site in CaAHAS is much more rigid and ready to receive inhibitors with only minor conformational changes, a feature of importance for the rational design of alternative CaAHAS inhibitors.

Effect of P188A and A191L Substitutions in CnAHAS. To understand why some SU herbicides are potent inhibitors of CaAHAS but fail to inhibit CnAHAS we performed in silico mutation and energy minimization of P188A and A191L on the structures of uninhibited CaAHAS and for the CE and MT complexes. These models show that in the P188A substitution key nonpolar interactions (including CH–π interactions; see SI Appendix, Fig. S7) formed between P188 and the aromatic ring of the SUs are no longer present due to the smaller alanine side chain (SI Appendix, Fig. S2C). However, the side chain of leucine in the A191L substitution protrudes toward the binding cavity, reducing its volume; therefore, conformational adjustments are required to accept the herbicide (SI Appendix, Fig. S2C). Although extra contacts may be formed in the presence of this substitution, the overall effect of the P188A and A191L substitutions is negative as witnessed by the increase in the K i value of CE and resistance in the presence of SM, BSM, and ES. In contrast, the K i values of the TPs are less affected by these substitutions (Table 1). In the light of these results, this appears to be because these herbicides rely less on the interactions formed with P188 than on the additional contacts that may occur with the A191L substitution (SI Appendix, Fig. S2D).

Fungal Growth Inhibition by AHAS-Inhibiting Herbicides. The five classes of commercial herbicide that target AHAS were assessed for their ability to prevent the growth of five Candida species (C. albicans, Candida parapsilosis, C. glabrata, Candida tropicalis, and C. krusei), C. neoformans, and S. cerevisiae in culture (SI Appendix, Table S2). CE was found to display broad-spectrum antifungal activity, being highly effective in preventing the growth of C. albicans, C. parapsilosis, C. glabrata, and S. cerevisiae with MIC values as low as 0.03 μg/mL (72.32 nM), 0.003 μg/mL (7.23 nM), 0.005 μg/mL (12.05 nM), and 0.008 μg/mL (19.29 nM), respectively (Table 2), while other herbicides, including MT, are also effective at preventing growth, but not with the same level of broad-spectrum activity (SI Appendix, Table S2). Independent of the strain tested, some herbicides reduced growth for 24 h, whereas CE, among others, maintained potency for up to 72 h (Fig. 5A and SI Appendix, Table S2). CE has a minimum fungicidal concentration (MFC) for C. albicans that is four times MIC, and thus is considered fungicidal rather than fungistatic (Table 2). Table 2. MIC and MFC of CE for fungal pathogen growth in cell culture Fig. 5. Fungal growth inhibition by herbicide. (A) Percentage of C. albicans cell growth vs. log 10 [CE] or [PS] after 24, 48, and 72 h. The solid lines represent the best fit to the data using the modified Gompertz function (57). (B) Isobolograms showing the interactions between CE (Top) or PS (Bottom) and three front-line antifungal drugs against C. albicans growth. The FIC CE are defined as the MIC of CE in combination with an antifungal drug (itraconazole, fluconazole, or amphotericin B) divided by the MIC of CE alone. A straight line represents an additive interaction (CE–amphotericin B combination), a concave curve indicates synergy (CE–itraconazole combination), and a convex curve shows antagonism (CE–fluconazole interaction). The FIC values for PS and each antifungal drug were determined in the same way. AmB, amphotericin B; Flu, fluconazole; Itr, itraconazole. (C) Fungal growth inhibition by CE in presence of different nitrogen sources: (i) 10 mM proline (blue lines); (ii) 2.5 mM proline, 2.5 mM leucine, 2.5 mM valine, and 2.5 mM isoleucine (red lines); (iii) 3.3 mM leucine, 3.3 mM valine, and 3.3 mM isoleucine (black lines); and (iv) 4 g/L BSA (magenta lines). The MIC values were determined at 48-h incubation using the modified Gompertz function. Note that the substitution of ammonium sulfate for proline, as the nitrogen source, does not produce a significant change in the MIC values reported in SI Appendix, Table S2. (D) Inhibition of C. albicans growth by CE in presence different combinations of BCAAs. YNB media was supplemented with 5 mM valine, 5 mM leucine, and/or 5 mM isoleucine in the presence or absence of 10 mM ammonium sulfate or 10 mM proline. (E) C. albicans growth in the presence of different combinations of BCAAs (5 mM each) and in the absence of herbicide. Error bars represent the SD of the mean (SEM) (n = 3).

Antifungal Activity of CE Is Enhanced by Itraconazole. To test if the current first-line antifungal compounds, fluconazole, itraconazole, or amphotericin B, can influence the activity of CE in reducing the growth of C. albicans, a grid experiment was performed. This showed that CE in combination with itraconazole had a synergistic effect, resulting in a fractional inhibitory concentration (FIC) value of 0.63 (Fig. 5B, Top). In addition, an additive interaction was observed when CE and amphotericin B were combined (FIC = 1). In contrast, the combination of CE with fluconazole shows an antagonistic interaction (FIC = 1.5). It is worth noting that fluconazole (a fungistatic agent) is an antagonist of other antifungal drugs, for example amphotericin B (a fungicidal) (28), an interaction usually observed when the drugs have different mechanisms of action. A method that plants adopt to detoxify AHAS-inhibiting herbicides is to use P450 enzymes. This is achieved by adding or removing functional groups, thereby preventing binding to the enzyme (29, 30). Azoles are potent inhibitors of the fungal cytochrome P450 lanosterol 14-α-demethylase (CYP51A1). They also have activity toward human P450 enzymes (CYP3A4 and CYP2C9), increasing the serum concentration of several drugs (31). Therefore, we hypothesize itraconazole may inhibit CE degradation via P450s, thus maintaining the concentration of the compound in the cell for a longer time period. This implies existing fungal P450s can play a role in reducing the antifungal activity of those herbicides that are susceptible to modification. To answer this question, a synergy grid experiment was performed using PS, a TP that has a low MIC value for C. albicans growth of 6.41 μg/mL after 48 h, but which loses activity after 72 h (MIC = 185.5 μg/mL) (Fig. 5A), in combination with fluconazole, itraconazole, or amphotericin B. The results show that only itraconazole enhances the longevity of antifungal activity of PS (synergistic effect, FIC = 0.31) (Fig. 5B, Bottom), resulting in a >15-fold reduction in MIC value for PS after 72 h. However, adding fresh PS alone after 24, 48, and 72 h to concentrations equal to those used in the microdilution tests or additive self-drug combination of PS did not produce an improvement in the MIC values. These data suggest that itraconazole could be coadministered to minimize the effect of herbicide metabolism.

Effect of BCAA Supplementation on the Antifungal Activity of CE. Next, we investigated whether BCAA supplementation affects the efficacy of CE in reducing the growth of five Candida species and C. neoformans in culture media. Compared with the control [yeast nitrogen base (YNB) broth supplemented with 10 mM proline as nitrogen source], the addition of BCAAs (10 mM; >15-fold the concentration found in human blood) as sole nitrogen source produces a shift in the MICs of CE for all five Candida strains. C. neoformans is unable to grow under these conditions (15) (Fig. 5C). The effect of BCAAs on C. albicans growth in the presence of CE is significantly lower (2.3-fold increase of MIC) compared with that of the other Candida strains (>20-fold increase of MIC). The inclusion of proline along with BCAAs partially restores the antifungal activity of CE against C. albicans, C. parapsilosis, and C. tropicalis (Fig. 5C). In this situation, C. neoformans growth was viable (15) with a twofold increase in the MIC of CE. Given that serum albumin could be a potential source of BCAAs during fungal infections, we next investigated the effect of BSA (4 g/L) on the performance of CE. These results showed that only C. glabrata and C. parapsilosis can partially bypass AHAS inhibition through scavenging BCAAs from BSA (8.3- and 2.9-fold increase of MIC, respectively). Remarkably, the MIC of CE against C. neoformans was reduced a further 5.5-fold compared with the control under this assay condition (Fig. 5C). To identify the requirements of these fungal pathogens to circumvent AHAS inhibition in the presence of BCAAs, we next assessed the growth of C. albicans in YNB broth with leucine, valine, isoleucine, proline, and/or ammonium sulfate as the only nitrogen source or in combinations, and in the presence of different concentrations of CE (Fig. 5D). These experiments showed that the addition of 10 mM valine, leucine, or isoleucine as the sole nitrogen source does not affect the efficacy of CE in preventing C. albicans growth. Indeed, the MICs of CE determined under these conditions are lower compared with the MIC determined using YNB media supplemented with 10 mM ammonium sulfate or proline. However, the combination of valine and isoleucine suppresses significantly the antifungal activity of CE (MIC = 5.6 μg/mL), while this effect is nullified by addition of leucine, proline, or ammonium sulfate (Fig. 5D). Amino acid transport is highly regulated by the nitrogen catabolite repression or the Ssy1p-Ptr3p-Ssy5p systems, the intracellular and extracellular amino acid ratio, and the presence of nitrogenated molecules in the environment (32, 33). Ammonium diminishes BCAA transport via Gap1 (Gap2 in C. albicans) and Bap2 repression (34, 35), a mechanism which explains why ammonium sulfate restores the antifungal activity of CE in the presence of BCAAs. In agreement with this result, 100 μg/mL eugenol, an inhibitor of BCAA permeases (36), synergizes with CE (FIC 0.38; 48-h MIC CE = 0.125 μg/mL) against C. albicans when BCAAs are present in the growth media (SI Appendix, Fig. S10). However, while it has been shown that proline derepresses BCAA transport (34), here we observed that it also diminishes the ability of C. albicans to bypass AHAS inhibition by taking up BCAAs from the media. In addition, we observed when leucine, valine, and isoleucine are added in combination in the media, growth is reduced (Fig. 5E). This could be due to reciprocal competition as the three BCAAs try to use the same method of transport into the cell (37) (Fig. 5E). It has been shown that absence of valine is highly detrimental for C. albicans AHAS (ilv2Δ) mutant growth (14). Here, we show valine increases the growth of C. albicans and that both leucine and isoleucine compete with valine, as seen by a 15% reduction in fungal growth when these are in combination (Leu–Val or Ile–Val). Isoleucine also inhibits growth in presence of leucine and the combination of the three BCAAs yields 35% less growth compared with that observed when valine is the nitrogen source (Fig. 5E). These results suggest that, even though C. albicans is able to scavenge BCAAs from the environment and, to some extent, bypass AHAS inhibition by herbicide to support growth, the intricate interactions between substrates and amino acids limit the absorption of BCAAs and therefore add further support for the concept of using AHAS-inhibiting herbicides to treat human fungal infections.