Design and optimization of highly-selective, broad spectrum fungal CYP51 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.06.037

While the orally-active azoles such as fluconazole and posaconazole are effective antifungal agents, they potently inhibit a broad range of off-target human cytochrome P450 enzymes (CYPs) leading to various safety issues (e.g., drug-drug interactions, liver, and reproductive toxicities). Recently we described the rationally-designed, antifungal agent VT-1161 that is more selective for fungal CYP51 than related human CYP enzymes such as CYP3A4. Herein, we describe the use of a homology model of Aspergillus fumigatus to design and optimize a novel series of highly selective, broad spectrum fungal CYP51 inhibitors. This series includes the oral antifungal VT-1598 that exhibits excellent potency against yeast, dermatophyte, and mold fungal pathogens.

Full text of DOI:10.1016/j.bmcl.2017.06.037

Accepted Manuscript
Design and Optimization of Highly-Selective, Broad Spectrum Fungal CYP51
Inhibitors
Christopher M. Yates, Edward P. Garvey, Sammy R. Shaver, Robert J.
Schotzinger, William J. Hoekstra
PII:
S0960-894X(17)30639-X
http://dx.doi.org/10.1016/j.bmcl.2017.06.037
BMCL 25071
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 May 2017
12 June 2017
13 June 2017
Please cite this article as: Yates, C.M., Garvey, E.P., Shaver, S.R., Schotzinger, R.J., Hoekstra, W.J., Design and
Optimization of Highly-Selective, Broad Spectrum Fungal CYP51 Inhibitors, Bioorganic & Medicinal Chemistry
Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.06.037
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Christopher M. Yates, Edward P. Garvey, Sammy R. Shaver, Robert J. Schotzinger, and William J. Hoekstra

Design and Optimization of Highly-Selective, Broad Spectrum Fungal CYP51 Inhibitors
Christopher M. Yates*, Edward P. Garvey, Sammy R. Shaver, Robert J. Schotzinger, and William J. Hoekstra
Viamet Pharmaceuticals Inc., Durham, NC 27703, USA
Abstract: While the orally-active azoles such as fluconazole and posaconazole are effective antifungal agents,
they potently inhibit a broad range of off-target human cytochrome P450 enzymes (CYPs) leading to various
safety issues (e.g., drug-drug interactions, liver, and reproductive toxicities). Recently we described the
rationally-designed, antifungal agent VT-1161 that is more selective for fungal CYP51 than related human
CYP enzymes such as CYP3A4. Herein, we describe the use of a homology model of Aspergillus fumigatus to
design and optimize a novel series of highly selective, broad spectrum fungal CYP51 inhibitors. This series
includes the oral antifungal VT-1598 that exhibits excellent potency against yeast, dermatophyte, and mold
fungal pathogens.
Metalloenzymes present a major challenge to the development of potent and selective inhibitors due to
the high homology common in the area around the metal. Many metalloenzyme inhibitors consist of two
chemical components: the metal-binding group (MBG), the portion of the inhibitor designed to bind to the
metal, and the scaffold, the portion of the inhibitor recognized by the amino acid residues that form the
substrate-binding site of the metalloenzyme. The MBG interaction is a key component to developing potent
inhibitors of metalloenzymes but if the metal/MBG interaction is too strong, unintended interactions with
related metalloenzymes can occur as well. The approach of attenuating the metal/MBG interaction in
conjunction with optimization of the scaffold has been successful in the development of potent and selective
cytochrome P450 enzyme inhibitors, 1,2 including fungal CYP51 (lanosterol 14 α-demethylase).1
Historically the azole class of antifungal drugs have inhibited fungal CYP51 activity through strong,
reversible binding to the heme-iron located in the enzyme active site utilizing 1-imidazole and 1-(1,2,4-
triazole) MBGs.3 Unfortunately, while the use of these MBGs has provided potent antifungal drugs, these
drugs are hampered by liver and reproductive toxicities and drug-drug interactions.4,5 Significant advances
have been made in the discovery of selective fungal CYP51 inhibitors through utilizing a 1-tetrazole as a
preferred MBG.1 This led to the antifungal compound VT-1161 (Figure 1) which has demonstrated excellent
yeast and dermatophyte CYP51 inhibitory potency while maintaining selectivity towards human CYP51 and
human hepatic CYP enzymes.6 VT-1161 has recently completed two separate Phase 2b studies for treatment
of onychomycosis (NCT02267356) and recurrent vulvovaginal candidiasis (NCT02267382).
Figure 1. Clinical and marketed antifungal agents.
_______
* Corresponding author. Tel: 919-467-8539, ext. 315; email: cyates@viamet.com

Although many fungal infections are treated with acute therapy, many invasive fungal infections persist
and require chronic therapy for weeks, months, or even years. Chronic fungal infections such as cryptococcal7
or coccidioidal8 meningitis are life-threatening, while other infections such as chronic pulmonary
aspergillosis9 and refractory pneumonia caused by endemic fungi8 seriously impact quality of life. In addition,
infections such as chronic mucocutaneous candidiasis are less serious but nonetheless require chronic
therapy.10 An ideal drug for the long-term treatment of such infections would have a broad spectrum that
covers yeast, mold, and endemic fungi, would have a patient-friendly dosing regimen (e.g., oral once-daily),
and would be very safe with few if any side effects and drug-drug interactions. Although many efficacious
antifungal drugs have been approved, no current drug has all the above attributes. For example, many of the
approved azoles, such as posaconazole (Figure 1), have sufficiently broad coverage of fungal pathogens, but
are highly prone to side effects due to inhibition of human CYP enzymes.4,5 These side effects not only limit
patient tolerability towards therapy, but they can also limit the dose of drug and thereby limit efficacy in
treating these very serious infections.
To aid in the development of a broad spectrum fungal CYP51 inhibitor, A. fumigatus-CYP51 was chosen
as an initial target due to the lack of mold activity in the previous discovery program. As there were no A.
fumigatus crystal structures available to provide structural guidance upon initiation of this program, we
created a series of homology models suitable for guiding our medicinal chemistry efforts. The homology
models were created using Molecular Operating Environment (MOE).11,12,13 To enable development of a
useful homology model, a series of models were constructed using seven protein crystal structures available
from the Protein Data Bank (PDB codes: 2BZ9, 3G1Q, 3K1O, 2X2N, 3KHM, 2WV2, 1EA1) as templates.14
The preliminary homology models were evaluated by docking a series of internal azole compounds, as well as
fluconazole and posaconazole.15 The models were rank ordered by their performance in correctly predicting
relative activity, using binding affinity as a surrogate. As compounds were synthesized and assayed, the
accuracy of the models was continually monitored and refined. Many of the models constructed from
templates with no ligand or small ligands were not useful due to the access channel being collapsed between
the active site and the surface of the protein. The models chosen for continued development were constructed
using 3K1O, a Trypanosoma cruzi crystal structure with posaconazole bound in the active site.16,17

Figure 2. Overlay of VT-1161 (green) and posaconazole (blue) in an A. fumigatus-CYP51 homology model.
The grid lines show the active site near the near the heme-iron and the access channel towards the surface of
the protein. The grid colors, purple and green representing hydrophilic and lipophilic, respectively, show the
predominantly lipophilic characteristics of the CYP51 access channel. The shorter VT-1161 is not able to
reach the bend in the channel and fill the lipophilic space near the entrance.
Our previous lead antifungal compound, VT-1161, was utilized for initial evaluation of the A. fumigatus
homology model and to aid in the design of a new series of compounds for synthesis. When comparing the
docked views of VT-1161 in the Candida albicans crystal structure and the new A. fumigatus homology
model, it could be seen that the linear portion of the A. fumigatus access channel is extended before it bends
out to the surface of the protein. This was further confirmed by docking posaconazole in the A. fumigatus
model and overlaying it with the VT-1161 docking (Figure 2). It was hypothesized that extended analogs
would have improved interactions with the hydrophobic access channel. A series of extended analogs with
varied linkers, flexibility, and trajectories were designed and docked to provide prioritization for synthesis.
The ethyne series showed significant potential in the homology model and was therefore chosen for initial
evaluation. A series of aryl ethyne analogs, 6a-g, maintaining several of the key components developed in
VT-1161, including the 1-tetrazole MBG, was prepared using Sonogashira or Stille couplings (Scheme 1;
Table 1).18
Scheme 1

The 4-fluorophenyl ethyne 6a showed promise in regards to maintaining potent yeast activity but the
larger lipophilic groups, demonstrated by 6b and 6c, showed a drop in antifungal activity. Antifungal activity
was also diminished when the halo group was shifted to the meta-position of the terminal phenyl ring, 6d and
6e. The extended ether and aniline based compounds 6f and 6g showed modest anti-A. fumigatus activity
against mold while retaining the strong anti-yeast activity, but 6g also showed a decrease in selectivity ratio in
regards to inhibition of CYP3A4. Previous experience in the development of VT-1161 and historical data in
the azole class of antifungal agents have demonstrated that the active site of CYP51 is sensitive in regards to
stereochemistry near the heme-iron. To test the impact of chirality of the ethyne series, example 6a was
selected for resolution by chiral HPLC. The resulting enantiomers were submitted for screening to furnish the
modest boost in antifungal activity of 6a(+) against A. fumigatus (6a(-) was inactive against both C. albicans
and A. fumigatus, data not shown).
Table 1. Antifungal Activity and CYP3A4 Potency of Aryl Ethyne Inhibitors.
A. fumigatus
c
C. albicans MICa
CYP3A4 IC50
Compound
R
MICb
>64
16
4-F
4-F
4-Cl
4-CF3
3-F
3-Cl
<0.001
<0.001
0.004
0.016
0.004
100
198
6a
6a(+)
6b
6c
6d
>64
>64
>64
>64
64
>200
>200
>200
>200
60
0.031
<0.001
6e
6f
4-OCH2CF3

4-NHCH2CF3
<0.001
0.015
0.5
4
10
0.075
32
6g
-
-
-
0.062
>64
>64
Posaconazole
Fluconazole
VT-1161
<0.001
65
a. Minimum concentration that achieved 50% inhibition of fungal growth; MIC units in µg/mL.19 b. Minimum
concentration that achieved 100% inhibition of fungal growth; MIC units in µg/mL.19 c. Inhibition of
CYP3A4 measured biochemically using microsomes obtained from pooled human hepatocytes; IC50 units in
µM.20
Further homology modeling of 6a(+) showed the compound filled the access channel in a similar manner
to VT-1161 but the extended analogs, 6f and 6g, were able to follow the bend and begin to fill the lipophilic
channel towards the surface (Figure 3). Encouraged by the activity and potential of the ether series to both
maintain selectivity and improve activity against A. fumigatus, a series of benzyl ethers was synthesized to
extend towards the surface. The compounds were synthesized in modest yields by alkylating 4-bromophenol
with benzyl bromides. The resulting aryl bromides were coupled with 4 and the epoxide opening was
performed with tetrazole to obtain the desired targets 7a-g in a manner analogous to Scheme 1.18
Figure 3. CYP51 active site view of 6f (yellow) docked into A. fumigatus-CYP51 homology model and
overlaid with posaconazole (blue).
Table 2. Antifungal Activity and CYP3A4 Potency of Benzyl Ethers.

C. albicans A. fumigatus CYP3A4
Compound
R
c
MICa
0.25
MICb
>64
>64
>64
32
IC50
4-OCF3
4-CF3
4-CONHMe
4-F
>200
>200
122
>200
>200
>200
>200
>200
>200
7a
7b
7c
7d
7e
7e(+)
7f
7f(+)
7g
0.125
0.031
0.0625
0.0625
0.031
0.031
0.0078
0.016
4-CHF2
4-CHF2
4-CN
4-CN
3-CN
4
2
8
0.25
>64
a. Minimum concentration that achieved 50% inhibition of fungal growth; MIC units in µg/mL.19 b. Minimum
concentration that achieved 100% inhibition of fungal growth; MIC units in µg/mL.19 c. Inhibition of
CYP3A4 measured biochemically using microsomes obtained from pooled human hepatocytes; IC50 units in
µM.20
A variety of moieties were explored with the primary focus on electron withdrawing groups in the 4-
position of the terminal aryl group near the surface due to size limitations in the access channel and to aid in
stabilizing the benzyl group from metabolism (Table 2). The 4-trifluoromethyl ether (7a), 4-trifluoromethyl
(7b), and 4-acetamide (7c) showed no antifungal activity against A. fumigatus. The 4-fluoro (7d), 4-
difluoromethly (7e), and 4-cyano (7f) compounds showed promise as racemates, therefore the two more
potent inhibitors, 7e and 7f, were resolved as single isomers to provide compounds with improved activity,
7e(+) and 7f(+). Compound 7f(+) demonstrated significant improvement in anti-A. fumigatus activity.
Interestingly, although the homology modeling showed space for substitution near the surface of the protein,
shifting the cyano to the meta position, 7g, led to a loss of A. fumigatus antifungal activity. The series overall
maintained good anti-yeast activity and excellent selectivity in regards to CYP3A4.
Since 7f showed promising activity, additional analogs were synthesized maintaining the cyano
portion of the molecule (Table 3). Unfortunately, altering the aryl group in 8 led to loss of anti-mold activity,
as exemplified by 8c and 8d. A variety of moieties were explored in combination with cyano on the terminal
phenyl group, with only the 3-fluoro, 4-cyano benzyl ether 8a showing promising activity; however, its
resolved enantiomer 8a(+) fell short of the activity of 7f(+). We also experimented with the inverse ether 9,
but this led to diminished activity.
Table 3. Activity profile of 7f analogs.

C. albicans A. fumigatus CYP3A4
Compound
R
Ar
c
MICa
0.031
0.004
0.16
MICb
1
IC50
3-F, 4-CN
3-F, 4-CN
2-F, 4-CN
4-CN
2,4-DiF
2,4-DiF
2,4-DiF
2-F, 4-Cl
2,5-DiF
-
>200
>200
>200
>200
>200
>200
8a
8a(+)
8b
8c
8d
0.5
>64
>64
>64
>64
0.031
0.016
0.031
4-CN
-
9
a. Minimum concentration that achieved 50% inhibition of fungal growth; MIC units in µg/mL.19 b. Minimum
concentration that achieved 100% inhibition of fungal growth; MIC units in µg/mL.19 c. Inhibition of
CYP3A4 measured biochemically using microsomes obtained from pooled human hepatocytes; IC50 units in
µM.20
Further evaluation of the representative ethynyl ether VT-1598 (7f(+)) demonstrated a marked affinity
for A. fumigatus-CYP51 (Kd = 13 nM), and a co-crystal structure with VT-1598 and A. fumigatus CYP51B
has been obtained.21,22 In an initial study of the breadth of intrinsic antifungal activity, VT-1598 had broad
coverage, similar to posaconazole (Table 4). Whereas posaconazole was slightly more potent against mold
isolates, VT-1598 was slightly more potent against yeast isolates, and both were much more potent than
fluconazole against all species tested. In terms of selectivity of inhibiting fungal CYP51, when tested against a
panel of key human CYP450 enzymes, VT-1598 showed weak or no inhibition up to the top concentration
tested (Table 5). In contrast, both posaconazole and fluconazole showed inhibition potencies that have been
proven to be clinically relevant in terms of unwanted side effects. For example, these inhibition potencies
against CYP2C9, 2C19, and 3A4 can be directly correlated with drug-drug interactions.6 Finally, VT-1598 had
robust pharmacokinetics when dosed orally in mice (5 mg/kg).23 The mean time to reach maximum measured
plasma concentration (Tmax), Cmax, AUClast, and t1/2 values were 4 hours, 2.5 µg/mL, 67 µg∙hr/mL, and 24
hours, respectively. VT-1598 concentrations were also measured in disease relevant tissues, brain, lung, and
kidney, to determine tissue levels. Relative to plasma, VT-1598 exposures were higher in kidney with Cmax
and AUC48 values 270% and 268% of plasma, respectively. In the lung, Cmax and AUC48 values were 116%
and 94% of plasma, respectively, and in the brain, Cmax and AUC48 values were 58% and 65% of plasma,
respectively. The t1/2 for each tissue was similar to plasma half-life, ranging from 16 to 19 hours. Given its
overall profile, VT-1598 has been progressed into advanced antifungal screening and in vivo models,24,25
which will be the subject of future publications.
Table 4. Broad Spectrum Antifungal Activity of VT-1598 (7f(+)).

C. albicans C. glabrata C. neoformans A. fumigatus A. flavus T. rubrum
Compound
MICa
0.0078
0.015
0.5
MICa
0.12
0.12
8
MICa
0.0019
0.0078
0.25
MICb
0.25
MICb
0.5
MICa
0.25
0.25
16
VT-1598
Posaconazole
Fluconazole
0.062
>64
0.25
>64
a. Minimum concentration that achieved 50% inhibition of fungal growth; MIC units in µg/mL.19 b. Minimum
concentration that achieved 100% inhibition of fungal growth; MIC units in µg/mL19
Table 5. Biochemical Inhibition of Human CYP Enzymes by Fungal CYP51 Inhibitors.
IC50, µM19
Human CYP 2C9
2C19
138
7.2
3A4
>200
0.075
32
11B1
>100
0.31
30
11B2
>100
0.021
13
17lyase 17OHase
19
>100
8.4
>200
109
34
38
0.042
>200
>100
1.8
VT-1598
Posaconazole
Fluconazole
13
>200
22
The development of homology models for the A. fumigatus-CYP51 has guided our effort in the
development of a novel, 1-tetrazole-based series of antifungal compounds. Building off the potent and
selective antifungal VT-1161, a series of modifications led to the discovery of a promising ethynyl ether series
that has provided compounds with strong anti-A. fumigatus activity in addition to retaining high potency
against yeast and dermatophytes. VT-1598 is a promising drug candidate which has broad antifungal activity
in combination with an excellent selectivity and pharmacokinetic profile. As such, VT-1598 may possess the
ideal attributes for a safe, broad-acting antifungal to treat serious chronic invasive fungal infections.
References and Notes
1. Hoekstra, W.J.; Garvey, E.P.; Moore, W.R.; Rafferty, S.W.; Yates, C.M.; Schotzinger, R.J. “Design and
Optimization of Highly-Selective Fungal CYP51 Inhibitors” Bioorg. Med. Chem. Lett. 2014, 24, 3455-
3458.
2. Rafferty, S.W.; Eisner, J.R.; Moore, W.R.; Schotzinger, R.J.; Hoekstra, W.J. “Highly-Selective 4-(1,2,3-
Triazole)-Based P450c17a 17,20-Lyase Inhibitors” Bioorg. Med. Chem. Lett. 2014, 24, 2444-2447.
3. Podust, L.M.; Poulos, T.L.; Waterman, M.R. “Crystal structure of cytochrome P450 14α-sterol
demethylase (CYP51) from Mycobacterium tuberculosis in complex with azole inhibitors” Proc. Nat.
Acad. Sci. 2001, 98(6), 3068-3073.
4. Pitman, S.K.; Drew, R.H.; Perfect, J.R. “Addressing current medical needs in invasive fungal infection
prevention and treatment with new antifungal agents, strategies and formulations” Expert Opin. Emerging
Drugs 2011, 16(3), 559-586.
5. Obach, R.S.; Walsky, R.L.; Venkatakrishnan, K.; Gaman, E.A.; J. Brian Houston, J.B.; Tremaine, L.M.
“The Utility of in Vitro Cytochrome P450 Inhibition Data in the Prediction of Drug-Drug Interactions” J
Pharmacol. Exp Ther 2006, 316(1), 336-348.

6. Warrilow, A.G.S.; Hull, C.M.; Parker, J.E.; Garvey, E.P.; Hoekstra, W.J.; Moore, W.R.; Schotzinger, R.J.;
Kelly, D.E.; Kelly, S.L. The Clinical Candidate VT-1161 is a Highly Potent Inhibitor of Candida albicans
CYP51 but Fails to Bind the Human Enzyme. Antimicrob. Agents Chemother. 2014, 58(12): 7121-7127.
7. Saag, M.S.; Graybill, R.J.; Larsen, R.A.; Pappas, P.G.; Perfect, J.R.; Powderly, W.G.; Sobel, J.D.;
Dismukes, W.E. Practice guidelines for the management of cryptococcal disease. Infectious Diseases
Society of America. Clin. Infect. Dis. 2000, 30(4), 710-718.
8. Galgiani, J.N.; Ampel, N.M.; Blair, J.E.; Catanzaro, A.; Geertsma, F.; Hoover, S.E.; Johnson, R.H.; Kusne,
S.; Lisse, J.; MacDonald, J.D.; Meyerson, S.L.; Raksin, P.B.; Siever, J.; Stevens, D.A.; Sunenshine, R.;
Theodore, N. Executive Summary: 2016 Infectious Diseases Society of America (IDSA) Clinical Practice
Guideline for the Treatment of Coccidioidomycosis. Clin. Infect. Dis. 2016, 63(6), 717-722.
9. Denning, D.W.; Cadranel, J.; Beigelman-Aubry, C.; Ader, F.; Chakrabarti, A.; Blot, S.; Ullmann, A.J.;
Dimopoulos, G.; Lange, C.; European Society for Clinical Microbiology and Infectious Diseases and
European Respiratory Society. Chronic pulmonary aspergillosis: rationale and clinical guidelines for
diagnosis and management. Eur. Respir. J. 2016, 47(1), 45-68.
10. Eyerich, K.; Eyerich S.; Hiller J.; Behrendt H.; Traidl-Hoffmann C. Chronic mucocutaneous candidiasis,
from bench to bedside. Eur. J. Dermatol. 2010, 20(3), 260-265.
11. Chemical Computing Group, Inc., 1010 Sherbrooke St. West, Suite #910, Montreal QC, Canada H3A 2R7
12. Relevant precedence for homology modeling of fungal CYP51: Sheng, C., et. al. J. Biomol. Struct. Dyn.
2004, 22(1), 91-99; Rupp, B., et. al. J. Comput.-Aided Mol. Des. 2005, 19, 149-163; Sheng, C., et. al.
Antimicrob. Agents Chemother. 2009, 53(8), 3487-3495; Sheng, C., et. al. ChemMedChem 2010, 5, 390-
397.
13. The homology models were prepared by selecting a protein crystal structure available from the RCSB
Protein Data Bank, aligning the selected pdb file with the sequence of human CYP51A (UniProtKB,
Q16850), then running the homology modeler program within MOE using Amber 99 as the forcefield
while selecting the heme and ligand atoms as an environment for induced fit. 10 intermediate models are
created with sidechain sampling at 300K followed by final scoring using the GB/VI (generalized
Born/volume integral) forcefield at a RMS gradient of 0.5. Ramachandran plot analysis of the final
homology model shows 8 residues as outliers (1.68% of 475 residues).
14. RCSB Protein Data Bank http://www.rcsb.org/pdb/home
15. The binding energies in the optimized homology model for fluconazole, VT-1161, and posaconazole were
-9.43, -11.67, and -14.98, respectively. The binding energy for VT-1598 was -12.72 kcal/mol.
16. The structure of posaconazole represented in the published pdb file, 3K1O, is incorrect. The correct
structure of posaconazole was used in all homology work described herein and was based upon the
following reference: Bennett, F; Saksena, A.K.; Lovey, R.G.; Liu, Y.T; Patel, N.M; Pinto, P.; Pike, R.;
Jao, E.; Girijavallabhan, V.M. Ganguly, A.K.; Loebenberg, D.; Wang, H.; Cacciapuoti, A.; Moss, E.;
Menzel, F.; Hare, R.S.; Nomeir, A. Hydroxylated analogues of the orally active broad spectrum antifungal,
Sch 51048 (1), and the discovery of posaconazole [Sch 56592; 2 or (S,S)-5], Bioorg. Med. Chem. Lett.
2006, 16(1), 186-190.
17.Lepesheva, G.I.; Hargrove, T.Y.; Anderson, S.; Kleshchenko, Y.; Furtak, V.; Wawrzak, Z.;
Villalta, F.; Waterman, M.R. “Structural Insights into Inhibition of Sterol 14α-Demethylase
in the Human Pathogen Trypanosoma cruzi” J. Biol. Chem. 2010, 285, 25582-25590.
18. Hoekstra, W.J.; Yates C.M. PCT Int. Appl. 2013, WO2013090210.
19. Minimum inhibitory concentrations (MIC) for standard ATCC isolates were determined under the CLSI
guidelines M27-A3 (with yeast endpoints being 50% inhibition of growth at 48 hours) and M38-A2 (with
mold endpoints being 100% inhibition of growth at 48 hours and dermatophyte endpoints being 100%
inhibition at 168 hours) (Eurofins Panlabs, Taiwan).
20. IC50 values for CYP enzymes were determined in biochemical assays using either human hepatocyte
microsomes (2C9, 2C19, 3A4) or recombinant enzymes (11B1, 11B2, 17, 19) with each substrate at its
measured Km value. Substrates were: diclofenac (2C9), omeprazole (2C19), testosterone (3A4),
deoxycortisol (11B1), deoxycorticosteroid (11B2), 17a-hydroxypregnenolone (17 lyase), pregnenolone
(17 hydroxylase), and testosterone (19). Reactions were analyzed for product using HPLC/MS/MS

methods and IC50 values (in µM) were determined by fitting a 4-parameter logistical fit to the dose
response data (OpAns, LLC).
21. Hargrove, T.; Garvey, E.P.; Hoekstra, W.J.; Yates, C.M.; Wawrzak, Z.; Rachakonda, G.; Villalta, F.;
Lepesheva, G.I. "Crystal structure of the new investigational drug candidate VT-1598 in complex with
Aspergillus fumigatus sterol 14α-demethylase provides insights into its broad-spectrum antifungal
activity" Antimicrob. Agents Chemother. (accepted manuscript) doi:10.1128/AAC.00570-17
22. The x-ray structure of VT-1598 in complex with Aspergillus fumigatus-CYP51 can be found at
www.rcsb.org under accession code 5FRB.
23. For the PK study in male Swiss albino mice, VT-1598 was dosed by oral gavage at 5 mg/kg in 20%
cremophor EL (in water) (N=3 mice). Time points collected were pre-dose, 0.5, 1, 2, 4, 8, 24, and 48
hours. Plasma levels were measured by HPLC/MS/MS methods and pharmacokinetic parameters were
generated by using Phoenix® WinNonLin® Version 6.4.
24. Shubitz, L.F.; Trinh, H.; Roy, M.; Garvey, E.P.; Brand, S.R.; Hoekstra, W.J.; Schotzinger, R.J. “Novel
CYP51 Inhibitors for the Treatment of Coccidioidomycosis.” The Proceedings of the 60th Annual
Coccidioidomycosis Study Group, April 9, 2016, 45.
25. Garvey, E.P.; Sharp, A.D.; Warn, P.A.; Wiederhold, N.P.; Yates, C.M.; Schotzinger, R.J. “VT-1598 is a
Highly Potent Inhibitor of Cryptococcus spp. In Vitro and In Vivo.” 10th International Conference on
Cryptococcus and Cryptococcosis, March 26-30, 2017.