Scientific Rationale

Modulators of the fungal stress response could greatly improve the ability to control otherwise lethal fungal infections in immunocompromised hosts, especially cancer patients undergoing high-dose chemotherapy and/or bone marrow transplant procedures. Although Hsp90 and calcineurin inhibitors under current development appear to be well-tolerated in early phase cancer clinical trials, compromise of host chaperone protein function could have very deleterious effects in the context of active infection and the associated stresses of fever and cardiovascular instability. For example, host immune response is compromised by treatment with nonselective calcineurin inhibitors such as cyclosporin A and FK506. An obvious way to avoid this problem would be the identification of fungal-specific inhibitors that do not impair the host chaperone protein. To address these concerns, this project seeks to identify new fungal-selective chemosensitizers. In particular, the design of the screening cascade will also allow capture of compounds targeting still other, as yet unknown, components of fungal stress response pathways that enable the emergence and maintenance of resistance to current antifungal drugs.

Several compounds have been previously identified as chemosensitizers, increasing the susceptibility of various C.albicans strains to fluconazole treatment (2,3,4,5). Cernicka et al. previously reported that the compound 7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine (CTBT, Figure 1) was capable of chemosensitizing C.albicans strains to fluconazole (3). Against fluconazole-susceptible C.albicans strain 90028 and fluconazole-resistant C.albicans strain Gu5, CTBT was effective with an MIC value of 2.4 μM when combined with fluconazole. In the absence of fluconazole, CTBT demonstrated no activity against C.albicans strain 90028, but did inhibit growth of C.albicans strain Gu5 at concentrations greater than 2.4 μM. The anti-arrhythmic drug amiodarone was recently demonstrated to act synergistically with fluconazole in C.albicans with MIC values ranging between 1.6 μM to 18.8 μM (4). Plagiochin E, a natural product isolated from liverwort, increased yeast susceptibility to fluconazole at 2.4 μM (5). These agents were not considered for the current project because of their documented single-agent antifungal activity (amiodarone, an MIC₅₀ value of 3.1 μM [6]; plagiochin E, an IC₅₀ value of 3.8 μM [5]).

Figure 1

Chemosensitizing Agents for Reversing Fluconazole Resistance.

The most potent compounds currently known are several HDAC inhibitors previously reported by Mai et al. (7). As depicted in Figure 2, compounds 4 and 5 are uracil-derived hydroxamic acids that exhibited MIC values ranging from 1.2 μM to 1.4 μM when combined with fluconazole. When tested independently, neither compound demonstrated activity against C. albicans at concentrations up to 368 μM. When compounds 4 and 5 were evaluated in a biochemical binding assay with murine HDAC1, their IC₅₀ values were measured at 37 and 51 nM, respectively (7). In addition, closely related analogs inhibit human HDAC1 and HDAC4 and were further shown to possess antiproliferative and cytotoxic effects against several human cell lines (8,9). These findings strongly suggest that compounds 4 and 5 would not be particularly selective for fungal protein targets, diminishing their potential as fungal-selective chemosensitizers. At the present time, compounds 4 and 5 have not been registered with MLSMR, are not sold commercially, and as such, are not available for evaluation in the current investigation. Ideally, screening the MLPCN compound collection would yield novel chemical hits that work by new modes of action that could be investigated by the Assay Provider, but one backup option that is available is to identify fungal selective HDAC inhibitors.

Figure 2

Uracil-derived HDAC Inhibitors Capable of Reversing Antifungal Drug Resistance.

2.1. Assays

A summary listing of completed assays and corresponding PubChem AID numbers is provided in Appendix A (Table A1). Refer to Appendix B for the detailed assay protocols.

2.2. Probe Chemical Characterization

After preparation as described in Section 2.3, the probe (CID7694069/ML229, SID 103023254) was analyzed by UPLC, ¹H and ¹³C NMR spectroscopy, and high-resolution mass spectrometry. The data obtained from NMR and mass spectroscopy were consistent with the structure of the probe, and UPLC indicated an isolated purity of greater than 98%. The architecture of the tetracyclic core was confirmed by X-ray analysis of a closely related analog. The associated spectroscopic data is provided in Appendix C, and the crystal structure is provided in Appendix E.

The observed solubility of the probe (CID7694069/ML229) was calculated to be 95.4 μM in PBS solution. The probe appears to be exceptionally stable in PBS solution (>99% remaining after a 48 hour incubation). The data from the PBS stability assay is provided in Figure 4. Plasma protein binding (PPB) was determined to be 87% bound in human plasma. The probe is stable in human plasma with greater than 99% remaining after a 5-hour incubation period. The solubility, PPB, and plasma stability results are summarized in Section 3.4 (entry 9, Table 1). The in vivo metabolic and pharmacokinetic (DMPK) properties of ML229 have not been investigated.

Figure 4

Stability of the Probe (CID7694069/ML229) in PBS at 23 °C.

Table 1

Evaluation of 16 Synthetic Ester and Amide Analogs.

The probe (CID7694069/ML229) and five additional analogs were submitted to the SMR collection MLS003390980 (probe), MLS003390979 (CID7693333), MLS003390981 (CID17581618), MLS003390982 (CID1507946), MLS003390983 (CID7708820), and MLS003390984 (CID17581348).

2.3. Probe Preparation

The probe compound 8 (CID7694069/ML229) was prepared in one step by N-alkylation of the commercially available heterocyclic core 6 with α-chloroamide 7 (Scheme 1). Alternatively, this compound may be obtained from a commercial vendor (e.g., Vitas-M Laboratory, Ltd.; Catalog No. STK582969) Full experimental details are provided in this section.

Scheme 1

Synthesis of Probe (CID7694069/ML229).

General details. All reagents and solvents were purchased from commercial vendors and used as received. NMR spectra were recorded on a Bruker 300 MHz or Varian 500 MHz spectrometer using CDCl₃, acetone-d₆, CD₃OD, or DMSO-d₆ solvents, as indicated. Proton and carbon chemical shifts are reported in ppm (δ) relative to tetramethylsilane (¹H δ 0.00) or residual chloroform in CDCl₃ solvent (¹H δ 7.24, ¹³C δ 77.0). NMR data are reported as follows: chemical shifts, multiplicity (obs. = obscured, br = broad, s = singlet, d = doublet, t = triplet, m = multiplet); coupling constant(s) in Hz; integration.

Unless otherwise indicated, NMR data were collected at 25 °C. Flash chromatography was performed using 40–60 μm Silica Gel (60 Å mesh) on a Teledyne Isco Combiflash R_f system. Tandem Liquid Chromatography/Mass Spectrometry (LC/MS) was performed on a Waters 2795 separations module and 3100 mass detector. Analytical thin layer chromatography (TLC) was performed on EM Reagent 0.25 mm silica gel 60-F plates. Visualization was accomplished with UV light and aqueous potassium permanganate (KMnO₄) stain followed by heating. High-resolution mass spectra were obtained at the MIT Mass Spectrometry Facility with a Bruker Daltonics APEXIV 4.7 Tesla Fourier Transform Ion Cyclotron Resonance mass spectrometer. X-ray crystallography was performed at the MIT Department of Chemistry X-Diffraction Facility with a Siemens three-circle Platform diffractometer, coupled to a Bruker-APEX CCD detector.

Compound purity and identity were determined by UPLC-MS (Waters, Milford, MA). Purity was measured by UV absorbance at 210 nm. Identity was determined on an SQ mass spectrometer by positive electrospray ionization. Mobile Phase A consisted of either 0.1% ammonium hydroxide or 0.1% trifluoroacetic acid in water, while mobile Phase B consisted of the same additives in acetonitrile. The gradient ran from 5% to 95% mobile Phase B over 0.8 minutes at 0.45 ml/min. An Acquity BEH C18, 1.7 μm, 1.0 × 50 mm column was used with column temperature maintained at 65°C. Compounds were dissolved in DMSO at a nominal concentration of 1 mg/ml, and 0.25 μl of this solution was injected.

Step 1. Preparation of 2-chloro-1-(pyrrolidin-1-yl)ethanone (7): Finely-ground potassium carbonate (6.9 g, 50 mmol, 3.8 equiv) was suspended in anhydrous tetrahydrofuran (13 ml) while under nitrogen and cooled to 0 °C before adding 2-chloroacetyl chloride (1.0 ml, 13 mmol, 1.0 equiv). A solution of pyrrolidine (1.0 ml, 13 mmol, 1.0 equiv) in tetrahydrofuran (26 ml) was then added dropwise via syringe. The reaction was removed from the ice bath and stirred for 17 hours at room temperature. At this point, the suspension was filtered through Celite with additional tetrahydrofuran. The filtrate was concentrated under reduced pressure to approximately 5 ml, and this solution was purified by column chromatography over silica gel (hexanes/ethyl acetate: 100/0 to 0/100), yielding the title compound as a white solid (590 mg, 32% yield).

¹H NMR (300 MHz, CDCl₃): δ 4.03 (s, 2H), 3.52 (apparent q, J = 6.4, 4H), 1.98 (m, 4H); MS (ESI⁺): 149.9.

Step 2. Preparation of 6-(2-oxo-2-(pyrrolidin-1-yl)ethyl)-2,3-dihydrothiazolo-[3′,2′:1,2]-pyrimido-[5,4-b]-indol-5(6H)-one (8): 2,3-dihydrothiazolo-[3′,2′:1,2]-pyrimido-[5,4-b]-indol-5(6H)-one 6 (Vitas-M Laboratory, Ltd.; Catalog No. STK780784, 31 mg, 0.13 mmol, 1.0 equiv) and powdered potassium hydroxide (11 mg, 0.19 mmol, 1.5 equiv) was added to a vial with stir bar. The vial was sealed with a septum cap and flushed with nitrogen, before anhydrous dimethyl sulfoxide (600 μl) was added. The mixture was stirred for 30 minutes at room temperature, then a solution of 2-chloro-1-(pyrrolidin-1-yl)ethanone 7 (30 mg, 0.20 mmol, 1.5 equiv) in dimethyl sulfoxide (0.3 ml) was added via syringe. An additional 0.3 ml of dimethyl sulfoxide was used to ensure complete transfer of 7. The reaction was stirred at room temperature for 14 hours, generating a fine, white precipitate. Water (0.5 ml) was added to the vial, and the reaction was thoroughly mixed. The fine precipitate was filtered out through a cotton plug with additional water (1 ml). The collected solid was then dissolved with 1:1 (v/v) methanol/dichloromethane (approximately 3 ml) and adsorbed on silica gel before being purified by column chromatography over silica gel (methanol/dichloromethane: 0/100 to 10/90) to give the title compound as a white solid (33 mg, 74% yield).

¹H NMR (500 MHz, CDCl₃): δ 8.10 (d, J = 8.0, 1H), 7.48 (t, J = 7.7, 1H), 7.36 (d, J = 8.4, 1H), 7.25 (t, J = 5.0, 1H), 5.42 (s, 2H), 4.54 (t, J = 7.4, 2H), 3.59 (t, J = 6.8, 2H), 3.52 (t, J = 6.3, 2H), 3.49 (t, J = 6.3, 2H), 2.08 (apparent dt, J = 6.8, 6.8, 2H), 1.92 (apparent dt, J = 6.9, 6.9,2H); ¹³C NMR (125 MHz, CDCl₃): δ 165.8, 155.4, 155.0, 141.0, 140.5, 127.9, 121.2, 120.8, 120.5, 119.0, 110.0, 48.2, 46.7, 46.2, 45.7, 27.4, 26.3, 24.0; HRMS (ESI): calculated mass for C₁₈H₁₈N₄O₂S [M+H] 355.1223, found 355.1231.

2.4. Additional Analytical Analysis

Plasma Protein Binding. Plasma protein binding was determined by equilibrium dialysis using the Rapid Equilibrium Dialysis (RED) device (Pierce Biotechnology, Rockford, IL) for both human and mouse plasma. Each compound was prepared in duplicate at 5 μM in plasma (0.95% acetonitrile, 0.05% DMSO) and added to one side of the membrane (200 μl) with PBS pH 7.4 added to the other side (350 μl). Compounds were incubated at 37 °C for 5 hours in a 250-rpm orbital shaker. Following the incubation, the samples were analyzed by UPLC-MS (Waters, Milford, MA) with compounds detected by SIR detection on a single quadrupole mass spectrometer.

Plasma Stability. Plasma stability was determined at 37 °C for 5 hours in both human and mouse plasma. Each compound was prepared in duplicate at 5 μM in plasma diluted 50/50 (v/v) with PBS pH 7.4 (0.95% acetonitrile, 0.05% DMSO). The compounds were incubated at 37 °C for 5 hours with a 250-rpm orbital shake with time points taken at 0 hours and 5 hours. The samples were analyzed by UPLC-MS (Waters, Milford, MA) with compounds detected by SIR detection on a single quadrupole mass spectrometer.

Probe attributes

• Compounds that inhibit yeast growth in the presence, but not in the absence of 8 μg/ml fluconazole.
• Compounds that show at least 10-fold selectivity between the primary Candida test strain and mammalian cells.
• Compounds that show activity toward resistant clinical isolates at an IC₅₀ < 50 μM.
• IC₅₀ ≤ 1 μM in primary or resistant screen cell line.

3.1. Summary of Screening Results

Figure 6. displays the critical path for probe development.

Figure 6

Critical Path for Probe Development.

A high-throughput screen (HTS) of 302,509 compounds (PubChem AID 1979) was performed in duplicate in the fluconazole-resistant C. albicans strain CaCi-2 in the presence of a sublethal concentration of fluconazole. Using a screening active cutoff of ≥75% inhibition at a screening concentration of 9.5 μM, 1893 hits were identified as Candida CaCi-2 growth inhibitors in the presence of fluconazole, and 1654 hits were available as cherry picks. These picked compounds were retested in dose against the C. albicans strain CaCi-2 to confirm their inhibitory activity and determine an IC₅₀ value. Of these, there were 622 compounds that met the criterion of inhibitory activity of less than or equal to 1 μM.

An orthogonal screen of these 1654 cherry picks against a more resistant C. albicans strain, CaCi-8, in the presence of a sublethal concentration of fluconazole (AID 2423) selected for compounds that were active with an IC₅₀ value less than 50 μM. There were 836 compounds that met this criterion, and 403 of these compounds were among in the 622 actives from the CaCi-2 retest (see Figure 5).

Figure 5

Bar Chart of the Antifungal Screening Campaign. Sequential results of primary, secondary, and orthogonal assays, resulting in 296 compounds that passed all criteria.

Murine 3T3 fibroblasts provided an assay for overt compound toxicity to mammalian cells. Of the 1654 cherry picks, 1012 compounds were inactive in this assay, indicating fungal selectivity, of which 350 also met the criteria in the prior CaCi-2 and CaCi-8 assays.

To eliminate cherry pick compounds that intrinsically inhibit Candida growth, an additional secondary screen of the 1654 cherry picks in the absence of fluconazole was included, with a 10 μM IC₅₀ cutoff. Of the 350 compounds of interest, 296 compounds met this criterion.

Two ancillary secondary assays were run to bin the remaining 296 compounds into three classes: Hsp90 inhibitors, calcineurin inhibitors, or other. The Hsp90 test used a Saccharomyces cerevisiae strain engineered to express beta-galactosidase driven by glucocorticoid response element. The glucocorticoid hormone receptor depends heavily on Hsp90 for function. Of the 296 compounds of interest, 17 compounds were active as defined by a 10 μM upper threshold of inhibition.

The second binning assay for calcineurin inhibition was evaluated in a yeast carrying a construct encoding calcineurin-dependent response elements (CDRE) driving expression of beta-galactosidase. Reporter activity with or without the prior addition of test compounds was measured following challenge with the stressor CaCl₂. Of the 296 compounds of interest, two compounds were active as defined by a 10 μM upper threshold of inhibition. The remaining 277 compounds were binned as “Other.”

Thirty (30) compounds were chosen for initial dry powder confirmation studies from the 296 identified above by first clustering into small groups of related analogs, and then picking representative analogs from each of those families. After re-testing these dry powders in the test cascade, three compounds were chosen as potential probe candidates, and a first round of 31 analogs (plus three probe candidates) were obtained and assayed. Using the results from these assays as guidance, a second round of analogs (plus the probe candidate) was prepared for SAR analysis. Section 4.2 describes the mechanism of action studies and categorization of the probe (i.e., “Other” mechanism).

3.2. Dose Response Curves for Probe

Figure 7Dose-dependent Activity of the Probe (ML229) Against Various Cell Lines

C.albicans CaCi-2 in the presence of fluconazole (IC₅₀ = 960 nM, AID 504502) (A); C.albicans CaCi-8 in the presence of fluconazole (IC₅₀ = 1390 nM, AID 504504) (B); Murine 3T3 fibroblasts in the absence of fluconazole (inactive, AID 504525) (C); C.albicans CaCi-2 in the absence of fluconazole (inactive, AID 504499) (D).

3.3. Scaffold/Moiety Chemical Liabilities

A search of PubChem for the probe (CID7694069/ML229) indicated that the probe has been previously evaluated in 216 bioassays but was determined to be inactive in all of them. A structure-based search in SciFinder and Reaxys did not identify any publications or patents in which the probe appeared. There are no obvious chemical liabilities associated with the new probe compound.

3.4. SAR Tables

In order to investigate the activity of this structural class, a collection of 32 structurally related analogs were synthesized and evaluated for their ability to reverse fluconazole resistance in the C.albicans test strains. Figure 8 depicts the diversification points selected for modification. Three diversity points (highlighted in purple, blue, and green) were explored, and the number of analogs screened for each site is specified.

Figure 8

Summary of analogs prepared to investigate the SAR profile of the HTS hit. Key SAR findings for each site of diversification are provided in italics

The biological assay data and physical properties of these analogs are presented below in Table 1 through Table 3. Characterization data (¹H NMR spectra and UPLC chromatograms) of these analogs are provided in Appendix C.

Table 3

Evaluation of Analogs Bearing Expanded/Truncated Tetracycle Cores (4 analogs).

The initial hit from the HTS campaign described in Section 3.1 was 6-(2-(4-methylpiperidin-1-yl)-2-oxoethyl)-2,3-dihydrothiazolo[3′,2′:1,2]pyrimido[5,4-b]indol-5(6H)-one (entry 1, Table 1). Beginning with the unsubstituted tetracycle core, a number of alternate side chains were evaluated as replacements for the 4-methylpiperidyl amide in order to improve cellular potency and PBS solubility (Table 1). In the absence of the entire side chain, the parent tetracycle retained moderate activity against the less resistant CaCi-2 line, but was ineffective against the more resistant CaCi-8 isolate (entry 2, Table 1). Converting the amide to a methyl ester did not affect potency against either strain of C. albicans (entry 4, Table 1), while the free acid was an ineffective derivative (entry 3, Table 1).

Additional amide analogs were prepared, and most of these compounds were active with IC₅₀ values in the low micromolar range. No change in potency was observed when the 4-methyl substituent was removed from the original hit (entry 5, Table 1). The necessity of the piperidine moiety was further interrogated by preparing the morpholine and N-methyl piperazine analogs (entries 6 and 7, Table 1). While there was a marked improvement in aqueous solubility, this was not accompanied any increase in potency. The 4,4-difluoropiperidyl amide showed a small improvement over the hit compound (CID 7693498) but was still fairly insoluble in PBS solution (entry 8, Table 1). Replacing the piperidine with smaller cyclic amines yielded the largest increase in potency thus far. Both pyrrolidine and 2,5-dihydropyrrole exhibited nanomolar potency against CaCi-2 and low micromolar potency against CaCi-8; the pyrrolidine derivative benefited from higher PBS solubility (entries 9 and 10, Table 1). Incorporating fluorides into the pyrrolidine scaffold diminished plasma protein binding (PPB) levels as well as its bioactivity (entry 11, Table 1). No activity was observed when the even smaller azetidine system was examined (entry 12, Table 1). Acyclic amides were inactive against CaCi-8, although some demonstrated activity against CaCi-2 (entries 13–15, Table 1). Reduction of the amide to the corresponding amine eliminated the compound’s cellular activity (entry 16, Table 1).

The tetracyclic core was also obtained with different substitutions at the 9-position, and a series of analogs was prepared from the 9-fluorotetracycle (Table 2). Many of these derivatives were considerably more effective against the less resistant CaCi-2 strain than their nonfluorinated counterparts. Without the side chain, the 9-fluorotetracycle demonstrated low micromolar activity (entry 1, Table 2), as did the methyl acetate derivative (entry 3, Table 2). The free acid was inactive in all cellular assays (entry 2, Table 2); however, all amides derived from this fluorinated tetracycle were nanomolar growth inhibitors of the CaCi-2 line (entries 4–10, Table 2) except for the azepine-derived amide (entry 9, Table 2). While not as potent as the other amides, the azepine variant was still a low micromolar chemosensitizer, lying just above the nanomolar threshold of 1.00 μM (i.e., IC₅₀ = 1.16 μM).

Table 2

Evaluation of Synthetic Analogs Bearing Substituted Tetracycle Cores (12 analogs).

Complementing the 9-fluoro derivatives, methyl ether was also incorporated into the 9-position of the tetracycle (entries 11–12, Table 2), but neither compound was an effective chemosensitizing agent. Table 3 summarizes attempts to further perturb the tetracycle core. Three analogs contained an expanded D-ring (entries 1–3, Table 3), but only one of these compounds exhibited any activity in the cellular assays (entry 3, Table 3). When the entire tetracycle was replaced with the more common beta-carboline scaffold, the resulting compound was unable to chemosensitize any of the tested C.albicans strains to fluconazole (entry 4, Table 3).

The efficacy of these tetracyclic compounds against CaCi-2 was not generally reproduced when evaluated with the more resistant CaCi-8 strain. In this particular assay, several compounds demonstrated a pronounced Eagle effect (i.e., at higher concentrations several agents lost their chemosensitizing properties) (11–13), which complicated assessment of their in vivo potency. Data from all tested concentrations, including the higher dosages where activity diminishes, were included when calculating the IC₅₀ values reported in Tables 1–3, and compounds exhibiting the Eagle effect are noted. While some compounds in Table 1 possess the characteristic parabolic dose-response curve of the Eagle effect, this phenomenon did not significantly impact the associated IC₅₀ values. However, several 9-fluoro analogs were classified as inactive in the CaCi-8 cellular assay when examined over the full range of dosages. However, if the observed Eagle effect is ignored when determining IC₅₀ values, these same compounds demonstrate nanomolar cellular activity. Because of the profound impact of the Eagle effect on the 9-fluoro compounds’ apparent potency, these analogs were not considered probe candidates. The full dose-response curves (unmasked and masked) and adjusted IC₅₀ values for compounds exhibiting this effect are provided in Appendix F (Figure A1).

Figure A1

Representative Dose-Response Curves for Compounds Exhibiting the Eagle Effect.

Overall, chemistry efforts in this project led to roughly 3-fold improvement in potency and greater than 50-fold improvement in PBS solubility relative to the hit from HTS. The HTS hit (CID7693498) was 3.0 μM against CaCi-2 with 1.6 μM PBS solubility. The probe 6-(2-oxo-2-(pyrrolidin-1-yl)ethyl)-2,3-dihydrothiazolo[3′,2′:1,2]pyrimido[5,4-b]indol-5(6H)-one (CID7694069/ML229) is 0.96 μM against CaCi-2 with 95.4 μM PBS solubility.

3.5. Cellular Activity

All assays were performed in whole cells. A murine 3T3 fibroblast mammalian cell toxicity assay was also included as a secondary screen. Experimental details are provided above in Section 2.1.2. The probe (CID7694069/ML229) clearly met the established probe criteria specified for this project (Table 4).

Table 4

Comparison of the Probe to Project Criteria.

3.6. Profiling Assays

The probe (CID7694069/ML229) was evaluated for inhibitory activity against calcinuerin and Hsp90. Assay results indicated the probe was not an inhibitor of Hsp90 or calcinuerin. Additional details are provided in Section 4.2.

4.1. Comparison to Existing Art and How the New Probe is an Improvement

Investigation into relevant prior art entailed searching the following databases: SciFinder, Reaxys, PubChem, PubMed, US Patent and Trademark Office (USPTO), PatFT, AppFT, and World Intellectual Property Organization (WIPO). The search terms applied and hit statistics are provided in Table 5. The searches were performed on and are current as of February 7, 2011.

Table 5

Search Strings and Databases Employed in Prior Art Search.

Several compounds have been previously identified as chemosensitizers, increasing the susceptibility of various C.albicans strains to fluconazole treatment (2–7). Of these, the most potent belonged to a series of HDAC inhibitors reported by Mai et al. (7). Depicted in Figure 9, these compounds are uracil-derived hydroxamic acids and exhibited MIC values ranging from 1.2 μM to 2.8 μM when combined with fluconazole. When tested in the absence of fluconazole, neither compound demonstrated activity against C.albicans at concentrations up to 368 μM. However, the potent activity of these compounds against murine HDAC1 (IC₅₀ <51 nM), combined with the antiproliferative activity of similar analogs in human cells (8,9), indicates they would not possess any significant species selectivity. The absence of selectivity disqualified compounds 4 and 5 as probe candidates. In addition, neither compound could be readily obtained for direct comparison to ML229.

Figure 9

Chemosensitizers for Reversing Fluconazole Resistance (MIC=1.2–2.8 μM).

The probe (CID7694069/ML229) has a potency of 960 nM against the C.albicans strain CaCi-2, and does not exhibit any effect in the absence of fluconazole or against mammalian fibroblasts.

4.2. Mechanism of Action Studies

All compounds were binned into one of three categories: 1) Hsp90-inhibitors, 2) Calcineurin inhibitors, or 3) Other mechanism.

Potential inhibition of the Hsp90-based chaperone machinery was evaluated using yeast reporter assays involving the glucocorticoid hormone receptor and the tyrosine kinase, v-Src. Both of these well-established client proteins depend heavily on Hsp90 for their function. Refer to Section 2.1.4 for method details.

Potential inhibition of calcineurin function was evaluated in yeast carrying a construct encoding calcineurin-dependent response elements (CDRE) driving expression of a reporter enzyme. Reporter activity with or without the prior addition of test compounds was measured following challenge with the stressor CaCl₂.

The probe (CID7694069/ML229) was classified into the “Other” categorization, showing no inhibition of calcineurin or Hsp90 pathways.

4.3. Planned Future Studies

The goal of probe optimization is to generate potent, fungal-specific agents for future mechanistic studies and target identification projects. The activity of probes against drug-resistant yeast will make possible the use of the powerful genetic approaches available including genome-wide, overexpression, and deletion libraries, both in parallel arrayed format and pooled bar-coded format. To complement genetic approaches, affinity precipitation and proteomic approaches based on Stable Isotope Labeling with Amino acids in Cell culture (SILAC) technology, which are already being employed in other projects, may be used. The probe (CID7694069/ML229) could be studied in well-established mouse models of invasive, drug-resistant Candida fungemia, which are available and have been employed in the Whitehead Institute in conjunction with the laboratory of Gerald Fink. There is currently an IACUC-approved protocol in place to enable such experimentation in future work and to evaluate the therapeutic potential of probes generated by this screen.

Primary CaCi-2 (AID no. 1979) and CaCi-2 Dose-Response Retest (AID nos. 2467, 488836, 493081, 493150, 504502)

Orthogonal Resistant Strain Dose Response (AID nos. 2423, 488807, 493082, 493149, 504504)

Counterscreen Mammalian Cell Toxicity Dose Response (AID Nos. 2327,493147, 504525)

Secondary Single Agent (No Fluconazole) Activity Assay Protocol (AID Nos. 2387, 488802, 493134, 493157, 504499)

Hsp90 Binning (AID Nos.2400, 504390)

Spectroscopic Data for SAR Analogs

¹H NMR Spectrum (300 MHz, CDCl₃) of Analog CID 7693498 (Entry 1, Table 1)

UPLC-MS Chromatogram of Analog CID 7693498

¹H NMR Spectrum (300 MHz, DMSO-d₆) of Analog CID 1508416 (Entry 2, Table 1)

UPLC-MS Chromatogram of Analog CID 1508416

¹H NMR Spectrum (300 MHz, DMSO-d₆) of Analog CID 49843149 (Entry 3, Table 1)

UPLC-MS Chromatogram of Analog CID 49843149

¹H NMR Spectrum (300 MHz, CDCl₃) of Analog CID 49835884 (Entry 4, Table 1)

UPLC-MS Chromatogram of Analog CID 49835884

¹H NMR Spectrum (300 MHz, CDCl₃) of Analog CID 49766519 (Entry 5, Table 1)

UPLC-MS Chromatogram of Analog CID 49766519

¹H NMR Spectrum (300 MHz, CDCl₃) of Analog CID 49766513 (Entry 6, Table 1)

UPLC-MS Chromatogram of Analog CID 49766513

¹H NMR Spectrum (300 MHz, Acetone-d₆) of Analog CID 49843308 (Entry 7, Table 1)

UPLC-MS Chromatogram of Analog CID 49843308

¹H NMR Spectrum (300 MHz, DMSO-d₆) of Analog CID 49842942 (Entry 8, Table 1)

UPLC-MS Chromatogram of Analog CID 49842942

¹H NMR Spectrum (300 MHz, DMSO-d₆) of Analog CID 49843324 (Entry 10, Table 1)

UPLC-MS Chromatogram of Analog CID 49843324

¹H NMR Spectrum (300 MHz, DMSO-d₆) of Analog CID 49843090 (Entry 11, Table 1)

UPLC-MS Chromatogram of Analog CID 49843090

¹H NMR Spectrum (300 MHz, DMSO-d₆) of Analog CID 49843065 (Entry 12, Table 1)

UPLC-MS Chromatogram of Analog CID 49843065

¹H NMR Spectrum (300 MHz, CDCl₃) of Analog CID 1507946 (Entry 13, Table 1)

UPLC-MS Chromatogram of Analog CID 1507946

¹H NMR Spectrum (300 MHz, CDCl₃) of Analog CID 1507368 (Entry 14, Table 1)

UPLC-MS Chromatogram of Analog CID 1507368

¹H NMR Spectrum (300 MHz, CDCl₃) of Analog CID 17581618 (Entry 15, Table 1)

UPLC-MS Chromatogram of Analog CID 17581618

¹H NMR Spectrum (300 MHz, CD₃OD) of Analog CID 49868641 (Entry 16, Table 1)

UPLC-MS Chromatogram of Analog CID 49868641

¹H NMR Spectrum (300 MHz, DMSO-d₆) of Analog CID 1508612 (Entry 1, Table 2)

UPLC-MS Chromatogram of Analog CID 1508612

¹H NMR Spectrum (300 MHz, DMSO-d₆) of Analog CID 49868642 (Entry 2, Table 2)

UPLC-MS Chromatogram of Analog CID 49868642

¹H NMR Spectrum (300 MHz, CDCl₃) of Analog CID 49868637 (Entry 3, Table 2)

UPLC-MS Chromatogram of Analog CID 49868637

¹H NMR Spectrum (300 MHz, CDCl₃) of Analog CID 6498438 (Entry 4, Table 2)

UPLC-MS Chromatogram of Analog CID 6498438

¹H NMR Spectrum (300 MHz, DMSO-d₆) of Analog CID 49868634 (Entry 5, Table 2)

UPLC-MS Chromatogram of Analog CID 49868634

¹H NMR Spectrum (300 MHz, CDCl₃) of Analog CID 49868635 (Entry 6, Table 2)

UPLC-MS Chromatogram of Analog CID 49868635

¹H NMR Spectrum (300 MHz, CDCl₃) of Analog CID 49766545 (Entry 7, Table 2)

UPLC-MS Chromatogram of Analog CID 49766545

¹H NMR Spectrum (300 MHz, CDCl₃) of Analog CID 49868641 (Entry 8, Table 2)

UPLC-MS Chromatogram of Analog CID 49868641

¹H NMR Spectrum (300 MHz, CDCl₃) of Analog CID 7708613 (Entry 9, Table 2)

UPLC-MS Chromatogram of Analog CID 7708613

¹H NMR Spectrum (300 MHz, CDCl₃) of Analog CID 1508304 (Entry 10, Table 2)

UPLC-MS Chromatogram of Analog CID 1508304

¹H NMR Spectrum (300 MHz, CDCl₃) of Analog CID 7708097 (Entry 11, Table 2)

UPLC-MS Chromatogram of Analog CID 7708097

¹H NMR Spectrum (300 MHz, CDCl₃) of Analog CID 17581348 (Entry 12, Table 2)

UPLC-MS Chromatogram of Analog CID 17581348

¹H NMR Spectrum (300 MHz, CDCl₃) of Analog CID 7708820 (Entry 1, Table 3)

UPLC-MS Chromatogram of Analog CID 7708820

¹H NMR Spectrum (300 MHz, CDCl₃) of Analog CID 1508322 (Entry 2, Table 3)

UPLC-MS Chromatogram of Analog CID 1508322

¹H NMR Spectrum (300 MHz, CDCl₃) of Analog CID 7693333 (Entry 3, Table 3)

UPLC-MS Chromatogram of Analog CID 7693333

¹H NMR Spectrum (300 MHz, CDCl₃) of Analog CID 49766506 (Entry 4, Table 3)

UPLC-MS Chromatogram of Analog CID 49766506