bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Synergistic and Antagonistic Drug Interactions in the Treatment of Systemic Fungal Infections

Morgan A. Wambaugh, Steven T. Denham, Brianna Brammer, Miekan Stonhill, and Jessica C. S. Brown

Division of Microbiology and Immunology, Pathology Department, University of Utah School of Medicine, Salt Lake City, UT 84132, USA

[email protected]; [email protected]

Keywords Cryptococcus neoformans, overlap2 method (O2M), synergy, antagonism, drug interactions, fluconazole, dicyclomine hydrochloride, berbamine hydrochloride, nafcillin sodium, Candida species

Summary Invasive fungal infections cause 1.6 million deaths annually, primarily in immunocompromised individuals. Mortality rates are as high as 90% due to limited number of efficacious drugs and poor drug availability. The azole class antifungal, fluconazole, is widely available and has multi-species activity but only inhibits fungal cell growth instead of killing fungal cells, necessitating long treatments. To improve fluconazole treatments, we used our novel high-throughput method, the overlap2 method (O2M), to identify drugs that interact with fluconazole, either increasing or decreasing efficacy. Although serendipitous identification of these interactions is rare, O2M allows us to screen molecules five times faster than testing combinations individually and greatly enriches for interactors. We identified 40 molecules that act synergistically (amplify activity) and 19 molecules that act antagonistically (decrease efficacy) when combined with fluconazole. We found that critical frontline beta-lactam antibiotics antagonize fluconazole activity. A promising fluconazole-synergizing anticholinergic drug, dicyclomine, increases fungal cell permeability and inhibits nutrient intake when combined with fluconazole. In vivo, this combination doubled the time-to-endpoint of mice with disseminated Cryptococcus neoformans infections. Thus, our ability to rapidly identify synergistic and antagonistic drug interactions can potentially alter the patient outcomes.

Introduction Invasive fungal infections are an increasing problem worldwide, contributing to 1.6 million deaths annually (Almeida et al., 2019; Bongomin et al., 2017; Brown et al., 2012). These problematic infections are difficult to treat for many reasons. Delayed diagnoses, the paucity and toxicity of antifungal drugs, and the already immunocompromised state of many patients result in mortality rates of up to 90% (Brown et al., 2012; Pianalto and Alspaugh, 2016; Scorzoni et al., 2017). To date, there are only four classes of antifungals, which primarily target the fungal cell envelope (cell wall and plasma membrane) (Coelho and Casadevall, 2016; Odds et al., 2003; Pianalto and Alspaugh, 2016; Scorzoni et al., 2017). The population of immunocompromised individuals is growing due to medical advancements, such as immunosuppression for transplants and chemotherapy. Emerging fungal pathogens are simultaneously increasing in both clinical burden and the number of causal species due to human activity such as agricultural drug use (Berger et al., 2017) and global warming (Almeida et al., 2019; Garcia-Solache and Casadevall, 2010). Thus, the need for more and better antifungal therapeutics is evident. bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Among the most common invasive mycoses is cryptococcosis, which causes 220,000 cases and 180,000 deaths per year (Rajasingham et al., 2017). Cryptococcus neoformans and Cryptococcus gattii are the etiological agents of cryptococcosis, though nearly 95% of cases are caused by C. neoformans (Brown et al., 2012; Maziarz and Perfect, 2016). As C. neoformans is globally distributed throughout the environment, most individuals are exposed by two years of age (Goldman et al., 2001). However, systemic disease primarily occurs in the immunocompromised, particularly those with decreased T helper-1 cell reactions (Maziarz and Perfect, 2016). Accordingly, HIV/AIDS patients account for 80% of cryptococcal cases (Maziarz and Perfect, 2016; Rajasingham et al., 2017). The primary treatment for cryptococcosis involves three different classes of antifungals. Standard care is a combination of amphotericin B (polyene class) and 5-fluorocytosine (5-FC; pyrimidine analog) for two weeks, followed by high dose azole treatment (e.g. fluconazole (FLZ)) for at least 8 weeks, and finally a low dose oral FLZ for at least 6 months (Cox and Perfect, 2018; Mourad and Perfect, 2018). Despite this, mortality rates remain as high as 80% for cryptococcal meningitis (Rajasingham et al., 2017). This is mainly due to the difficulty of obtaining ideal treatment standards. 5-FC is unavailable in 78% of countries, mostly due to licensing issues (Kneale et al., 2016; Mourad and Perfect, 2018). Without the inclusion of 5-FC in the treatment regiment, mortality increases by up to 25% (Kneale et al., 2016). Amphotericin B is administered intravenously, so treatment requires hospitalization, which is particularly challenging in areas such as sub-Saharan Africa, which has the highest burden of cryptococcal disease (Rajasingham et al., 2017). Due to these therapeutic hurdles, many patients are treated with FLZ alone, which decreases survival rates from 75% to 30% in high burden areas (Kneale et al., 2016). Additional treatment options are thus needed to prevent these unnecessary deaths. One theoretical approach to improve treatment is synergistic combination therapy. Synergistic interactions occur when the combined effect of two drugs is greater than the sum of each drug’s individual activity (Cokol et al., 2011; Kalan and Wright, 2011). This is a powerful treatment option which has been utilized for a variety of infections (Kalan and Wright, 2011; Robbins et al., 2015; Spitzer et al., 2011; Zheng et al., 2018). Amphotericin B and 5-FC act synergistically, and mortality rates increase dramatically when one is unavailable (Beggs, 1986; Kneale et al., 2016; Schwarz et al., 2006). Synergistic interactions can also cause fungistatic drugs to switch to fungicidal, providing a more effective treatment option (Cowen et al., 2009). Additionally, molecules can interact antagonistically to decrease therapeutic efficacy (Caesar and Cech, 2019; Roberts and Gibbs, 2018). Antagonistic interactions further complicate this already challenging infection (Khandeparkar and Rataboli, 2017; Vadlapatla et al., 2014), since immunocompromised patients are frequently treated with multiple drugs. 56% of AIDS patients experience polypharmacy, or greater than five medications (Siefried et al., 2018). Polypharmacy doubles the risk of antiviral therapy nonadherence to 49% of HIV+ patients (Lohman et al., 2018) and increases mortality by 68% in HIV+ and 99% in HIV- patients (Cantudo-Cuenca et al., 2014). Better understanding of the molecular mechanisms underlying both synergistic and antagonistic drug interactions will allow us to improve identification and selection for or against these interactions. The overlap2 method (O2M) uses at least one known synergistic drug pair and a large scale chemical-genetics dataset to predict synergistic and antagonistic drug interactions rapidly and on large scales (Brown et al., 2014; Wambaugh and Brown, 2018; Wambaugh et al., 2017). Each molecule of the synergistic pair induces a growth phenotype in a precise set of mutants (enhanced or reduced growth). Since these mutants exhibit the same phenotype in the presence of both molecules in a synergistic pair, we hypothesize that any other molecule eliciting the same phenotype in those mutants will also synergize with each molecule in the original pair. This method can be used against multiple microbes and applied to any published chemical-genetics dataset (Brown et al., 2014; Wambaugh et al., 2017). bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

In this study, we utilized previously identified synergy prediction mutants (Brown et al., 2014) to screen a library of small molecules enriched for Federal Drug Administration (FDA-)- approved molecules. We non-discriminately identified 59 molecules that interact with FLZ, either synergistically or antagonistically. When validating these new combinations, we found that even though the analysis used a C. neoformans dataset (Brown et al., 2014), our synergistic and antagonistic combinations acted against pathogenic fungi from multiple phyla. These include C. deuterogattii, Candida species, and multiple clinical and environmental strains of C. neoformans, as well as clinical isolates of the increasingly problematic and multi-drug resistant species Candida auris (Chowdhary et al., 2017). Furthermore, we elucidated molecular mechanisms underlying the interaction with FLZ for a few of our most clinically relevant combinations. We also demonstrate these effects in an in vivo model of cryptococcosis. A particularly promising synergistic combination, dicyclomine hydrochloride and FLZ, almost doubled time-to-endpoint in a murine infection model. In sum, our high-throughput method, O2M, identifies FLZ interacting molecules with potential clinical impacts.

Results

Synergy prediction mutants for fluconazole allow for high-throughput screening of small molecule interactions We previously demonstrated that O2M identifies genes whose knockout mutants, termed synergy prediction mutants, exhibit phenotypes that are indicative of synergistic interactions between small molecules (Brown et al., 2014; Wambaugh et al., 2017). O2M requires a chemical-genetics dataset, in which a library of knockout mutants is grown in the presence of >100 small molecules. We calculated quantitative growth scores (slower or faster growth) for each mutant/molecule combination. This produces the “chemical genetic signature” for each molecule in the dataset. We then used these “signatures” from a known synergistic combination to identify additional combinations, the rationale being that similarities between chemical-genetic signatures of known synergistic pairs contain information that is indicative of the interaction. When we compare the chemical-genetic signatures of a pair of small molecules already known to act synergistically, we identify a subset of mutants with similar growth scores and term them “synergy prediction mutants” (Fig. 1A). We hypothesize that any molecule eliciting the same growth phenotype in these mutants would also act synergistically with either molecule in the known synergistic pair. This was completed and tested in our previous publication (Brown et al., 2014) using FLZ and its known synergistic interacting partners fenpropimorph and sertraline (Jansen et al., 2009; Zhai et al., 2012). O2M identified three gene deletion mutants (cnag_00573Δ, cnag_03664Δ, and cnag_03917Δ) as synergy prediction mutants for identifying interactions with FLZ (Brown et al., 2014). Using these gene mutants, we performed a high-throughput screen for synergistic interactions. Our assay is simple: differential growth between wild-type and synergy prediction mutants is indicative of a synergistic interaction with FLZ or any other starting drug. It does not require multi-drug assays, as the “synergy prediction mutant” substitutes for one of the small molecules in the interaction, phenocopying the FLZ-small molecule interaction to produce synthetic lethality. We screened the Microsource Spectrum Collection, a small-molecule library of 2,000 compounds enriched for FDA-approved molecules. We grew C. neoformans wild-type and synergy prediction mutants (cnag_00573Δ and cnag_03917Δ) in the presence of each small molecule (1 µM), identifying those that caused a significant difference in growth between the wild-type and both synergy prediction mutants after 48 hours of growth (Fig. 1B). The mutant cnag_03664Δ was not used due to its inherent slow growth. Using these synergy prediction mutants, we identified 313 putative FLZ synergistic molecules (Table S1). We validated potential synergistic interactions in checkerboard assays, for which serial dilutions of each drug are crossed in a 96-well plate (Fig. 1B). Synergistic interactions are bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

defined as a ≥ 4-fold decrease in the minimum inhibitory concentration (MIC) of each small molecule in the pair, resulting in a fractional inhibitory concentration index (FICI) of ≤ 0.5 (Johnson et al., 2004; Odds, 2003). We tested the 129 molecules with single agent efficacy against C. neoformans growth in the preferred checkerboard assay. We found that 40 molecules were synergistic with FLZ, meaning 31% of these molecules were correctly predicted by O2M (Fig. 2A). However, checkerboard assays require that both small molecules in the pair are able to inhibit growth of C. neoformans individually, which was not the case with all our putative synergistic molecules. In those cases, we performed Bliss Independence, which identifies whether molecules enhance the action of FLZ (Tang et al., 2015). In a 96-well plate, we created a gradient of FLZ combined with 10 µM and 100 nM concentrations of the 55 small molecules that could not inhibit C. neoformans alone. We found 6 of these molecules enhanced the action of FLZ at both 10 µM and 100 nM concentrations and were deemed synergistic (Fig. S1). The FLZ-synergistic molecules belonged to a wide range of bioactive categories including antidepressants, adrenergic agonists, as well as antiinfectives (Fig. 2B and Table 1). Additionally, our screen identified antagonistic interactions. These interactions are defined by a minimum 4-fold increase in MICs causing increased fungal growth (Cetin et al., 2013). We identified 19 antagonistic interactions with FLZ (Fig. 2C). Of note, many antagonists were documented antiinfectives, including some antifungals (Fig. 2D and Table 1). The remaining 70 molecules did not interact with FLZ and represent false positives of our screen (Fig. S2A). Overall, the O2M screen predicted that 16% of the library’s small molecules would interact with FLZ. Of the predicted interactions, 46% were validated by checkerboard assay to truly interact with FLZ (synergistic or antagonistic) (Fig. 2E). All small molecules that interact with FLZ and inhibit fungal cell growth (i.e. were tested in checkerboard assays) are listed with their MICs in Table 1. FLZ non-interacting molecules are listed in Table S2. The remaining 129 molecules predicted to interact were not tested due to unavailability, or known toxicities that would have made them impractical treatments.

Identification of general anti-cryptococcal molecules by O2M During the screening process, wild-type C. neoformans is grown in each of the small molecules alone, allowing us to identify general anti-C. neoformans molecules (Fig. 1B). These molecules had MICs ranging from 16 nM to 760 µM, and were mostly listed as antifungals (Table S3). However, the phenotype of a general anti-C. neoformans molecule can overshadow any synthetic-lethal phenotypes in the synergy prediction mutant. Therefore, we also tested these molecules for synergistic interactions in the standard checkerboard assay. Two of the general anti-C. neoformans molecules, sulconazole nitrate and tacrolimus, were synergistic with FLZ (Fig. S2B).

Fluconazole-synergizing and -antagonizing responses are conserved across fungal species We next focused on several promising synergistic molecules based on either low MICs or interesting bioactivity, with the intent of testing them against additional C. neoformans strains and other medically important fungi. We also selected potentially important antagonistic interactions based on bioactivities relevant to cryptococcosis patient populations. We tested these interactions against the C. neoformans lab strain KN99 and 10 additional environmental or clinical C. neoformans isolates (Chen et al., 2015). We also tested our combinations against Cryptococcus deuterogattii, Candida albicans, Candida glabrata, and two strains of Candida auris. Our interacting small molecules displayed similar MICs across these different C. neoformans strains and fungal species (Table S4). Of the FLZ-interacting pairs, benzalkonium chloride, berbamine hydrochloride (HCl), clomipramine HCl, dicyclomine HCl, and sertraline HCl synergistically inhibited the growth of most of the strains/species (Fig. 3B, C, E, G, J). bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Additionally, nafcillin sodium was antagonistic with FLZ in most of the strains/species (Fig. 3M). The other synergistic or antagonistic molecule pairs had more variable results among the different strains/species (Fig. 3), highlighting the importance of testing putative antimicrobials against multiple strains when considering therapeutic relevance.

Molecular structure predicts additional synergistic interactions Upon identifying new FLZ-based synergistic pairs, we sought to predict other FLZ synergizing molecules based on structure alone. We chose four of our newly identified synergistic molecules that contained large ring structures (a commonality amongst our synergists): dicyclomine HCl, desipramine HCl, sertraline HCl, and diphenhydramine HCl. We used both ChemSpider (Pence and Williams, 2010) and ChemMine MSC Similarity Tool to identify structurally similar molecules (Backman et al., 2011). We chose three molecules (proadifen, drofenine, and naftidrofuryl) with ≥50% structure similarity to dicyclomine HCl, and all were synergistic with FLZ in checkerboard assays (Fig. S3A-D, M). We tested three molecules (impramine, mianserine, and lofepramine) with >40% structural similarity to desipramine, two of which were synergistic with FLZ (Fig. S3E-H, M). Lofepramine, which was not synergistic, is the prodrug to desipramine. Sibutramine, shares >40% structural similarity with sertraline, and is synergistic with FLZ as well (Fig. S3I, J, M). Lastly, we tested citalopram, which shares >45% structural similarity to diphenhydramine, but was not synergistic with FLZ (Fig. S3K-M). Overall, 75% of the structurally similar molecules proved to be synergistic with FLZ, demonstrating that structure can serve as a powerful basis to predict additional synergistic combinations prior to elucidating mechanism of action.

Exposure to beta-lactam antibiotics increases ergosterol levels and antagonizes fluconazole activity Among the antagonists, we were particularly interested in the interaction between nafcillin sodium (nafcillin) and FLZ that emerged in multiple fungal strains and species (Fig. 3M). Nafcillin is a common penicillinase-resistant penicillin antibiotic (Letourneau, 2019; Nathwani and Wood, 1993). Furthermore, the cryptococcosis patient population, which consists of mainly HIV/AIDS patients, is at high risk for multiple infections, increasing the likelihood that they could require overlapping treatments for bacterial and fungal infections (Kaplan et al., 2009). Thus, we first wanted to determine if other beta-lactam antibiotics would also antagonize fluconazole activity (Fig. 4A-M). Antagonism with FLZ was not a universal attribute among penicillin antibiotics, but was evident for oxacillin and methicillin (Fig. 4N). We also tested a first- generation cephalosporin, cefazolin, that is often prescribed in place of nafcillin (Letourneau, 2019; Miller et al., 2018) and a second-generation cephalosporin, cefonicid. We found that both of these molecules also antagonize FLZ (Fig. 4N). Of the beta-lactams tested, those that are often used to treat Staphylococcus aureus were antagonistic with FLZ (Fowler Jr. and Holland, 2018; Letourneau, 2019). Due to this, we decided to also test alternative treatment molecules for S. aureus infections, particularly vancomycin and linezolid (Fowler Jr. and Holland, 2018; Lowy, 2019), both of which we found to have no interaction with FLZ (Fig. 4N). To investigate the mechanism of antagonism, we looked to other known drug interactions involving nafcillin. In particular, nafcillin antagonizes warfarin and other drugs in vivo by inducing cytochrome P450 enzymes through an unknown mechanism, which increases warfarin metabolism (King et al., 2018; Wungwattana and Savic, 2017). FLZ inhibits a fungal cytochrome P450 enzyme, 14α- demethylase, which halts ergosterol biosynthesis and fungal growth (Fig. 4O) (Odds et al., 2003). We hypothesized that nafcillin may also induce cytochrome P450 enzymes, such as 14α- demethylase, in C. neoformans, counteracting FLZ’s mechanism of action. To test this bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

hypothesis, we examined whether nafcillin affects ergosterol biosynthesis. We extracted sterols from C. neoformans cells grown in the presence of nafcillin, FLZ, nafcillin + FLZ, or vehicle. We found a concentration-dependent increase in ergosterol with nafcillin treatment (Fig. 4P). Ergosterol levels in nafcillin + FLZ are not statistically different from the control treatment (Fig. 4P). Finally, we tested whether nafcillin is synergistic with the antifungal amphotericin B. Since amphotericin B kills target cells by binding and extracting ergosterol from the plasma membrane (Anderson et al., 2014), we hypothesized that if nafcillin increases ergosterol levels in C. neoformans, nafcillin would act synergistically with amphotericin B by increasing amphotericin B binding sites (i.e. ergosterol). Indeed, amphotericin B was synergistic with nafcillin in checkerboard assays (Fig. 4Q), further suggesting that nafcillin increases ergosterol levels in C. neoformans.

The synergistic interaction between dicyclomine HCl and FLZ affects cell permeability and nutrient uptake Finally, we investigated the drug dicyclomine HCl (dicyclomine), an anticholinergic agent (Table 1). This FDA-approved drug, also known as Bentyl, is used to treat urinary incontinence (Malone and Okano, 1999; Page and Dirnberger, 1981). In humans it targets a G-protein coupled receptor (GPCR) encoded by the CHRM1 gene (Consortium, 2018; Kilbinger and Stein, 1988). C. neoformans does not have a CHRM1 ortholog, but there are a large number GPCR in fungi that could be potential targets (Xue et al., 2008). When we screened a deletion library of C. neoformans for mutants resistant to dicyclomine, we found that 44% of the annotated dicyclomine-resistant mutants were involved in transport and trafficking, suggesting that those processes may be related to dicyclomine’s mechanism (Fig. 6A and Table S5). Thus, we hypothesized that dicyclomine alters Golgi transport. In Saccharomyces cerevisiae, simultaneously inhibiting Golgi trafficking and blocking ergosterol synthesis leads to mislocalization of essential plasma membrane transporters (Estrada et al., 2015). We hypothesized that the combination of dicyclomine and FLZ will phenocopy this effect in C. neoformans (Fig. 6B). Using propidium iodide internalization as a measure of cell permeability, we observed that dicyclomine, similar to FLZ, permeabilizes fungal cells at high doses (Fig. 6C, D, F, G). Furthermore, we saw a greater than additive increase in permeability when fungal cells were treated with low concentrations of dicyclomine and FLZ in combination (Fig. 6E and H). dicyclomine-induced permeability appeared to be independent of significant changes to cell wall chitin (Fig. S4A-F). We next tested if dicyclomine + FLZ disrupted nutrient transporter function by measuring uptake of amino acids. If amino acid permeases are not localized to the plasma membrane, fungal cells are resistant to toxic amino acid analogs (Roberg et al., 1997). Using the same low doses of FLZ and DIC that alone do not permeabilize cells, C. neoformans is susceptible to the effect of either 5-fluoroanthranilic acid (5-FAA) or 5-methyl-tryptophan (5-MT). When the dose is combined, cells now show resistance (Fig. 6I-L and Fig. S4G-J). This demonstrates that certain amino acid transporters’ function is decreased and they thus may be mislocalized, conferring resistance to toxic forms of tryptophan.

Dicyclomine + FLZ act synergistically in vivo and enhances survival of mice with cryptococcosis We intranasally inoculated CD-1 outbred mice (Charles River) with C. neoformans and allowed the infection to progress 8 days. Colony forming unit data indicated that at this point 100% of the mice exhibited fungal dissemination to the liver, and 40% exhibited dissemination to the brain (Fig. S5). A disseminated infection is consistent with human patients at treatment onset, as patients often don’t seek treatment until C. neoformans has disseminated to the brain bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

(Zhu et al., 2010). From 8 days post-inoculation (d.p.i) until 40 d.p.i., we administered dicyclomine, FLZ, dicyclomine + FLZ or PBS (vehicle) intraperitoneally. We used doses of both FLZ and dicyclomine which were within the range of doses given to humans (Lexicomp, 1978- 2019a, b). We sacrificed mice when they reached 80% of their initial mass (survival endpoint). Dicyclomine at either a low or high dose did not affect mouse survival compared to PBS- treatment. However, dicyclomine in combination with FLZ significantly improved survival over FLZ alone in a dose-dependent manner (Fig. 6M), indicating that dicyclomine may not be effective at treating cryptococcosis on its own, but could well be of therapeutic benefit when combined with FLZ.

Discussion

Synergistic combination therapies are increasingly important clinical options, especially for drug resistant microbes (Cowen and Lindquist, 2005; Kalan and Wright, 2011; Uppuluri et al., 2008; Zheng et al., 2018). Traditionally, synergistic drug pairs were discovered serendipitously, but new methods are improving our ability to uncover important interactions (Brown et al., 2014; Cokol et al., 2011; Cokol et al., 2018; Jansen et al., 2009; Robbins et al., 2015; Spitzer et al., 2011; Wambaugh et al., 2017; Wildenhain et al., 2015). In this study, we identified a wide variety of molecules that interact synergistically with the antifungal FLZ to inhibit fungal growth. We do so without the use of noisy multi-drug assays, allowing for rapid and scalable screening. We also identify and investigate antagonistic interactions, which are clinically important (Khandeparkar and Rataboli, 2017; Vadlapatla et al., 2014) but have not been investigated in a systemic manner. Of the 59 FLZ interacting molecules we identified, 10 have been previously described in various fungi, though 3 of those were reported as synergists while our testing revealed them to be antagonists (Ahmad et al., 2010; Butts et al., 2014; Cardoso et al., 2016; Eldesouky et al., 2018; Kang et al., 2010; Li et al., 2018; Marchetti et al., 2000; Quan et al., 2006; Robbins et al., 2015; Spitzer et al., 2011; Zhai et al., 2012). Additionally, we discovered synergistic molecules with a wide variety of bioactivities, suggesting that O2M identifies synergistic molecules with molecular mechanisms that can differ from that of the starting synergistic pair. O2M analysis for FLZ synergizers used fenpropimorph and sertraline. Fenpropimorph inhibits ergosterol biosynthesis (Marcireau et al., 1990) and sertraline inhibits protein translation (Zhai et al., 2012). Furthermore, we identified broad spectrum interactions. All the combinations tested showed efficacy against multiple clinical and environmental isolates of C. neoformans, as well as C. deuterogattii, a related species which can cause disease in apparently immunocompetent individuals (Applen Clancey et al., 2019). We also tested our combinations against common Candida species that often develop multi-drug resistance (Colombo et al., 2017), including the emerging MDR pathogen Candida auris (Fig. 3). Our data demonstrate that FLZ synergizers and antagonizers exhibit broad activities against multiple species and isolates. Another key component of drug discovery is the ability to improve upon the efficacy of known drugs through synthetic modification and/or identification of structurally related molecules. We found that structural similarity predicts synergistic interactions (Fig. S3), just as drugs of similar structure have similar function. These data will open up our ability to rapidly identify many more synergizers and antagonizers from a single example. We investigated the antagonistic interaction between FLZ and nafcillin, as nafcillin is a commonly used beta-lactam antibiotic for Staphylococcus aureus and other difficult to treat bacterial infections (Letourneau, 2019). Patients with these infections include some of the same patients at risk for cryptococcosis (HIV and cancer patients) (Kaplan et al., 2009; Utay et al., 2016) and other fungal infections, including C. auris (Rudramurthy et al., 2017). When we examined nafcillin-related molecules, we found that methicillin and oxacillin also antagonize FLZ. Furthermore, two cephalosporins often used in place of nafcillin, cefonicid and cefazolin, bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

also unexpectedly antagonistize fluconazole activity. Nafcillin has previously been shown to adversely affect patients on the drug warfarin due to nafcillin’s induction of cytochrome P450 (King et al., 2018), which decreases warfarin concentration. This has been seen in a similar combination of FLZ and the antibiotic rifampicin (Panomvana Na Ayudhya et al., 2004). Rifampicin is a potent inducer of drug metabolism due to elevation of hepatic cytochrome P450 through increased gene expression (Bolt, 2004). This combination did indeed lower the serum levels of FLZ (Panomvana Na Ayudhya et al., 2004), which resulted in relapse of cryptococcal meningitis (Coker et al., 1990). We hypothesize that an analogous process occurs when nafcillin is combined with FLZ, with nafcillin increasing ergosterol biosynthesis enzymes, counteracting FLZ’s activity. In a recent autopsy study, 10 or 16 patients who died of cryptococcosis were administered either a penicillin or a cephalosporin (Hurtado et al., 2019). We recommend that these patients receive linezolid or vancomycin instead, since these drugs are used for similar bacterial targets but do not antagonize fluconazole activity (Fig. 4N). Our data demonstrate that O2M identifies promising new antifungal treatments that can rapidly move into the clinic. Our new combination of dicyclomine + FLZ almost doubled the median time-to-endpoint of mice treated with human dosages of dicyclomine (Fig. 5M), which is lower than dicyclomine’s fungal MIC. Dicyclomine is orally bioavailable and able to cross the blood brain barrier (Das et al., 2013; Koerselman et al., 1999), which makes it particularly promising for fungal meningitis treatment. Since dicyclomine, like many of our FLZ synergizers, is approved by the FDA for other indications, it could rapidly move into the clinic. In sum, O2M considerably streamlined the identification of important drug interactions affecting C. neoformans growth. These interactions are both synergistic and antagonistic among multiple fungal species capable of causing disease in humans. We focused on FDA-approved molecules to bypass the time and considerable expense it takes to develop a new drug (Pushpakom et al., 2018). However, our method would work equally well on any library of small molecules or biologic drugs to discover new antifungals. We showed that identifying these drug interactions can quickly lead to additional interacting pairs by examining structure (Fig. S3) or by investigating underlying mechanism (Wambaugh et al., 2017). Finally, our newly discovered interaction of dicyclomine and FLZ exhibited therapeutic potential in vivo, demonstrating the clinical potential of fluconazole-containing synergistic pairs in the clinic.

Acknowledgements We thank the University of Utah Metabolomics Core for sterol quantification, Jerry Kaplan, Ph.D. for advice on sterol extraction, and members of the Brown and Mulvey labs for helpful discussion and feedback. This work was supported by NIH grant R01AI137331 and funds from the Pathology Department at the University of Utah to JCSB.

Author Contributions MAW and JCSB designed experiments. MAW, STD, BB, MS conducted experiments. MAW wrote the paper with input from JCSB. MAW, STD, and JCSB edited the paper.

Declaration of Interests The authors declare no competing interests.

Figure 1. High-throughput screening for fluconazole interacting molecules using synergy prediction mutants. A) Outline of overlap2 method (O2M), which is also presented in Brown et al. and Wambaugh et al. O2M requires a chemical-genetic dataset which can be generated by growing a collection of mutants in the presence of > 100 small molecules individually. Growth scores are then calculated for each small molecule + mutant combination. In the heatmaps, the vertical line represents a different mutant. Blue represents slower growth compared to wild-type cells and yellow represents faster growth compared to wild-type cells. Comparing our starting bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

drug (FLZ) and known synergistic molecules, we can identify genes whose knockout mutants show similar growth scores to the starting drug and all known synergistic partners. These are the synergy prediction mutants. B) Screening method to identify molecules that synergize with FLZ as well as anti-C. neoformans molecules. These molecules are then validated in a checkerboard assay.

Figure 2. Synergistic and antagonistic molecules identified from high-throughput screen. A) Fractional inhibitory concentration index (FICI) score of synergistic molecules identified from our high-throughput screen. Color of bar corresponds with bioactivities listed in C. B) Categories of bioactivities of synergistic molecules with the corresponding number of molecules in each category. C) FICI scores of antagonistic molecules from screen. Colors correspond with bioactivities listed in D. D) Categories of bioactivities of antagonistic molecules with corresponding number of molecules. All bioactivities came from Microsource Spectrum molecule list which is also seen in Table 1. E) Representation of percentage of molecules predicted from the entire library (top), molecules tested in various assays (middle), and molecules yielding an interaction from checkerboards (bottom). * represents FICI for 50%inhibition of C. neoformans all other scores listed are the FICI for 90% inhibition (FICI90).

Figure 3. Synergistic and antagonistic combinations affect other fungal strains and species. Fractional inhibitory concentration index (FICI) scores of synergistic and antagonistic combinations with FLZ in other fungal strains/species for A) 3-Amino-beta-pinene B) Benzalkonium Cl C) Berbamine HCl D) Bismuth Subsalicylate E) Clomipramine HCl F) Dehydroepiandrosterone G) Dicyclomine H) Estriol I) Fluocinolone Acetonide J) Sertraline HCl K) Xylometazoline HCl L) Ivermectin M) Nafcillin Sodium. * represents FICI for 50% inhibition all other scores listed are the FICI90. Strains/species listed on left. CM18 (top) represents original result. Green bars represent FICI scores ≤ 0.5 yielding a synergistic result. Violet bars represent FICI scores ≥ 4 yielding an antagonistic result. No interactions are in grey bars. HCl = hydrochloride, Cl = chloride.

Figure 4. Nafcillin Sodium affects ergosterol levels. Molecular structures of beta-lactam antibiotics shown for A) Nafcillin Sodium B) Cefazolin C) Cefonicid D) Cefoxitin E) Methicillin F) Aztreonam G) Oxacillin H) Carbenicillin J) Azlocillin K) Ampicillin L) Amoxicillin M) Amdinocillin. N) FICI scores for 50% inhibition of C. neoformans of various antibiotics related to nafcillin sodium tested with fluconazole. Violet bars over the red line illustrate a FICI score of ≥ 4 indicating antagonism. O) Ergosterol biosynthesis pathway illustrating cytochrome P450 enzymes. P) Ergosterol quantification from cell treated with Nafcillin (NAF), FLZ, or NAF+FLZ. Data normalized to control treated. Q) FICI for 50% inhibition of C. neoformans treated with nafcillin sodium in combination with amphotericin B. * = p value is 0.0268 (Mann-Whitney test).

Figure 5. Dicyclomine affects permeability and nutrient transporters. A) Pie chart with processes of deletion mutants that were resistant to dicyclomine. Numbers represent number of mutants. B) Prediction for dicyclomine (DIC) + FLZ synergy mechanism. C- E) Representative flow plots of propidium iodide staining. F-H) Quantification of propidium iodide staining. I-L) Growth curves of C. neoformans with and without various concentrations of 5-FAA in addition to control, dicyclomine, fluconazole, or synergy treatment. M) Survival of CD-1 outbred mice given FLZ (8 mg/kg), DIC low (1.15 mg/kg), DIC high (2.30 mg/kg), Synergy (SYN) low (FLZ + 1.15 mg/kg DIC), SYN high (FLZ + 2.30 mg/kg) or PBS treatments. N=10. * = p value is 0.0286 (Mann-Whitney test); ** = p value is 0.0036 (Mantel-Cox test). bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Table 1. Minimum inhibitory concentrations for fluconazole interacting molecules. All values are against C. neoformans strain CM18. MIC for 90% inhibition (MIC90) listed when possible. MIC50 = MIC for 50% inhibition.

Figure S1. Bliss independence scores of non-single agent molecules. Average bliss independence score of molecules at 10 µM (A) and 100 nM (B). Molecules were considered synergistic if they exhibited a negative score in both concentrations the majority of times tested (green labels).

Figure S2. FICI scores of non-interacting molecules and general antifungals. A) Fractional inhibitory concentration index (FICI) of molecules identified from our high- throughput screen that did not interact with FLZ. B) FICI scores of molecules identified as general anti-C. neoformans molecules from our high-throughput screen. Synergistic interactions with FLZ labeled in green and non-interacting molecules labeled in grey. * represents FICI for 50% inhibition of C. neoformans all others are FICI for 90% inhibition.

Figure S3. FICI scores of structurally similar molecules. Chemical structure of dicyclomine HCl (A) and structurally similar molecules proadifen (B), drofenine (C) and naftidrofuryl (D). Chemical structure of desipramine HCl (E) and structurally similar molecules impramine (F), mianserine (G), and lofepramine (H). Chemical structure of sertraline HCl (I) and structurally similar sibutramine (J). Chemical structure of diphenhydramine HCl (K) and structurally similar citalopram (L). M) Fractional inhibitory concentration index (FICI) of structurally similar molecules. Synergistic interactions with FLZ labeled in green and non- interacting molecules labeled in grey. * represents FICI for 50% inhibition of C. neoformans all others listed are FICI for 90% inhibition.

Figure S4. Dicyclomine additional effects on fungal chitin staining and nutrient intake. A- C) Representative flow cytometry of Calcofluor white staining with various concentrations of Dicyclomine (DIC) (blue), FLZ (yellow), or synergy (green). D-F) Quantification of average fluorescence intensity of calcofluor white staining flow cytometry. G-J) Growth curves of C. neoformans with or without 0.4 mg/mL of 5-MT after treatment with control (G), dicyclomine (H), FLZ (I), or Synergy (SYN) (J).

Figure S5. C. neoformans disseminates by 8 days in CD-1 outbred mice. Fungal burden of C. neoformans in CD-1 outbred mice at 8 days post infection.

Table S1. Small molecules predicted to synergize with fluconazole by O2M. Small molecules predicted to interact with FLZ. Bioactivity and Status determined by Microsource Spectrum Library. INN, International Nonproprietary Names; USAN, United States Accepted Name; BAN, British Approved Names; JAN, Japanese Adopted Name; USP, United States Pharmacopeia; NF, National Formulary.

Table S2. Minimum inhibitory concentrations of non-interacting molecules. Minimum inhibitory concentration for 50% inhibition (MIC 50) and 90% inhibition (MIC 90) of small molecules predicted to interact with FLZ but resulted in no interaction.

Table S3. Minimum inhibitory concentrations of general anti-C. neoformans molecules. Minimum inhibitory concentration 90% inhibition (MIC 90) for small molecules able to inhibit wild-type C. neoformans growth.

Table S4. Minimum inhibitory concentrations for various fungal strains/species. bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Minimum inhibitory concentration for 50% inhibition (MIC 50) and 90% inhibition (MIC 90) of small molecules that interacted with FLZ in various fungal species. N/A represents molecules that did not have an MIC.

Table S4. Gene deletion mutants resistant to Dicyclomine. Gene knockouts in KN99 resistant to dicyclomine at 1.65 mg/mL.

Methods

Fungal strains Screening, validation, and structurally similar assays were performed with CM18 lab strain of C. neoformans. Screening with synergy prediction mutants (CNAG_00573Δ and CNAG_03917Δ) was in the CM18 background. Mechanistic studies were tested using the KN99 lab strain of C. neoformans. Clinical and environmental isolates of C. neoformans tested were a gift from Dr. John R. Perfect. C. deuterogattii strain R265 was purchased from ATCC. Candida albicans reference strain SC5314 and Candida glabrata reference strain CBS138 were used. Candida auris strains AR0383 and AR0384 were from the CDC.

Microsource Spectrum library screen We inoculated either CM18 wild-type or CNAG_00573Δ or CNAG_03917Δ cells at 1000 cells per well of YNB + 2% glucose, then added small molecule to a final concentration of 1µM. Plates were incubated for 48 hours at 30 °C. OD600 was measured on the BioTek plate reader model Synergy H1. Small molecules that altered growth by absolute value 0.22 in both synergy prediction mutants was considered significant.

C. neoformans growth and small-molecule assays All assays were performed in 1x YNB + 2% glucose. To determine MICs, an overnight culture was grown at 30 °C with rotation, diluted to OD600 = .02925 and 1000 cells were added to each well (2 µL of culture into 100 µL of media per well). Plates were incubated at 30 °C unless otherwise stated. Small molecule gradients were diluted in 2-fold dilution series. MIC values were calculated after 48 hours of incubation.

Checkerboard Assay and FICI calculations We followed previously published methods (Hsieh et al., 1993; Orhan et al., 2005). Starting inoculation of either fungal strain was 2µL of an OD600 = 0.02925 (1000 cells per well of 100 µL medium). Plates were grown statically for 48 hours at 30 °C with minor shaking/resuspension of cells at 24 and 48 hours. Checkerboards were read at 0 and 48 hours on a BioTek plate reader model Synergy H1 (Candida albicans and Candida glabrata were read at 0, 24, and 48 hours). Growth inhibition was assessed and FICIs for 50% and 90% inhibition were determined. Repeated results were averaged for the average FICI.

Bliss independence Assay We created a gradient of fluconazole in a 96-well plate, then added small molecules at 10 µM or 100 nM final concentrations or vehicle. CM18 wild-type was added at 1000 cells per well of YNB+ 2% glucose. Percent growth was calculated for fluconazole, combinations, or small molecules alone. We then determined if growth inhibition caused by the combination was equal or greater than growth inhibition of the small molecules alone. The most repeatable result of multiple experiments was then averaged for average Bliss Independence Score.

Sterol extraction bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

KN99 culture was grown overnight in YNB + 2% glucose. Cells were sub-cultured into various treatments (vehicle control was YNB + 2% glucose). 6 ODs of each culture were harvested and lyophilized overnight. Pellets were resuspended in 25% alcoholic potassium hydroxide, vortexed, and incubated at 85 °C water bath for 1 hour. Water and n-heptane were added to each tube, vortexed, and the n-heptane layer was transferred to borosilicate glass tubes.

Metabolomics Metabolomics analysis was performed at the Metabolomics Core Facility at the University of Utah which is supported by 1 S10 OD016232-01, 1 S10 OD021505-01, and 1 U54 DK110858- 01.

Resistance to dicyclomine screen YNB + 2% glucose agar plates with or without 1.65 mg/mL of dicyclomine were made. Deletion mutants in KN99 strain were grown in YNB + 2% glucose then pinned to YNB plates. Plates were assessed at 1, 2, and 3 days for resistance.

Cell permeability assay An overnight culture of KN99 was grown in YNB + 2% glucose. This was then sub-cultured into the various treatments (vehicle control was either 0.1% DMSO or only YNB + 2% glucose). Cultures were grown at 30 °C with rotation for 24 hours. Cultures were washed twice and resuspended in PBS. 3 µL of propidium iodide (stock concentration = 1 mg/mL) was added to the FACS tube. After 1 min flow cytometry was performed. Voltage gates used were as follows: FSC: 500; SSC: 310; PE: 496.

Resistance to toxic amino acids An overnight culture of KN99 was grown in YNB + 2% glucose. This was sub-cultured into either dicyclomine (0.3 mg/mL), FLZ (3E-4 mg/mL), Synergy, or Vehicle (YNB + 2% glucose) and grown at 30 °C with rotation for 24 hours. Cells were then sub-cultured again into honeycomb plates with those previous treatments (dicylomine, FLZ, Synergy, or Vehicle) with either 20, 10, 5, 2.5 ug/mL of 5-FAA or 0.4 mg/mL 5-MT or vehicle (3.2% DMSO). Plates were read on a Bioscreen machine at 30 °C with OD600 measured every 30 minutes for 48 hours.

Calcofluor white flow cytometry An overnight culture of KN99 was grown in YNB + 2% glucose then sub-cultured into either dicylomine, FLZ, Synergy, or Vehicle (YNB + 2% glucose) and grown at 30 °C with rotation for 24 hours. Cells were washed with PBS and calcofluor white added to a final concentration of 50 µg/mL and stained for 5-15 minutes. Cells were washed once more with PBS and assessed by flow cytometry. Voltage gates as follows: FSC: 500; SSC: 317; BV421-A: 185. Significance determined with Mann-Whitney test.

Murine model ~8-week-old female CD-1® IGS outbred mice (Charles River Laboratories) were intranasally inoculated with C. neoformans strain KN99 and monitored for survival as follows. The inoculum was prepared by culturing C. neoformans overnight in liquid YNB+2% glucose medium. C. neoformans cells collected from the overnight culture were washed twice in 1XPBS, counted, and suspended at a concentration of 5x105 cells / mL. Mice were anesthetized with ketamine/dexmedetomidine hydrochloride (Dexdomitor) administered intraperitoneally (i.p.) and suspended by their front incisors on a line of thread. The inoculum was delivered intranasally by pipetting 50 μL (2.5x104 cells / mouse) dropwise into the nostrils. After 10 minutes, mice were removed from the thread and were administered atipamezole (Antisedan) i.p. as a reversal bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

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Table 1. Minimum inhibitory concentrations for fluconazole interacting molecules Small Molecule Bioactivity MIC 50 MIC 90 Result 3,4'-Dihydroxyflavone N/A 1.40 mM 8.33 mM synergistic 3',4'-Dihydroxyflavone N/A 8.93 mM N/A synergistic 3-Amino-beta-Pinene N/A 11.7 mM 50.1 mM synergistic 4'-Methoxychalcone N/A 94.2 µM N/A synergistic Amiodarone adrenergic agonist, coronary 2.61 µM 6.98 µM synergistic Hydrochloride vasodilator, Ca channel blocker Anthracene-9-Carboxylic Cl transport inhibitor 0.182 mM 1.67 mM antagonistic Acid Benzalkonium Chloride antiinfective (topical) N/A 14.5 µM synergistic Berbamine antihypertensive, skeletal muscle N/A 63.5 µM synergistic Hydrochloride relaxant antiarrhythmic, alpha2 agonist, cholinesterase, anticonvulsant, Berberine Chloride antiinflammatory, antibacterial, 1.43 mM 2.61 mM antagonistic antifungal, antitrypanosomal, antineoplastic, immunostimulant Bergapten antipsoriatic, antiinflammatory 1.18 mM 2.36 mM antagonistic Bismuth Subsalicylate antidiarrheal, antacid, antiulcer 1.49 mM N/A synergistic antibiotic, antifungal; LD50 (rat po) Blasticidin S 0.230 mM 0.717 mM synergistic 16mg/kg Bromhexine expectorant N/A 15.1 mM synergistic Hydrochloride Cadaverine Tartrate N/A 2.27 mM 5.82 mM synergistic Carbimazole antithyroid 0.215 mM 0.859 mM antagonistic antineoplastic, antiinflamatory, NO Celastrol synthesis inhibitor, chaperone 68.6 µM 0.229 mM antagonistic stimulant Chloroguanide antimalarial N/A 1.03 mM synergistic Hydrochloride Chloroxine chelating agent 2.02 µM 6.91 µM synergistic Clomipramine antidepressant 0.650 mM 0.780 mM synergistic Hydrochloride antibacterial, leprostatic, dermatitis Dapsone 29.2 mM N/A synergistic herpetiformis suppressant Dehydroepiandrosterone N/A 1.50 mM 12.0 mM synergistic Desipramine antidepressant N/A 1.89 mM synergistic Hydrochloride antibacterial, antifungal, antineoplastic, Diallyl Sulfide antihypercholesterolaemic, 7.18 mM 32.9 mM synergistic hepatoprotectant Dicyclomine anticholinergic N/A 4.85 mM synergistic Hydrochloride Dihydromyristicin GSH transferase inducer 0.283 M N/A synergistic Diphenhydramine antihistaminic 17.5 mM N/A synergistic Hydrochloride Disulfram alcohol antagonist N/A 7.38 µM antagonistic Epiandrosterone N/A 1.18 mM 3.15 mM synergistic Estriol estrogen N/A 24.8 mM synergistic Ethacrynic Acid diuretic 0.350 mM 1.14 mM antagonistic analgesic (topical), antiseptic, Eugenol 2.40 mM 14.3 mM antagonistic antifungal Fluocinolone Acetonide glucocorticoid, antiinflammatory 1.84 mM 3.69 mM synergistic Glyburide antihyperglycemic 0.248 mM 1.99 mM synergistic bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Gossypetin N/A 1.62 mM N/A synergistic antioxidant, inhibits lipid peroxidation inhibitor, antiinflammatory, Guaiazulene 3.47 mM 10.8 mM antagonistic hepatoprotectant; LD50(rat) 1550 mg/kg po Heptaminol vasodilator 6.16 mM N/A synergistic Hydrochloride Hexetidine antifungal 50.7 µM 0.253 mM synergistic Ivermectin antiparasitic N/A 1.91 mM antagonistic Lapachol antineoplastic, antifungal 36.2 mM N/A antagonistic Linalool (+) N/A 0.783 mM 3.14 mM synergistic Mebeverine muscle relaxant (smooth) 4.44 mM 8.88 mM synergistic Hydrochloride Metergoline analgesic, antipyretic N/A 0.160 mM synergistic Nafcillin Sodium antibacterial 2.37 mM N/A antagonistic Nerol weak estrogen receptor blocker 0.165 mM 0.709 mM antagonistic Octopamine adrenergic agonist 24.6 mM N/A synergistic Hydrochloride Osthol N/A 0.490 mM 1.80 mM antagonistic antiinflammatory, Phenylbutyric Acid 0.719 mM 2.87 mM antagonistic antihyperammonemic (Na salt) Piplartine anti-asthma, antibronchitis 3.30 mM 0.188 M antagonistic Primaquine Diphosphate antimalarial 3.73 mM N/A synergistic Ribostamycin Sulfate antibacterial 9.35 mM 18.7 mM antagonistic acaricide, ectoparasiticide, Rotenone N/A 1.64 µM antagonistic antineoplastic, mitochondrial poison anesthetic (topical) and antiseptic, Safrole 20.1 mM 41.0 mM synergistic pediculicide NO synthesis (inducible) inhibitor, Scopoletin 2.89 mM 10.8 mM antagonistic anticoagulant Sertraline Hydrochloride antidepressant, 5HT uptake inhibitor 0.209 mM 0.313 mM synergistic Tanshinone IIA Sulfonate free radical scavenger N/A 1.16 mM synergistic Sodium Tolfenamic Acid antiinflammatory, analgesia 0.772 mM 7.74 mM synergistic Toremiphene Citrate antineoplastic, anti-estrogen N/A 27.6 mM synergistic Tulobuterol bronchodilator, beta adrenergic agonist 23.2 mM N/A synergistic Xylomethazoline adrenergic agonist, nasal 2.44 mM N/A synergistic Hydrochloride decongestant

bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was Figure 1. not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A. B. screen knockout knock out synergy library with small prediction genes molecules

O O

O N + + O N calculate genetic mutants screen wild-type and growth FLZ prediction strains with drug library compare FLZ FLZ gene list wild-type cnag_ and gene 00573∆ / 03917∆ synergistic syn. #1 list molecules gene syn. #2 list O O O N slower faster O N O N O growth growth + + + compare responding mutants for known synergistic drugs wild-type wild-type wild-type compare overlap O O O

in responding genes O N N + + O + O N identify cnag_00573∆ synergy prediction cnag_03917∆ mutants cnag_ cnag_ cnag_ 00573∆ 03917∆ 00573∆ / 03917∆ outcome #1: outcome #2: putative FLZ synergizing general anti- molecule C. neoformans molecule

no growth growth FLZ bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was Figure 2. not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A. Metergoline* C. Rotenone Hexetidine Diphenhydramine Hydrochloride Lapachol* Bromhexine Hydrochloride Celastrol* Berbamine Hydrochloride Osthol* Amiodarone Hydrochloride Desipramine Hydrochloride Nafcillin Sodium* Mebeverine Hydrochloride Piplartine* Linalool* Chloroguanide Hydrochloride Nerol Tulobuterol* Berberine Chloride* Gossypetin* Epiandrosterone* Eugenol Diallyl Sulfide* Ivermectin* Dehydroepiandrosterone* 3,4'-Dihydroxyflavone Anthracene-9-Carboxylic Acid Tanshinone IIA Sulfonate Sodium* Guaiazulene* Glyburide H eptaminol Hydrochloride* Ethacrynic Acid* Toremiphene Citrate Carbimazole Octopamine Hydrochloride* Phenylbutyric Acid* Clomipramine Hydrochloride Cadaverine Tartrate* Scopoletin* Benzalkonium Chloride Disulfiram* Blasticidin S* Dicyclomine Hydrochloride Bergapten Estriol* Ribostamycin Sulfate* 4'-Methoxychalcone* Dihydromyristicin 2 4 6 8 10 60 80 Tolfenamic Acid* 100 120 140 Fluocinolone Acetonide* FICI Score Safrole Estrogen BlockerChart Title D. Primaquine Diphosphate* 1 3-Amino-beta-Pinene* Sertraline Hydrochloride Xylometazoline Hydrochloride* Dapsone Antiinfective 6 Other Bismuth Subsalicylate* 10 Chloroxine* 3',4'-Dihydroxyflavone*

0.0 0.2 0.4 0.6 Antineoplastic

FICI Score not Other An�neoplas4 �c An�infec�ve Estrogen-blockertested b E. B. not predicted 84% N/A Other 9 16% not tested 14 predicted 41% bliss Adrenergic Agonist 18% 4 checkerboard 41% Antiinfective Antineoplastic Estrogen 8 2 1 Antidepressant antagonist no interaction 3 Adrenergic Agonist An�neoplas�c An�depressant 15% 54% An�infec�ve estrogen synergist 31% Figure 3. bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was A. 3-Amino-beta-pinenenot certified by peer review)B. is theBenzalkonium author/funder. All Cl rights reserved.C. Berbamine No reuse allowed HCl withoutD. permission.Bismuth Subsalicylate CM18 * * KN99 * MUC 416-4 MUC 402-1 NRHc.5013.ENR * MUC 418-1 Ftc 555-1 NRHc.5027.ENR.CLIN1 * Ftc 327-1 * MUC 437-1 PMHC.1050.ENR.CLIN1 * NRHc.5011.ENR C. deuterogattii * C. auris 1 C. auris 2 C. albicans * * C. glabrata

3 4 5 6 7 8 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.2 0.4 0.6 0.8 0 1 2 3 4 6 8 10 0.00.5 1.01.52.0 FICI Score FICI Score FICI Score FICI Score E. F. G. H. Clomipramine HCl Dehydroepiandrosterone Dicyclomine HCl Estriol CM18 * KN99 * * MUC 416-4 MUC 402-1 * NRHc.5013.ENR * * MUC 418-1 Ftc 555-1 * * NRHc.5027.ENR.CLIN1 * Ftc 327-1 * N/A MUC 437-1 * PMHC.1050.ENR.CLIN1 * * NRHc.5011.ENR * * C. deuterogattii * C. auris 1 * * C. auris 2 * C. albicans * C. glabrata *

0.0 0.2 0.4 0.6 0 1 2 3 4 6 8 0.0 0.2 0.4 0.6 0.8 10 12 0.00.5 1.01.52.0 10 20 30 40 50 FICI Score FICI Score FICI Score FICI Score I. J. K. L. Fluocinolone Acetonide Sertraline HCl Xylometazoline HCl Ivermectin CM18 * * * KN99 * * MUC 416-4 * N/A MUC 402-1 N/A * NRHc.5013.ENR N/A * MUC 418-1 * * Ftc 555-1 * * NRHc.5027.ENR.CLIN1 * * Ftc 327-1 * * MUC 437-1 * * * PMHC.1050.ENR.CLIN1 * * NRHc.5011.ENR * * C. deuterogattii * C. auris 1 N/A * C. auris 2 * N/A * C. albicans * * * C. glabrata * * * 0 1 2 3 4 0.0 0.1 0.2 0.3 0.4 0.0 0.5 1.0 1.5 2.0 0 2 4 6 8 FICI Score FICI Score FICI Score FICI Score M. Nafcillin Sodium CM18 * KN99 * MUC 416-4 * MUC 402-1 * NRHc.5013.ENR * MUC 418-1 * Ftc 555-1 * NRHc.5027.ENR.CLIN1 * Ftc 327-1 * MUC 437-1 * PMHC.1050.ENR.CLIN1 * NRHc.5011.ENR * C. deuterogattii * C. auris 1 * C. auris 2 * C. albicans * C. glabrata *

0 5 10 15 FICI Score Figure 4. bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A. E. J. NH N O O O H H O N N NH S S O H O O N N N S O O O OH N O O O HO B. OH O N F. S NH K. N N H NH N S N N H O N N N S N S O O O N O O HN N S CH O O OH O H OH N O C. O SOH L. O G. NH S SOH N H N N H O HO S OH N N S H O O N N N S COOH NN O O O N O O H O O H. O M. D. N S HO H O H S O N O NH H N N N O S S H O O O OH N N O O OH OH N. O O Linezolid P. Vancomycin 2.0 * Cefazolin * Cefonicid 1.5 Cefoxitin

Methicillin 1.0 Aztreonam Oxacillin Carbenicillin 0.5

Azlocillin ergosterol/control Ampicillin Amoxicillin Amdinocillin 0.0

0 5 10 15 20 Control O. FICI 50 Squalene 1.0 mg/mL2.0 NAFmg/mL3.0 NAFmg/mL NAF 0.001 mg/mL FLZ FLZ+2.0FLZ+3.0 mg/mL NAFmg/mL NAF FLZ + 1.0 mg/mL NAF

Lanosterol Ergosterol Q. CYP51 FLZ 4,4-dimethyl-cholesta- NAF Amphotericin B 8,12,24-trienol

5,7,22,24(28)-Ergosta-tetraenol 0.0 0.2 0.4 0.6 FICI 50 CYP61 5,7,24(28)-Ergosta-trienol bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was Figure 5. not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Chart Title A. translation & other 4 B. transcription FLZ FLZ FLZ 3 DIC DIC DIC

enzymes 8 hypothetical proteins 27 trafficking & transport DIC DIC DIC 12 increased mislocalization of hypothe�cal trafic ENZY transl other permeability transporters C. Fluconazole D. Dicyclomine E. Synergy

unstained unstained unstained control control control 1/300 MIC 1/6 MIC FLZ

1/100 MIC Count 1/4 MIC 1/100 MIC Count Count FLZ + DIC 1/30 MIC 1/2 MIC DIC 1/3 MIC 1/4 MIC

F. Fluconazole G. Dicyclomine H. Synergy 100000 50000 80000 * *

80000 40000 60000 60000 30000 40000 40000 20000 20000 PE-AIntensity PE-AIntensity PE-AIntensity 20000 10000 0 0 0

Control 1/3 MIC Control1/6 MIC1/4 MIC1/2 MIC Control Synergy 1/30 MIC Unstained 1/100 MIC Unstained Unstained DIC 1/4 MIC FLZ 1/100 MIC I. J. K. 0.6 Control 0.6 Dicyclomine 0.6 Fluconazole Vehicle Vehicle Vehicle

0.4 20 µg/mL 0.4 20 µg/mL 0.4 20 µg/mL 600 600 10 µg/mL 600 10 µg/mL 10 µg/mL OD OD 0.2 OD 0.2 5 µg/mL 0.2 5 µg/mL 5 µg/mL

2.5 µg/mL 2.5 µg/mL 2.5 µg/mL 0.0 0.0 0.0 0 12 24 36 48 0 12 24 36 48 0 12 24 36 48 Time Time Time L. M. 100 0.6 Synergy Vehicle FLZ 0.4 20 µg/mL DIC low 600 10 µg/mL 50 DIC high ** OD 0.2 5 µg/mL SYN low Percent survival 2.5 µg/mL SYN high 0.0 PBS 0 12 24 36 48 0 Time 0 10 20 30 40 50 d.p.i. bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Table S1. Small Molecule cas # Bioactivity Status Assay Used Manufacturer 1,3-Dideacetyldeoxykhivorin N/A N/A Undetermined activity not tested N/A 1,3-Dideacetylkhivorin N/A N/A Undetermined activity not tested N/A 12-Hydroxy-4,4-Bisnor-4,8,11,13-Podocarpatetraen-3-One N/A N/A Undetermined activity not tested N/A 12-Methoxy-4,4-Bisnor-5alpha-8,11,13-Podocarpatrien-3-ol N/A N/A Undetermined activity not tested N/A 14-Methoxy-4,4-Bisnor-8,11,13-Podocarpatrien-3-One N/A N/A Undetermined activity not tested N/A 18alpha-Glycyrrhetinic Acid N/A antiinflammatory Experimental not tested N/A 1-Hydroxy-3,6,7-trimethoxy-2,8-diprenylxanthone 15404-76-9 N/A Undetermined activity not tested N/A 1-Methylxanthine 6136-37-4 diuretic, adenosine antagonist Experimental bliss independence VWR 1-Monopalmitin 542-44-9 N/A Undetermined activity checkerboard Sigma Chemical 1-Phenylbiguanide Hydrochloride 55-57-2 5HT3 receptor agonist Experimental bliss independence Sigma Chemical 2',2'-Bisepigallocatechin Digallate N/A N/A Undetermined activity not tested N/A 2,3,4'-Trihydroxy-4-Methoxybenzophenone N/A N/A Undetermined activity not tested N/A 2',3-Dihydroxy-4,4',6'-Trimethoxychalcone 38186-71-9 N/A Undetermined activity not tested N/A 2',4'-Dihydroxychalcone 4'-Glucoside N/A anthelmintic & antiulcerocenic Experimental not tested N/A precursor in corticoid 21-Acetoxypregnenolone 566-78-9 Undetermined activity bliss independence Santa Cruz biosynthesis, derivative prostaglandin synthetase 2-Hydroxy-3,4-Dimethoxybenzoic Acid N/A Experimental checkerboard Sigma Chemical inhibitor 2-Methoxyresorcinol 29267-67-2 N/A Undetermined activity checkerboard Santa Cruz 3,4'-Dihydroxyflavone 14919-49-4 N/A Undetermined activity checkerboard Fisher Scientific 3',4'-Dihydroxyflavone 4143-64-0 N/A Undetermined activity checkerboard Fisher Scientific 3,4-Dimethoxycinnamic acid 2316-26-9 N/A Undetermined activity checkerboard Fisher Scientific 3,4-Dimethoxydalbergione 41043-20-3 induces dermatitis Undetermined activity not tested N/A 3,7-Epoxycaryophyllan-6-One N/A N/A Undetermined activity not tested N/A 3-Acetoxypregn-16-En-12,20-Dione N/A N/A Undetermined activity not tested N/A 3alpha-Acetoxydihydrodeoxygedunin N/A N/A Undetermined activity not tested N/A 3alpha-Hydroxy-4,4-Bisnor-8,11,13-Podocarpatriene N/A N/A Undetermined activity not tested N/A 3-alpha-Hydroxydeoxygedinin N/A N/A Undetermined activity not tested N/A 3-Amino-1,2,4-Triazole 61-82-5 catalase inhibitor Experimental checkerboard Fisher Scientific 3-Amino-beta-Pinene 18172-67-3 N/A Undetermined activity checkerboard Sigma Chemical 3beta-Chloroandrostanone N/A N/A Undetermined activity not tested N/A 3-Bromo-7-Nitroindazole 74209-34-0 NO synthetase inhibitor Experimental checkerboard Fisher Scientific 3-Deacetylkhivorin N/A N/A Undetermined activity not tested N/A 3-Deoxo-3beta-Acetoxydeoxydihydrogedunin N/A N/A Undetermined activity not tested N/A 3-Deoxo-3beta-Hydroxymexicanolide 16-Enol Ether N/A N/A Undetermined activity not tested N/A 3-Hydroxytryramine 62-31-7 dopaminergic Experimental not tested N/A antioxidant, lipoxygenase 3-Methyl-1-Phenyl-2-Pyrazolin-5-One (MCI-186) N/A Experimental checkerboard Santa Cruz inhibitor 4'-Methoxychalcone 22966-19-4 N/A Undetermined activity checkerboard VWR 5,7-Dichlorokynurenic Acid 190908-40-8 NMDA receptor antagonist (gly) Experimental bliss independence Fisher Scientific 5beta-12-Methoxy-4,4-Bisnor-8,11,13-Podocarpatrien-3-One N/A N/A Undetermined activity not tested N/A 5-Fluoroindole-2-Carboxylic Acid 399-76-8 NMDA receptor antagonist (gly) Experimental Not tested N/A 5-Hydroxyiminoisocaryophyllene N/A N/A Undetermined activity not tested N/A 6alpha-Methylprednisolone Acetate N/A glucocorticoid Experimental not tested N/A vascular protectant, 7,8-Dihydroxyflavone 38183-03-8 Experimental bliss independence Santa Cruz antihaemorrhagic 7-Deacetoxy-7-Oxokhivorin 15004-51-0 N/A Undetermined activity not tested N/A 7-Desacetoxy-6,7-Dehydrogedunin 26927-01-5 N/A Undetermined activity checkerboard Sigma Chemical 8-Cyclopentyltheophylline 35873-49-5 A1 adenosine agonist Experimental bliss independence Fisher Scientific 8-Hydroxy-15,16-Bisnor-11-labden-13-One N/A N/A Undetermined activity not tested N/A 8-Hydroxycarapinic Acid N/A N/A Undetermined activity not tested N/A 8-Iodocatechin Tetramethyl Ether N/A N/A Undetermined activity not tested N/A glucose uptake stimulant; AMPK Acadesine 2627-69-2 Experimental bliss independence Cayman Chemicals activator Acamprosate Calcium 77337-73-6 alcohol antagonist USAN, INN, BAN checkerboard Fisher Scientific Acetyltryptophan 1218-34-4 antidepressant Experimental checkerboard Fisher Scientific Aconitic Acid 585-84-2 N/A Undetermined activity checkerboard Fisher Scientific adrenergic agonist, Adrenaline Bitartrate 51-42-3 bronchodilator, antiglaucoma USP, JAN checkerboard Fisher Scientific agent Aliskiren Hemifumarate 173334-57-1 renin inhibitor USAN, INN bliss independence VWR antihyperuricemia, antigout, Allopurinol 315-30-0 USP, INN, BAN, JAN bliss independence Fisher Scientific antiurolithic Aloin 5133-19-7 cathartic, laxative BAN checkerboard Cayman Chemicals Aminohydroxybutyric Acid 924-49-2 antiepileptic JAN bliss independence Santa Cruz adrenergic agonist, coronary Amiodarone Hydrochloride 1951-25-3 USAN, INN, BAN, JAN checkerboard Fisher Scientific vasodilator, Ca channel blocker Amprolium 121-25-5 coccidiostat USP, INN, BAN bliss independence VWR Anabasine Hydrochloride 13078-04-1 insecticide agricultural use not tested N/A Ancitabine Hydrochloride 10212-25-6 antineoplastic INN, JAN checkerboard Santa Cruz Andrographolide 5508-58-7 N/A Undetermined activity bliss independence Cayman Chemicals Androsta-1,4-Dien-3,17-Dione 897-06-3 N/A Undetermined activity not tested N/A Angolensic Acid, Methyl Ester 2629-14-3 N/A Undetermined activity not tested N/A Anhydrobrazilic Acid N/A N/A Undetermined activity not tested N/A Anthothecol 10410-83-0 N/A Undetermined activity not tested N/A Anthracene-9-Carboxylic Acid 723-62-6 Cl transport inhibitor Experimental checkerboard Fisher Scientific Aphyllic Acid 642-67-1 N/A Undetermined activity not tested N/A Apiin 26544-34-3 N/A Undetermined activity bliss independence Santa Cruz Artenimol 81496-81-3 antimalarial, antiinflammatory INN checkerboard Sigma Chemical Arthonioic Acid 25556-24-5 N/A Undetermined activity not tested N/A wound healing, Experimental Asiatic acid 464-92-6 Experimental checkerboard Sigma Chemical carcinogen Astaxanthin 71772-51-5 N/A Undetermined activity checkerboard Cayman Chemicals oxidative phosphorylation Atractyloside Potassium 17754-44-8 Experimental not tested N/A inhibitor Avocadyne 34524-38-4 antibacterial, antifungal Experimental not tested N/A beta adrenergic agonist, Bambuterol Hydrochloride 81732-46-9 bronchodilator, cholinesterase INN, BAN bliss independence Cayman Chemicals inhibitor Batyl Alcohol 544-62-7 N/A Undetermined activity not tested N/A USAN, NF, INN, BAN, Benzalkonium Chloride 8001-54-5 antiinfective (topical) checkerboard VWR JAN bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

antihypertensive, skeletal muscle Berbamine Hydrochloride 114784-59-7 Experimental checkerboard Cayman Chemicals relaxant antiarrhythmic, alpha2 agonist, cholinesterase, anticonvulsant, Berberine Chloride 633-65-8 antiinflammatory, antibacterial, JAN checkerboard Cayman Chemicals antifungal, antitrypanosomal, antineoplastic, immunostimulant Bergapten 484-20-8 antipsoriatic, antiinflammatory INN checkerboard Santa Cruz beta-Dihydrogedunol N/A N/A Undetermined activity not tested N/A beta-Escin 6805-41-0 membrane permeabilizer INN checkerboard Fisher Scientific beta-Peltatin 518-29-6 antineoplastic, cytotoxic Experimental not tested N/A Beta-Propiolactone 57-57-8 antiinfective USAN, INN not tested N/A Betonicine 515-25-3 N/A Undetermined activity checkerboard Santa Cruz Betulin 473-98-3 N/A Undetermined activity bliss independence Cayman Chemicals Bicuculline (+) 485-49-4 GABAa antagonist Experimental checkerboard Cayman Chemicals Biochanin A 491-80-5 phytoestrogen Experimental checkerboard Cayman Chemicals Biochanin A Diacetate N/A N/A Undetermined activity not tested N/A Bisabolol Acetate N/A N/A Undetermined activity not tested N/A Bismuth Subsalicylate 14882-18-9 antidiarrheal, antacid, antiulcer USP, JAN checkerboard Santa Cruz antibiotic, antifungal; LD50 (rat Blasticidin S 2079-00-7 agricultural use checkerboard ThermoFisher po) 16mg/kg Bleomycin 9041-93-4 antineoplastic USP, INN, BAN, JAN checkerboard VWR Bromhexine Hydrochloride 611-75-6 expectorant USAN, INN, BAN, JAN checkerboard VWR Bromo-3-Hydroxy-4-(Succin-2-YL)-Caryolane gamma-Lactone N/A N/A Undetermined activity not tested N/A Bucladesine 362-74-3 vasodilator INN, JAN bliss independence Cayman Chemicals Bussein 41060-14-4 N/A Undetermined activity not tested N/A Cadaverine Tartrate 462-94-2 N/A Undetermined activity checkerboard Santa Cruz Calcein 1461-15-0 chelating agent (Ca, Mg) Experimental checkerboard Cayman Chemicals Caperatic Acid 29227-64-3 antibacterial (tuberculostatic) Undetermined activity not tested N/A Capobenic Acid 21434-91-3 antiarrhythmic USAN, INN not tested N/A Carbimazole 22232-54-8 antithyroid INN, BAN checkerboard Santa Cruz Caryophyllene [t(-)] 87-44-5 N/A Undetermined activity checkerboard VWR Cearoin 52811-37-7 N/A Undetermined activity not tested N/A antineoplastic, antiinflamatory, Celastrol 34157-83-0 NO synthesis inhibitor, Experimental checkerboard Cayman Chemicals chaperone stimulant Cevadine 62-59-9 antihypertensive Experimental not tested Fisher Scientific Chaulmoogric Acid, Ethyl ester 623-32-5 antilepretic Experimental not tested N/A Chloroguanide Hydrochloride 637-32-1 antimalarial USP-XIV, INN, BAN checkerboard Sigma Chemical Chlorotrianisene 569-57-3 estrogen USP-XIII, INN, BAN not tested N/A Chloroxine 773-76-2 chelating agent USAN checkerboard Fisher Scientific Chlorzoxazone 95-25-0 muscle relaxant (skeletal) USP, INN, BAN, JAN checkerboard VWR Cholic Acid 81-25-4 N/A Undetermined activity checkerboard no interaction Cimcifugoside H1 N/A N/A Undetermined activity not tested N/A Cimcifugoside H2 161097-77-4 N/A Undetermined activity not tested N/A Citrulline 627-77-0 N/A Undetermined activity bliss independence Fisher Scientific Clomipramine Hydrochloride 17321-77-6 antidepressant USP, INN, BAN, JAN checkerboard Fisher Scientific Clovanediol Diacetate N/A N/A Undetermined activity not tested N/A Colistin Sulfate 1264-72-8 antibacterial USP, INN, BAN, JAN checkerboard Cayman Chemicals Convallatoxin 508-75-8 cardiotonic Experimental checkerboard Sigma Chemical Cortisone 53-06-5 antiinflammatory, glucocorticoid INN, BAN checkerboard Sigma Chemical Cotinine 486-56-6 antidepressant Experimental checkerboard Fisher Scientific Cresol 1319-77-3 antiinfectant USAN, NF, JAN checkerboard VWR antihypertensive, respiratory Crinamine 639-41-8 Experimental not tested N/A depressant, antineoplastic Cuneatin Methyl Ether 4253-00-3 N/A Undetermined activity not tested N/A Cycloveratrylene N/A N/A Undetermined activity not tested N/A antiinflammatory, respiratory Cytisine 485-35-8 INN bliss independence Fisher Scientific stimulent antibacterial, leprostatic, Dapsone 80-08-0 dermatitis herpetiformis USP, INN, BAN checkerboard Sigma Chemical suppressant Daunorubicin 20830-81-3 antineoplastic USAN, INN, BAN, JAN checkerboard Santa Cruz Decoquinate 18507-89-6 coccidiostat USP, INN, BAN bliss independence VWR Dehydro(11,12)Ursolic Acid Lactone N/A N/A Undetermined activity not tested N/A Dehydroepiandrosterone 53-43-0 N/A Undetermined activity checkerboard Cayman Chemicals Demethylnobiletin 2174-59-6 N/A Undetermined activity not tested N/A Derrusnin 14736-62-0 N/A Undetermined activity not tested N/A Desipramine Hydrochloride 58-28-6 antidepressant USP, INN, BAN, JAN checkerboard Sigma Chemical Dexpanthenol 81-13-0 cholinergic USP, INN, BAN checkerboard VWR antibacterial, antifungal, antineoplastic, Diallyl Sulfide 592-88-1 Experimental checkerboard Fisher Scientific antihypercholesterolaemic,hepat oprotectant Dictamnine 484-29-7 N/A Undetermined activity not tested N/A Dicyclomine Hydrochloride 67-92-5 anticholinergic USP, INN, BAN, JAN checkerboard Sigma Chemical Difucol Hexamethyl Ether 14262-07-8 N/A Undetermined activity not tested N/A Digitonin 11024-24-1 N/A Undetermined activity checkerboard Cayman Chemicals Digitoxin 71-63-6 inotropic, cardiotonic USP, INN, BAN, JAN bliss independence Santa Cruz Dihydroergotamine Mesylate 6190-39-2 vasoconstrictor, antimigraine USP, INN, BAN, JAN checkerboard VWR Dihydrofissinolide N/A N/A Undetermined activity not tested N/A Dihydrogambogic Acid N/A N/A Undetermined activity not tested N/A Dihydrogedunin N/A N/A Undetermined activity not tested N/A Dihydrojasmonic Acid 98674-52-3 plant growth regulator Experimental checkerboard Sigma Chemical Dihydromyristicin 607-91-0 GSH transferase inducer Experimental checkerboard Cayman Chemicals Dihydrorotenone N/A N/A Undetermined activity not tested N/A Dimethylcaffeic Acid 14737-89-4 N/A Undetermined activity not tested N/A antiinflammatory, Dimethylsulfone 67-71-0 agricultural use bliss independence Cayman Chemicals antiproliferative, antiparasitic Diphenhydramine Hydrochloride 147-24-0 antihistaminic USP, INN, BAN, JAN checkerboard Fisher Scientific Disulfram 97-77-8 alcohol antagonist USP, INN, BAN, JAN checkerboard VWR Dopamine Hydrochloride 62-31-7 cardiotonic, antihypotensive USP, INN, BAN, JAN bliss independence VWR bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

antioxidant, lipoxygenase Ebselen 60940-34-3 INN checkerboard Cayman Chemicals inhibitor, inhibits oxidation of LDL Embelin N/A anthelmintic, oral contraceptive Experimental bliss independence Cayman Chemicals Emodic Acid 578-45-5 cathartic, purgative Experimental not tested N/A Epiandrosterone N/A N/A Undetermined activity checkerboard Fisher Scientific Epicatechin Pentaacetate N/A N/A Undetermined activity not tested N/A Epitheaflavin Monogallate N/A N/A Undetermined activity not tested N/A Epoxygedunin N/A N/A Undetermined activity not tested N/A Ergosterol 57-87-4 N/A Undetermined activity bliss independence Cayman Chemicals USP-XX, INN, BAN, Estradiol Benzoate 50-50-0 estrogen Santa Cruz JAN Estriol 50-27-1 estrogen USP, INN, BAN, JAN checkerboard Fisher Scientific Estrone 53-16-7 estrogen USP, INN, BAN checkerboard Fisher Scientific antineoplastic, hypoxic cell Etanidazole 22668-01-6 USAN, INN not tested N/A radiosensitizer Ethacrynic Acid 58-54-8 diuretic USP, INN, BAN, JAN checkerboard Fisher Scientific Eucatropine Hydrochloride 536-93-6 anticholinergic (opthalmic) USP, INN, BAN not tested N/A analgesic (topical), antiseptic, Eugenol 97-53-0 USP checkerboard Fisher Scientific antifungal Euphorbiasteroid 28649-59-4 N/A Undetermined activity not tested N/A Filipin 480-49-9 antifungal USAN, INN checkerboard Sigma Chemical Fludrocortisone Acetate 514-36-3 mineralocorticoid USP, INN, BAN, JAN checkerboard Sigma Chemical Fluocinolone Acetonide 67-73-2 glucocorticoid, antiinflammatory USP, INN, BAN, JAN checkerboard Fisher Scientific Flurothyl 333-36-8 central stimulant, convulsant USP-XXI, INN, BAN VWR Fumarprotocetraric Acid 489-50-9 N/A Undetermined activity not tested N/A Gallamine Triethiodide 65-29-2 muscle relaxant (skeletal) USP, INN checkerboard Santa Cruz Gemfibrozil 25812-30-0 antihyperlipoproteinemic USP, INN, BAN checkerboard VWR Gentisic Acid 490-79-9 analgesic, antiinflammatory Experimental checkerboard Fisher Scientific Geraldol 21511-25-1 N/A Undetermined activity not tested N/A Glucitol-4-Gucopyanoside N/A N/A Undetermined activity not tested N/A Glucosaminic Acid N/A N/A Undetermined activity bliss independence Fisher Scientific Glyburide 10238-21-8 antihyperglycemic USP, INN, BAN, JAN checkerboard VWR Gossypetin 489-35-0 N/A Undetermined activity checkerboard Fisher Scientific antioxidant, inhibits lipid peroxidation inhibitor, Guaiazulene 49-84-9 antiinflammatory, Experimental checkerboard VWR hepatoprotectant; LD50(rat) 1550 mg/kg po Guaiol(-) 489-86-1 N/A Undetermined activity bliss independence Sigma Chemical Haematommic Acid 479-25-4 N/A Undetermined activity not tested N/A Harmalol Hydrochloride 6028-07-5 anthelmintic, narcotic agent Experimental bliss independence VWR Harmane 486-84-0 intercalating agent, sedative Experimental checkerboard Santa Cruz Hastatoside 50816-24-5 N/A Undetermined activity not tested N/A Hecogenin 467-55-0 antiinflammatory Experimental bliss independence VWR Hematein 475-25-2 N/A Undetermined activity not tested N/A Heptaminol Hydrochloride 543-15-7 vasodilator INN, BAN checkerboard Sigma Chemical Hetacillin Potassium 5321-32-4 antibacterial USP-XIII, JAN not tested N/A Hexetidine 141-94-6 antifungal BAN checkerboard Santa Cruz Hexylresorcinol 136-77-6 anthelmintic, topical antiseptic USP, BAN checkerboard VWR anticholinesterase, cognition Huperzine A 102518-79-6 Experimental bliss independence Cayman Chemicals enhancer Hydrocortisone Hemisuccinate 125-04-2 glucocorticoid USP, JAN bliss independence Santa Cruz Hydrocortisone Phosphate Triethylamine 3863-59-0 glucocorticoid USP, INN, BAN, JAN bliss independence Fisher Scientific Hypoxanthine 68-94-0 N/A Undetermined activity checkerboard VWR Inosine 58-63-9 cell function activator, cardiotonic INN, JAN checkerboard VWR Inositol 87-89-8 growth factor NF-XII checkerboard Fisher Scientific Iretol N/A N/A Undetermined activity not tested N/A Isopeonol 493-33-4 N/A Undetermined activity not tested N/A Isotectorigenin, 7-Methyl Ether N/A N/A Undetermined activity not tested N/A Ivermectin 70288-86-7 antiparasitic USP, INN, BAN checkerboard VWR Ketoconazole 65277-42-1 antifungal USP, INN, BAN, JAN checkerboard Fisher Scientific Khayanthone 25279-68-9 N/A Undetermined activity not tested N/A Khellin 82-02-0 vasodilator INN checkerboard Fisher Scientific Khivorin 2524-38-1 N/A Undetermined activity not tested N/A Koparin 65048-75-1 N/A Undetermined activity not tested N/A L(+/-)-Alliin 556-27-4 antibacterial, antioxidant Experimental checkerboard Sigma Chemical Lanosterol 79-63-0 N/A Undetermined activity checkerboard Cayman Chemicals Lapachol 84-79-7 antineoplastic, antifungal Experimental checkerboard Santa Cruz Lappaconitine 32854-75-4 analgesic, antiarrhythmic Experimental bliss independence Santa Cruz Larixinic Acid 118-71-8 N/A Undetermined activity checkerboard Fisher Scientific L-Deoxyalliin 21593-77-1 antineoplastic Experimental bliss independence VWR Leoidin Dimethyl Ether N/A N/A Undetermined activity not tested N/A Levalbuterol Hydrochloride 50293-90-8 bronchodilator, tocolytic USAN bliss independence Santa Cruz Linalool (+) N/A N/A Undetermined activity checkerboard Sigma Chemical Loganin 18524-94-2 N/A Undetermined activity checkerboard Cayman Chemicals Lupanyl Acid Hydrochloride N/A N/A Undetermined activity not tested N/A Lycopodine Perchlorate N/A N/A Undetermined activity not tested N/A Mangiferin 4773-96-0 MAO inhibitor, immunostimulant Experimental bliss independence Fisher Scientific Marmesin Acetate N/A N/A Undetermined activity not tested N/A Mebeverine Hydrochloride 2753-45-9 muscle relaxant (smooth) USAN, INN, BAN checkerboard Santa Cruz Melezitose N/A N/A Undetermined activity bliss independence Fisher Scientific Metameconine N/A N/A Undetermined activity not tested N/A Metergoline 17692-51-2 analgesic, antipyretic INN, BAN checkerboard Santa Cruz Metformin Hydrochloride 1115-70-4 antidiabetic USAN, JAN bliss independence VWR Methimazole 60-56-0 antihyperthyroid USP, INN, BAN, JAN checkerboard Fisher Scientific Methyl Robustone N/A N/A Undetermined activity not tested N/A Methylxanthoxylin 23121-32-6 N/A Undetermined activity not tested N/A Mexicanolide 1915-67-9 N/A Undetermined activity not tested N/A Morin 480-16-0 P450 and ATPase inhibitor Experimental bliss independence Fisher Scientific Mudulone 481-94-7 N/A Undetermined activity not tested N/A Nafcillin Sodium 985-16-0 antibacterial USP, INN, BAN checkerboard VWR N-Benzyltropan-4-ol N/A N/A Undetermined activity not tested N/A bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Nerol 106-25-2 weak estrogen receptor blocker Experimental checkerboard Fisher Scientific N-Methylanthranilic Acid 119-68-6 N/A Undetermined activity checkerboard Fisher Scientific N-Methylisoleucine 5125-98-8 N/A Undetermined activity not tested N/A Nonic Acid N/A N/A Undetermined activity not tested N/A Nonoxynol-9 26027-38-3 spermatocide, contraceptive USP, INN checkerboard Santa Cruz Obliquin N/A N/A Undetermined activity not tested N/A Octopamine Hydrochloride 104-14-3 adrenergic agonist INN checkerboard Cayman Chemicals Oleanolic Acid Acetate 4339-72-4 N/A Undetermined activity not tested N/A Orsellinic Acid, Ethyl Ester 2524-37-0 N/A Undetermined activity checkerboard Santa Cruz Osthol 484-12-8 N/A Undetermined activity checkerboard Cayman Chemicals o-Veratraldehyde 86-51-1 N/A Undetermined activity not tested N/A Oxolinic Acid 14698-29-4 antibacterial USAN, INN, BAN bliss independence Fisher Scientific Pachyrrhizin 10091-01-7 insecticide Experimental not tested N/A Paeonol 552-41-0 antibacterial Experimental checkerboard Cayman Chemicals Pamabrom 606-04-2 diuretic USP bliss independence VWR Pectolinarin 28978-02-1 N/A Undetermined activity checkerboard Sigma Chemical Pelletierine Hydrochloride N/A N/A Undetermined activity not tested N/A Peonol Methyl Ether 829-20-9 N/A Undetermined activity not tested N/A Phenacylamine Hydrochloride 5468-37-1 N/A Undetermined activity checkerboard Fisher Scientific

antiinflammatory, Phenylbutyric Acid 1821-12-1 Experimental checkerboard Fisher Scientific antihyperammonemic (Na salt)

induces experimental glucosuria, Phloridzin 60-81-1 Experimental bliss independence Cayman Chemicals antifeedant antineoplastic; 10% cytotoxicity Picropodophyllotoxin 477-47-4 Experimental bliss independence Sigma Chemical of podophyllotoxin Picropodophyllotoxin Acetate N/A N/A Undetermined activity not tested N/A K channel agonist, Pinacidil 85371-64-8 USAN, INN not tested Santa Cruz antihypertensive Piplartine 20069-09-4 anti-asthma, antibronchitis Experimental checkerboard Cayman Chemicals Podototarin N/A N/A Undetermined activity not tested N/A Primaquine Diphosphate 63-45-6 antimalarial USP, INN, BAN checkerboard Santa Cruz antihypertensive, emetic; Protoveratrine B 124-97-0 INN not tested N/A LD50(mouse) 0.21 mg/kg sc Pteryxin 13161-75-6 muscle relaxant Experimental not tested N/A Punctaporonin B 93697-36-0 N/A Undetermined activity not tested N/A xanthine oxidase inhibitor, Purpurogallin 569-77-7 Experimental not tested N/A antioxidant ornithine decarboxylase inhibitor, Putrescine Dihydrochloride 333-93-7 Experimental bliss independence Santa Cruz cell growth factor Pyrithyldione 77-04-3 hypnotic, sedative INN bliss independence Sigma Chemical Quebrachitol 642-38-6 N/A Undetermined activity bliss independence Santa Cruz gastric acid secretion inhibitor, Rabeprazole Sodium 117976-90-6 USAN, INN, BAN checkerboard VWR cell growth factor Resveratrol 501-36-0 antifungal, antibacterial Experimental bliss independence VWR Rhetsinine 526-43-2 N/A Undetermined activity not tested N/A Rhodinyl Acetate N/A N/A Undetermined activity not tested N/A Rhodocladonic Acid 26984-15-6 N/A Undetermined activity not tested N/A Ribostamycin Sulfate 25546-65-0 antibacterial INN, BAN, JAN checkerboard Santa Cruz Risedronate Sodium Hydrate 115436-72-1 calcium regulator USAN bliss independence Santa Cruz acaricide, ectoparasiticide, Rotenone 83-79-4 antineoplastic, mitochondrial agricultural use checkerboard Cayman Chemicals poison anesthetic (topical) and Safrole 9-59-7 Experimental checkerboard Sigma Chemical antiseptic, pediculicide Salsolidine 493-48-1 antihypertensive Experimental bliss independence Santa Cruz Salsoline 89-31-6 antihypertensive, antihistamine Experimental not tested N/A Sappanone A Trimethyl Ether N/A N/A Undetermined activity not tested N/A NO synthesis (inducible) inhibitor, Scopoletin 92-61-5 Experimental checkerboard VWR anticoagulant antidepressant, 5HT uptake Sertraline Hydrochloride 79559-97-0 USAN, INN, BAN checkerboard VWR inhibitor Smilagenin 126-18-1 N/A Undetermined activity not tested N/A Solidagenone 23534-56-7 N/A Undetermined activity not tested N/A Spermidine Trihydrochloride 334-50-9 ornithine decarboxylase inhibitor Experimental bliss independence Fisher Scientific Succinylacetone 51568-18-4 inhibitor of heme biosynthesis Experimental checkerboard Sigma Chemical Sulfamethizole 144-82-1 antibacterial USP, INN, BAN, JAN checkerboard Sigma Chemical Tanshinone IIA Sulfonate Sodium 568-72-9 free radical scavenger Experimental checkerboard Cayman Chemicals Terazosin Hydrochloride 70024-40-7 antihypertensive USP, INN, BAN, JAN bliss independence Fisher Scientific Thermopsine perchlorate 486-90-8 N/A Undetermined activity not tested N/A Tilorone 27591-69-1 antiviral USAN, INN bliss independence VWR Tolfenamic Acid 13710-19-5 antiinflammatory, analgesia INN, BAN, JAN checkerboard Cayman Chemicals Toremiphene Citrate 89778-27-8 antineoplastic, anti-estrogen USAN, INN, BAN checkerboard Santa Cruz Totarol-19-Carboxylic Acid, Methyl Ester N/A N/A Undetermined activity not tested N/A Tubocurarine Chloride 6989-98-6 muscle relaxant (skeletal) USP, INN, BAN, JAN bliss independence Santa Cruz bronchodilator, beta adrenergic Tulobuterol 41570-61-0 INN, BAN, JAN checkerboard VWR agonist Tylosin Tartrate 1405-54-5 antibacterial USP, INN, BAN checkerboard VWR Tyramine 51-67-2 adrenergic agonist Experimental checkerboard Cayman Chemicals Uncarine E 5171-37-9 N/A Undetermined activity not tested N/A Ursinoic Acid 30265-59-9 N/A Undetermined activity not tested N/A Ursocholanic Acid 546-18-9 N/A Undetermined activity checkerboard Santa Cruz Usnic Acid 125-46-2 antibacterial Experimental checkerboard Santa Cruz Utilin 31218-22-1 N/A Undetermined activity not tested N/A Valacyclovir Hydrochloride 124832-27-5 antiviral USAN, INN, BAN bliss independence Fisher Scientific Veratrine Sulfate 62-59-9 antihypertensive Experimental bliss independence Sigma Chemical Vincamine 1617-90-9 vasodilator INN, BAN checkerboard Cayman Chemicals CNS stimulant/depressant; Vindoline 2182-14-1 Experimental checkerboard Santa Cruz antihyperglycaemic Xylocarpus A N/A N/A Undetermined activity not tested N/A bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

adrenergic agonist, nasal Xylomethazoline Hydrochloride 1218-35-5 USP, INN, BAN checkerboard VWR decongestant Zolpidem 82626-48-0 sedative, hypnotic USAN, INN, BAN bliss independence Fisher Scientific bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Table S2. Small Molecule Bioactivity MIC 50 MIC 90 Result 1-Monopalmitin N/A 2.40 mM N/A no interaction 2-Hydroxy-3,4- prostaglandin synthetase 2.32 mM 4.64 mM no interaction Dimethoxybenzoic Acid inhibitor 2-Methoxyresorcinol N/A 14.8 mM N/A no interaction 3,4-Dimethoxycinnamic N/A 0.394 mM 7.89 mM no interaction Acid

3-Amino-1,2,4-Triazole catalase inhibitor 21.5 mM 43.1 mM no interaction 3-Bromo-7- NO synthetase inhibitor 7.11 mM 14.2 mM no interaction Nitroindazole 3-Methyl-1-Phenyl-2- antioxidant, lipoxygenase Pyrazolin-5-One (MCI- 0.408 mM 1.86 mM no interaction inhibitor 186) 7-Desacetoxy-6,7- N/A 0.563 mM 1.13 mM no interaction Dehydrogedunin Acamprosate Calcium alcohol antagonist 5.94 mM N/A no interaction Acetyltryptophan antidepressant 9.83 mM N/A no interaction Aconitic Acid N/A N/A 9.94 mM no interaction adrenergic agonist, Adrenaline Bitartrate bronchodilator, antiglaucoma 5.25 mM N/A no interaction agent Aloin cathartic, laxative 4.94 mM N/A no interaction Ancitabine antineoplastic 4.97 mM N/A no interaction Hydrochloride Artenimol antimalarial, antiinflammatory 0.682 µM N/A no interaction wound healing, Experimental Asiatic Acid N/A 18.4 µM no interaction carcinogen Astaxanthin N/A 2.00 mM N/A no interaction beta-Escin membrane permeabilizer 0.946 mM N/A no interaction Betonicine N/A 3.13 mM N/A no interaction Bicuculline GABAa antagonist N/A 3.54 mM no interaction Biochanin A phytoestrogen 5.54 mM N/A no interaction Bleomycin antineoplastic 0.952 µM 2.33 µM no interaction Calcein chelating agent (Ca, Mg) 0.765 mM N/A no interaction Caryophyllene N/A 0.4 M N/A no interaction Chlorzoxazone muscle relaxant (skeletal) N/A 1.30 mM no interaction Cholic Acid N/A 1.07 mM 1.93 mM no interaction Colistin Sulfate antibacterial N/A 0.167 mM no interaction Convallatoxin cardiotonic 10.7 mM N/A no interaction antiinflammatory, Cortisone 3.12 mM 6.24 mM no interaction glucocorticoid Cotinine antidepressant 33.8 mM 67.5 mM no interaction Cresol antiinfectant 0.775 mM 4.02 mM no interaction Daunorubicin antineoplastic N/A 3.52 mM no interaction Dexpanthenol cholinergic 0.135 M N/A no interaction bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Digitonin N/A N/A 5.86 µM no interaction Dihydroergotamine vasoconstrictor, antimigraine N/A 0.937 mM no interaction Mesylate Dihydrojasmonic Acid plant growth regulator 14.6 mM 58.3 mM no interaction antioxidant, lipoxygenase Ebselen inhibitor, inhibits oxidation of N/A 3.03 µM no interaction LDL Estrone estrogen N/A 5.30 mM no interaction Filipin antifungal N/A 11.9 mM no interaction Fludrocortisone mineralocorticoid 2.46 mM 7.35 mM no interaction Acetate Gallamine Triethiodide muscle relaxant (skeletal) 1.45 mM N/A no interaction Gemfibrozil antihyperlipoproteinemic 1.87 mM 6.16 mM no interaction Gentisic Acid analgesic, antiinflammatory N/A 11.7 mM no interaction Harmane intercalating agent, sedative N/A 1.33 mM no interaction Hexylresorcinol anthelmintic, topical antiseptic 36.8 µM 59.8 µM no interaction Hypoxanthine N/A N/A 11.0 mM no interaction cell function activator, Inosine 29.1 mM N/A no interaction cardiotonic Inositol growth factor 3.87 mM 7.49 mM no interaction Ketoconazole antifungal N/A 4.04 µM no interaction Khellin vasodilator 0.865 mM 2.31 mM no interaction L(+/-)-Alliin antibacterial, antioxidant 1.79 mM N/A no interaction Lanosterol N/A 0.445 mM N/A no interaction Larixinic Acid N/A 3.58 mM 7.18 mM no interaction Loganin N/A 2.67 mM N/A no interaction Methimazole antihyperthyroid 13.9 mM 27.8 mM no interaction N-Methylanthranilic N/A 0.278 mM 3.77 mM no interaction Acid Nonoxynol-9 spermatocide, contraceptive 0.119 M N/A no interaction Orsellinic Acid, Ethyl N/A 1.29 mM 2.94 mM no interaction Ester Paeonol antibacterial 11.0 mM 21.9 mM no interaction Pectolinarin N/A 0.546 mM 2.18 mM no interaction Phenacylamine N/A 15.9 mM N/A no interaction Hydrochloride gastric acid secretion inhibitor, Rabeprazole Sodium N/A 57.4 mM no interaction cell growth factor Succinylacetone inhibitor of heme biosynthesis 0.246 mM 1.43 mM no interaction Sulfamethizole antibacterial 14.4 mM 30.1 mM no interaction Tylosin Tartrate antibacterial 2.41 mM N/A no interaction Tyramine adrenergic agonist 11.6 mM 23.1 mM no interaction Ursocholanic Acid N/A N/A 4.92 mM no interaction Usnic Acid antibacterial 3.21 mM 3.95 mM no interaction Vincamine vasodilator 1.81 mM N/A no interaction bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

CNS stimulant/depressant; Vindoline 2.94 mM N/A no interaction antihyperglycaemic bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Table S3. Small Molecule Bioactivity MIC 90 Bithionate Sodium anthelmintic, antiseptic 3.70 µM Cloxyquin antibacterial, antifungal 947 nM Cycloheximide protein synthesis inhibitor 1.04 µM Fenbendazole anthelmintic 37.1 nM Hexachlorophene antiinfective (topical) 760 µM Miconazole antifungal (topical) 507 nM Oxiconazole Nitrate antifungal 34.9 nM Phenylmercuric Acetate antifungal 15.9 nM Pyrithione Zinc antibacterial, antifungal, antiseborrheic 22.9 µM Sulconazole Nitrate antifungal 206 nM Tacrolimus immune suppressant, antifungal 6.44 µM Thimerosal antiinfective, preservative 10.0 nM Thiram antifungal 38.4 nM bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Table S4. CM18 KN99 MUC 416-4 MUC 402-1 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 3-Amino-beta-Pinene 11.7 mM 50.1 mM 2.43 mM 4.51 mM 0.293 mM 0.879 mM 0.535 mM 0.967 mM Benzalkonium Cl N/A 14.5 µM 11.7 µM 23.3 µM 4.38 µM 8.74 µM 4.38 µM 8.74 µM Berbamine HCl N/A 63.5 µM 40.2 µM 80.5 µM N/A 80.5 mM N/A 80.5 µM Bismuth Subsalicylate 1.49 mM N/A 93.2 µM N/A 0.521 mM 1.63 mM 0.215 mM 1.11 mM Clomipramine HCl 0.650 mM 0.780 mM 0.157 mM 0.629 mM N/A 0.585 mM N/A 0.585 mM Dehydroepiandrosterone 1.50 mM 12.0 mM 1.43 mM 11.4 mM 0.880 mM 8.67 mM 3.29 mM 8.14 mM Dicyclomine HCl N/A 4.85 mM 2.42 mM 3.63 mM N/A 3.63 mM N/A 2.42 mM Estriol N/A 24.8 mM 12.4 mM 18.6 mM N/A 17.3 mM N/A 19.2 mM Fluocinolone Acetonide 1.84 mM 3.69 mM 0.345 mM 1.38 mM N/A 1.96 mM 0.460 mM 1.53 mM Sertraline HCl 0.209 mM 0.313 mM N/A 0.214 mM N/A 0.209 mM N/A 0.209 mM Xylometazoline HCl 2.44 mM N/A 2.44 mM N/A N/A N/A N/A N/A Ivermectin N/A 1.91 mM 0.158 mM 1.07 mM 0.317 mM 0.952 mM 1.07 mM N/A Nafcillin Sodium 2.37 mM N/A 1.56 mM N/A 3.46 mM N/A 3.21 mM N/A Fluconazole 25.0 µM 100 µM 24.5 µM 98.0 µM 18.4 µM 49.0 µM 12.8 µM 0.129 mM bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

NRHc.5013.ENR MUC 418-1 Ftc 555-1 NRHc.5027.EN.CLIN1 Ftc 327-1 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 0.879 mM 3.52 mM 2.40 mM 2.75 mM 1.44 mM 2.89 mM N/A 5.07 mM 1.93 mM 11.6 mM N/A 6.56 µM N/A 11.7 µM 11.7 µM 23.3 µM 11.7 µM 17.5 µM 11.7 µM 23.3 µM N/A 80.5 µM 40.2 µM 60.4 µM N/A 80.5 µM N/A 80.5 µM N/A 80.5 µM 0.128 mM 0.521 mM 0.272 mM 0.998 mM 0.544 mM 1.45 mM 0.284 mM 2.27 mM 0.340 mM 2.86 mM N/A 0.780 mM N/A 0.510 mM 0.390 mM 0.704 mM N/A 0.510 mM N/A 0.629 mM 2.12 mM 9.01 mM 3.22 mM 11.4 mM 4.10 mM 6.65 mM 3.82 mM 10.2 mM 1.95 mM 11.4 mM N/A 3.63 mM N/A 3.63 mM N/A 2.42 mM N/A 3.63 mM N/A 4.85 mM 14.7 mM 26.4 mM N/A 23.0 mM N/A 21.5 mM 24.8 mM N/A N/A N/A 1.46 mM 1.84 mM 0.767 mM 0.460 mM 0.345 mM 0.843 mM 1.33 mM 1.69 mM 0.576 mM 2.40 mM N/A 0.209 mM N/A 0.182 mM N/A 0.182 mM N/A 0.182 mM 0.107 mM 0.218 mM N/A N/A 2.90 mM N/A 2.79 mM N/A 2.00 mM N/A 26.7 mM N/A 0.218 mM N/A 0.326 mM 1.13 mM 2.17 mM N/A 0.190 mM 2.08 mM 0.358 mM 1.39 mM 3.16 mM N/A 2.20 mM N/A 3.08 mM N/A 3.76 mM N/A 3.46 mM N/A 6.14 µM 50.7 µM 15.2 µM 60.7 µM 11.7 µM 60.7 µM 25.1 µM 54.5 µM 7.96 µM 39.2 µM bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

MUC 37-1 PMHC.1050.ENR.CLIN1 NRHc.5011.ENR C. deuterogattii C. auris 1 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 N/A 70.8 mM N/A 2.90 mM N/A 3.65 mM N/A 0.967 mM N/A 9.69 mM N/A 5.83 µM N/A 11.7 µM N/A 23.3 µM N/A 5.83 µM N/A 46.8 µM N/A 82.5 µM 40.2 µM 80.5 µM N/A 40.2 µM N/A 80.5 µM 0.161 mM 0.322 mM 0.871 mM 1.27 mM 1.05 mM N/A 0.408 mM 0.816 mM 0.829 mM 2.32 mM 0.663 mM 2.32 mM N/A 0.532 mM N/A 0.629 mM N/A 0.629 mM N/A 0.629 mM 0.629 mM 1.26 mM 3.90 mM 10.2 mM 3.42 mM 9.30 mM 8.86 mM N/A 0.894 mM 1.79 mM 4.70 mM N/A N/A 2.42 mM N/A 4.85 mM N/A 3.63 mM N/A 3.63 mM 4.85 mM 9.66 mM 24.8 mM N/A 24.8 mM N/A 6.80 mM 26.4 mM N/A 24.8 mM 15.5 mM 18.6 mM 0.734 mM 1.38 mM 1.46 mM 2.08 mM 0.468 mM 1.15 mM 0.576 mM 1.38 mM N/A 0.756 mM N/A 0.200 mM N/A 0.214 mM N/A 0.214 mM N/A 0.214 mM 0.214 mM 0.429 mM 2.67 mM N/A 2.73 mM N/A 2.67 mM N/A 3.77 mM N/A N/A N/A 0.715 mM N/A 0.221 mM N/A 0.156 mM 0.207 mM 0.149 mM N/A 0.198 mM N/A 2.87 mM N/A 3.60 mM N/A 2.88 mM N/A 1.45 mM N/A 3.46 mM N/A 52.6 µM 0.111 mM 24.0 µM 90.8 µM 21.4 µM 85.9 µM 40.8 µM 81.6 µM 1.33 mM 4.00 mM bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

C. auris 2 C. albicans C. glabrata MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 4.83 mM 9.69 mM N/A 0.150 M N/A 0.150 M 31.1 µM 62.3 µM N/A 7.29 µM 5.38 µM 10.8 µM N/A 0.322 mM N/A 1.01 mM 0.126 mM 0.236 mM 1.57 mM N/A 0.497 mM 1.99 mM 0.491 mM 1.48 mM N/A 1.26 mM N/A 0.555 mM 0.660 mM 1.32 mM 2.86 mM 11.4 mM N/A 11.4 mM 0.888 mM 2.13 mM 4.85 mM 9.66 mM N/A 4.85 mM 4.74 mM 7.15 mM 6.98 mM 24.8 mM N/A 24.8 mM 6.13 mM 12.2 mM N/A 0.460 mM 3.69 mM N/A N/A 3.96 mM 0.214 mM 0.429 mM N/A 0.858 mM 1.41 mM N/A N/A N/A 2.44 mM N/A 3.13 mM N/A 0.119 mM N/A 1.54 mM N/A 0.296 mM 0.592 mM 3.16 mM N/A 3.16 mM N/A 2.43 mM N/A 1.16 mM 4.00 mM 6.25 µM 12.5 µM 0.564 mM 1.11 mM bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Table S5. CNAG Gene CNAG_00122 alpha-amylase CNAG_00185 hypothetical protein CNAG_00186 mitotic spindle organizing protein 1 ATP-binding cassette, subfamily D (ALD),peroxisomal long-chain fatty acid import CNAG_00651 protein CNAG_00696 alpha-mannosidase CNAG_00723 hypothetical protein CNAG_00727 mitochondiral protein with role in iron accumulation CNAG_00872 ATP-dependent DNA helicase CNAG_00875 rab family protein CNAG_01002 Hypothetical protein CNAG_01192 Hypothetical protein CNAG_01211 Hypothetical protein CNAG_01212 Hypothetical protein CNAG_01247 Hypothetical protein CNAG_01248 Vacuole morphology and inheritance protein 14 CNAG_01249 Hypothetical protein CNAG_01252 Thiosulfate/3-mercaptopyruvate sulfurtransferase CNAG_01256 Hypothetical protein CNAG_01585 Hypothetical protein CNAG_01975 Helicase CNAG_02016 hypothetical protein CNAG_02057 URE6; urease accessory protein CNAG_02058 Hypothetical protein CNAG_02108 GTPase activating protein CNAG_02409 LIV5; protein of unknown function CNAG_02416 hypothetical protein CNAG_02715 alpha-1,6-mannosyltransferase CNAG_02733 monosccharide transporter CNAG_03041 hypothetical protein CNAG_03043 hypothetical protein, variant 1 CNAG_03056 hypothetical protein CNAG_03460 phosphoglycerate dehydrogenase CNAG_03523 solute carrier family25, member 38 CNAG_03582 RIM20; pH-response regulator protein palA/RIM20 CNAG_03770 hypothetical protein CNAG_03828 aromatic amino acid aminotransferase I CNAG_03831 hypothetical protein CNAG_03871 phosphotyrosine protein phosphatase CNAG_03905 hypothetical protein CNAG_03908 wd-repeat protein CNAG_03938 nonmating-type specific pheromone GPCR; CPR2 CNAG_03945 hypothetical protein CNAG_03989 hypothetical protein CNAG_04155 small nuclear ribonucleoprotein E CNAG_04160 hypothetical protein, variant CNAG_04199 hypothetical protein bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

CNAG_04203 hypothetical protein CNAG_04311 charged multivesicular body protein 7 CNAG_04313 NADPH2 dehydrogenase CNAG_04618 Hypothetical protein, variant CNAG_05065 hypothetical protein CNAG_05075 solute carrier family 20 (sodium-dependent phosphate transporter) CNAG_05838 RGD1; rho GTPase activating protein homolog CNAG_06225 hypothetical protein bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was Figure S1. not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A. B. 1-Methylxanthine 1-Phenylbiguanide hydrochloride 21-Acetoxypregnenolone 5,7-Di chlorokynurenic Acid 7,8-Dihydroxyflavone 8-Cyclopentyltheophylline Acadesine Aliskiren Hemifumarate Allopurinol Aminohydroxybutyric Acid Amprolium Andrographolide Apiin Bambuterol Hydrochloride Betulin Bucladesine Citrulline Cytisine Decoquinate Digitoxin Dimethylsulfone Dopamine Hydrochloride Embelin Ergosterol Glucosaminic Acid Guaiol Harmalol Hydrochloride Hecogenin Huperzine A Hydrocortisone Hemisuccinate Hydrocor tisone Phosphate Triethylamine Lappaconitine L-Deoxyalliin Levalbuterol Hydrochloride Mangiferin Melezitose Metformin Hydrochloride Morin Oxolinic Acid Pamabrom Phloridzin Picropodophyllotoxin Putrescine Dihydrochloride Pyrithyldione Quebrachitol Resveratrol Risedronate Sodium Hydrate Salsolidine Spermidine Trihydrochloride Terazosin Hydrochloride Tilorone Tubocurarine Chloride Valacyclovir Hydrochloride Veratrine Sulfate Zolpidem DMSO Methanol

-0.5 0.0 0.5 -0.5 0.0 0.5 Bliss Score Bliss Score bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was Figure S2. not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A B Chlorzoxazone Bithionate Sodium MCI-186 Khellin Fenbendazole Filipin Calcein* Cycloheximide Bicuculline Cotinine Pyrithione Zinc Loganin Hexachlorophene 7-Desacetoxy-6,7-Dehydrogedunin Aloin* Phenylmercuric Acetate Gemfibrozil Digitonin Thimersal Vincamine* 2-Hydroxy-3,4-Dimethoxybenzoic Acid Cloxyquin Paeo nol Thiram Lanosterol* Inositol Oxiconazole Nitrate Inosine Ebselen Miconazole Asiatic Acid Tylosin Tartrate* Sulconazole Nitrate Dihydrojasmonic Acid Tacrolimus Astaxanthin 2-Methoxyresorcinol Hexylresorcinol 0 1 2 3 4 Artenimol* Ursocholanic Acid FICI Score Nonoxynol-9* Convallatoxin* Adrenaline Bitartrate* Rabeprazole Sodium* Caryophyllene* L-Alliin* 3,4-Dimethoxycinnamic Acid Tyramine Cortisone Biochanin A* Vindoline beta-Escin* Orsellinic Acid Dexpanthenol* Betonicine Gallamine Triethiodide* Bleomycin Ancitabine Hydro chloride* 1-Monopalmitin* Estrone Sulfamethizole Cresol Dihydroergotamine Mesylate Phenacylamine Hydrochloride* Fludrocortisone Acetate* N-Methylanthranilic Acid Cholic Acid Hypoxanthine 3-Amino-1,2,4-Triazole Methimazole Ketoconazole Larixinic Acid Succinylacetone Pectolinarin Acetyltryptophan Usnic Acid* Colistin Sulfate 3-Bromo-7-Nitroindazole Harmane Daunorubicin Acamprosate Calcium* Gentisic Acid Aconitic Acid

0 1 2 3 4 FICI Score bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Figure S3.

H CH N A. O E. K. I. N N CH O N O Cl Cl B. O NH N O J. N F. L. CH Cl N H C N F C. O N O O N N

G. N M. D. Proadifen N

O Drofenine

O N Naftidrofuryl* Impramine O H. Mianserine

N Cl Lofepramine Sibutramine

N Citaopram

O 0.0 0.2 0.4 0.6 0.8 FICI Score bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was Figure S4. not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A. Dicylclomine B. Fluconazole C. Synergy

unstained unstained unstained

control control control

Count 1/6 MIC 1/100 MIC 1/4 MIC DIC

1/4 MIC 1/30 MIC Synergy

1/2 MIC 1/3 MIC 1/100 MIC FLZ

D. Dicyclomine E. Fluconazole F. Synergy 20000 15000 15000 15000 10000 10000 10000 5000 5000 5000 BV421-A Intensity BV421-A BV421-A Intensity BV421-A BV421-A Intensity BV421-A 0 0 0

Control 1/3 MIC Control Synergy Control1/6 MIC1/4 MIC1/2 MIC 1/30 MIC Unstained 1/100 MIC Unstained Unstained DIC 1/4 MIC FLZ 1/100 MIC

G. 0.6 H. I. Control 0.6 Dicyclomine 0.6 Fluconazole

0.4 0.4 0.4 600 600 600 OD OD OD 0.2 Vehicle 0.2 Vehicle 0.2 Vehicle 0.4 mg/mL 0.4 mg/mL 0.4 mg/mL 0.0 0.0 0.0 0 12 24 36 48 0 12 24 36 48 0 12 24 36 48 Time Time Time

0.6 J. Synergy Vehicle 0.4

600 0.4 mg/mL

OD 0.2

0.0 0 12 24 36 48 Time bioRxiv preprint doi: https://doi.org/10.1101/843540; this version posted November 15, 2019. The copyright holder for this preprint (which was Figure S5. not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Fungal burden (8 d.p.i.) 107

106

105

104

103

102

101 colony forming units / organ / units forming colony 100

10-1

Liver Brain Lungs Spleen