The investigational drug VT-1129 is a highly potent inhibitor of Cryptococcus species CYP51 but only weakly inhibits the human enzyme

Article abstract of DOI:10.1128/AAC.00349-16

Cryptococcosis is a life-threatening disease often associated with HIV infection. Three Cryptococcus species CYP51 enzymes were purified and catalyzed the 14α-demethylation of lanosterol, eburicol, and obtusifoliol. The investigational agent VT-1129 bound tightly to all three CYP51 proteins (dissociation constant [K_d] range, 14 to 25 nM) with affinities similar to those of fluconazole, voriconazole, itraconazole, clotrimazole, and ketoconazole (K_d range, 4 to 52 nM), whereas VT-1129 bound weakly to human CYP51 (K_d, 4.53 μM). VT-1129 was as effective as conventional triazole antifungal drugs at inhibiting cryptococcal CYP51 activity (50% inhibitory concentration [IC₅₀] range, 0.14 to 0.20 μM), while it only weakly inhibited human CYP51 activity (IC₅₀, ～600 μM). Furthermore, VT-1129 weakly inhibited human CYP2C9, CYP2C19, and CYP3A4, suggesting a low drug-drug interaction potential. Finally, the cellular mode of action for VT-1129 was confirmed to be CYP51 inhibition, resulting in the depletion of ergosterol and ergosta-7-enol and the accumulation of eburicol, obtusifolione, and lanosterol/obtusifoliol in the cell membranes.

Full text of DOI:10.1128/AAC.00349-16

AAC Accepted Manuscript Posted Online 9 May 2016
Antimicrob. Agents Chemother. doi:10.1128/AAC.00349-16
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
VT-1129 and cryptococcal CYP51s
1
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The Investigational Drug VT-1129 is a Highly Potent Inhibitor
of Cryptococcus species CYP51 but only Weakly Inhibits the
Human Enzyme.
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8
9
Andrew G.S. Warrilowa, Josie E. Parkera, Claire L. Pricea, W. David Nesb, Edward P.
Garveyc, William J. Hoekstrac, Robert J. Schotzingerc, Diane E. Kellya and Steven L.
Kellya*
Centre for Cytochrome P450 Biodiversity, Institute of Life Science, Swansea University
10 Medical School, Swansea, Wales SA2 8PP, United Kingdoma; Center for Chemical
11 Biology, Department of Chemistry and Biochemistry, Texas Tech University, Lubbock,
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14
Texas 79409-1061, USAb; Viamet Pharmaceuticals, Inc., Durham, NC 27703, USAc
Running title: VT-1129 and cryptococcal CYP51s.
15 Keywords: CYP51, VT-1129, Cryptococcus, azole antifungal.
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19
*Corresponding author.
Mailing address: Institute of Life Science, Swansea University Medical School,
Swansea, Wales SA2 8PP, United Kingdom. Phone: +44 1792 292207 Fax: +44 1792
20 503430 Email: s.l.kelly@swansea.ac.uk
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22
1

VT-1129 and cryptococcal CYP51s
23
Cryptococcosis is a life-threatening disease often associated with HIV. Three
24 Cryptococcus species CYP51 enzymes were purified and catalyzed the 14α-
25 demethylation of lanosterol, eburicol and obtusifoliol. The investigational agent
26 VT-1129 bound tightly to all three CYP51 proteins (Kd 14 to 25 nM) with similar
27 affinity as fluconazole, voriconazole, itraconazole, clotrimazole and ketoconazole
28 (Kd 4 to 52 nM) whereas VT-1129 bound weakly to human CYP51 (Kd 4.53 μM). VT-
29 1129 was equally as effective as conventional triazole antifungal drugs at
30
31
inhibiting cryptococcal CYP51 activity (IC50 0.14 to 0.20 μM) while only weakly
inhibited human CYP51 activity (IC50 ~600 μM). Furthermore, VT-1129 weakly
32 inhibited human CYP2C9, CYP2C19, and CYP3A4 suggesting a low drug-drug
33 interaction potential. Finally, the cellular mode of action for VT-1129 was
34
35
36
37
38
confirmed to be CYP51 inhibition resulting in the depletion of ergosterol and
ergosta-7-enol and the accumulation of eburicol, obtusifolione and lanosterol /
obtusifoliol in the cell membranes.
2

VT-1129 and cryptococcal CYP51s
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40
41
Cryptococcosis is the most common systemic fungal infection in HIV/AIDS
immunocompromised patients and is caused by the opportunistic basidiomycete yeast
pathogen Cryptococcus neoformans (1) leading to infections of the lungs and brain.
42 Meningoencephalitis is the most lethal manifestation of cryptococcosis with a life
43 expectation of less than a month if untreated (2). Pathogenic Cryptococcus species
44
45
46
47
48
49
50
51
52
53
cause disease in almost one million people annually with over 620,000 deaths and a
third of all HIV/AIDS deaths are attributable to Cryptococcus species infection (1).
Current treatment options are limited to a handful of drugs, namely initial induction
therapy with a combination of amphotericin B and flucytosine followed by a maintenance
regime of fluconazole (2). Even after administering the recommended treatment, three-
month mortality rates of 10 to 20% are common (3; 4). In addition, adopting such
treatment is costly and often impractical (with amphotericin B requiring intravenous
administration), especially in developing countries where mortality rates can approach
100% (5; 6).
Three main C. neoformans varieties are observed in clinical infections. C.
54 neoformans var. grubii (primarily serotype A), ubiquitous in the environment especially in
55 soil, is globally distributed and is responsible for almost all cryptococcal infections in
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58
HIV/AIDS patients (6 - 8). C. neoformans var. neoformans (primarily serotype D) is less
likely to cause severe infection and is more commonly found in Europe (4). C.
neoformans var. gattii (primarily serotypes B and C), a tree-dwelling basidiomycete
59 yeast primarily located in the tropics and sub-tropics with localized outbreaks in
60
61
62
northeast America, is now considered a separate species (C. gattii) and is
predominantly a primary pathogen infecting healthy (immunocompetent) individuals but
will also infect immunocompromised patients if opportunity arises (9). Most
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VT-1129 and cryptococcal CYP51s
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65
Cryptococcus infections of humans and nearly all infections of HIV/AIDS patients are
caused by C. neoformans var. grubii, the most prevalent being the H99 strain, although
C. gattii infection is increasing in prevalence, especially in North America and Africa (9).
66 The taxonomy of Cryptococcus species is still evolving with Hagen et al (10) proposing
67 that C. neoformans var. neoformans and C. neoformans var. grubii are separate species
68 and that C. gattii consists of five distinct species based on phylogenetic analysis of 11
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70
genetic loci.
Azole resistance, especially towards fluconazole, amongst Cryptococcus species
71 in the clinic can be problematic due to prolonged maintenance treatment regimens (11).
72 Increased azole tolerance in Cryptococcus species has been attributed to point
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mutations in CYP51, including G468S and Y145F (12; 13), increased expression levels
of CYP51 and the transporter protein AFR1 (14) and the genome plasticity of
Cryptococcus species post infection (15). Recently an in silico three-dimensional model
76 of C. neoformans CYP51 has been published (16) with the aim of aiding new drug
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design. Because many of the marketed azole drugs are limited by a low therapeutic
index (17), a drug with a higher therapeutic index might be able to combat resistant
pathogens at plasma concentrations still below toxic levels.
In this study we compared the novel tetrazole antifungal VT-1129 (18; 19) (Fig. 1)
with clinical azole antifungal drugs in terms of its potency and selectivity of binding to
and inhibition of three recombinant cryptococcal CYP51 enzymes compared to human
CYP51, and also to human CYPs that are critical xenobiotic-metabolizing enzymes. In
addition, the in vivo mode of action for VT-1129 was demonstrated through sterol profile
analysis.
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VT-1129 and cryptococcal CYP51s
MATERIALS AND METHODS
87
88 Construction
of
pCWori+:CneoCYP51,
pCWori+:CgruCYP51
and
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90 gene (CneoCYP51 - UniProtKB accession number Q5KQ65), the C. neoformans var.
pCWori+:CgatCYP51 expression vectors. The C. neoformans var. neoformans CYP51
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92
93
grubii CYP51 gene (CgruCYP51 - Q09GQ2) and the C. gattii CYP51 gene (CgatCYP51
- E6QZS1) were synthesized by Eurofins MWG Operon (Ebersberg, Germany)
incorporating an NdeI restriction site at the 5' end and a HindIII restriction site at the 3'
94 end of the genes cloned into the pBSIISK+ plasmid. In addition the first eight amino
95
96
acids were changed to 'MALLLAVF' (20) and
a
four-histidine extension
(CATCACCATCAC) was inserted immediately before the stop codon. The cryptococcal
97 CYP51 genes were excised by NdeI / HindIII restriction digestion followed by cloning
98
99
into the pCWori+ expression vector. Gene integrities were confirmed by DNA
sequencing.
100
Heterologous expression and purification of recombinant cryptococcal
101 CYP51 proteins. The pCWori+:CYP51 constructs were transformed into competent
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104
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DH5α E. coli cells and expressed as previously described (21). Recombinant CYP51
proteins were isolated according to the method of Arase et al (22) except that 2%
(wt/vol) sodium cholate was used in the sonication buffer and Tween-20 was omitted.
The solubilized CYP51 proteins were purified by affinity chromatography using Ni2+-NTA
agarose as previously described (23; 21) prior to characterization. The Δ60 truncated
human CYP51 was expressed and purified as previously described (24) and was shown
to be comparable to the full-length human CYP51 in terms of binding azole antifungal
drugs. Protein purities were assessed by SDS polyacrylamide gel electrophoresis.
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VT-1129 and cryptococcal CYP51s
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111
112
Cytochrome P450 protein determinations. Reduced carbon monoxide
difference spectroscopy was performed (25) with carbon monoxide being passed
through the cytochrome P450 solution prior to addition of sodium dithionite to the
113 sample cuvette (light-path 10 mm). An extinction coefficient of 91 mM-1 cm-1 (26) was
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118
used to calculate cytochrome P450 concentrations from the absorbance difference
between 447 and 490 nm. Absolute spectra were determined between 700 and 300 nm
(light-path 10 mm). All spectral determinations were made using a Hitachi U-3310
UV/VIS spectrophotometer (San Jose, California).
Ligand binding studies. Stock 2.5 mM solutions of lanosterol, eburicol and
119 obtusifoliol were prepared in 40% (wt/vol) (2-hydroxypropyl)-β-cyclodextrin (HPCD)
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121
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123
using an ultrasonic bath. Sterol was progressively titrated against 5 μM CYP51 protein in
a quartz semi-micro cuvette (light-path 4.5 mm) with equivalent amounts of 40% (wt/vol)
HPCD added to the reference cuvette which also contained 5 μM CYP51. The
absorbance difference spectrum between 500 and 350 nm was determined after each
124 incremental addition of sterol (up to 75 μM). The sterol saturation curves were
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126
127
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132
constructed from ΔA390-425 derived from the difference spectra. The substrate
dissociation constants (Kd) were determined by non-linear regression (Levenberg-
Marquardt algorithm) using the Michaelis-Menten equation.
Binding of clotrimazole, fluconazole, voriconazole, itraconazole, ketoconazole
and VT-1129 to the cryptococcal CYP51 proteins were performed as previously
described (27; 21) using split-cuvettes with a 4.5 mm light-path. Stock 0.1 mg ml-1
solutions of the azole antifungal drugs were prepared in dimethylsulfoxide and
progressively titrated against 2 μM CYP51 in 0.1 M Tris-HCl (pH 8.1) and 25% (wt/vol)
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VT-1129 and cryptococcal CYP51s
133
glycerol. The difference spectra between 500 and 350 nm were determined after each
134 incremental addition of azole and binding saturation curves were constructed from
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136
ΔApeak-trough against azole concentration. The binding properties of VT-1129 with 5 μM
recombinant human CYP51 was also determined (24). The dissociation constants of the
137 enzyme-azole complex (Kd) were determined by non-linear regression (Levenberg-
138 Marquardt algorithm) using a rearrangement of the Morrison equation for 'tight' ligand
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140
141
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143
binding (28; 29). Tight binding occurs where the Kd for a ligand is similar or lower than
the concentration of the enzyme present (30).
CYP51 reconstitution assays. Cryptococcal CYP51 reconstitution assays (31;
32) contained 0.5 μM CYP51, 1 μM Aspergillus fumigatus cytochrome P450 reductase
(AfCPR - UniProtKB accession number Q4WM67), 50 μM lanosterol or 50 μM eburicol
144 or 50 μM obtusifoliol, 50 μM dilaurylphosphatidylcholine, 4% (wt/vol) (2-hydroxypropyl)-
145
146
147
148
149
150
151
152
153
β-cyclodextrin (HPCD), 0.4 mg ml-1 isocitrate dehydrogenase, 25 mM trisodium
isocitrate, 50 mM NaCl, 5 mM MgCl2 and 40 mM MOPS (pH ~7.2). Assay mixtures were
incubated at 37°C prior to initiation with 4 mM β-NADPHNa4 followed by shaking at 37°C
for 15 minutes. Human CYP51 reconstitution assays were performed as above except
0.5 μM soluble human CYP51 (24) and 2 μM human cytochrome P450 reductase
(P16435) were used and the reaction time reduced to 5 minutes at 37°C. Sterol
metabolites were recovered by extraction with ethyl acetate followed by derivatization
with N,O-bis(trimethylsilyl)trifluoroacetamide and tetramethylsilane prior to analysis by
gas chromatography mass spectrometry (33).
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VT-1129 and cryptococcal CYP51s
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IC50 determinations were performed using 50 μM lanosterol as substrate in which
various fluconazole, itraconazole, voriconazole and VT-1129 concentrations in 2.5 μl
dimethylsulfoxide were added prior to incubation at 37°C and addition of β-NADPHNa4.
Cryptococcus sterol analysis. C. neoformans var. neoformans (strain ATCC
MYA-565), C. neoformans var. grubii (strain ATCC 208821), and C. gattii (strain ATCC
MYA-4071) were grown in MOPS buffered RPMI (0.165 M MOPS), pH 7.0, at 37°C and
160 200 rpm. MOPS buffered RPMI, pH 7.0, in the absence (DMSO control, 1% vol/vol) and
161 presence of fluconazole or VT-1129 was inoculated at a final concentration of 2.5 x 104
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163
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cells ml-1. C. neoformans var. neoformans was grown in the presence of 0.2 µg ml-1
fluconazole or 0.0039 µg ml-1 VT-1129; C. neoformans var. grubii in the presence of 0.4
µg ml-1 fluconazole or 0.0039 µg ml-1 VT-1129; C. gattii 0.4 µg ml-1 fluconazole or 0.0078
165 µg ml-1 VT-1129. Cultures were grown for 2 days at 37°C, 200 rpm and nonsaponifiable
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170
lipids were extracted as previously reported (34).
Sterones were MOX-derivatized by the addition of 200 μl of methoxyamine-HCl
(2% wt/vol in anhydrous pyridine) incubated for 30 min at 70°C. Samples were mixed
with 2 ml of saturated NaCl and the lipids extracted in three sequential 2 ml volumes of
ethyl acetate. The combined ethyl acetate fractions were washed with 2 ml volumes of
171 NaCl saturated 0.1 M HCl, saturated NaCl, NaCl saturated 5% wt/vol sodium
172
173
174
bicarbonate solution and saturated NaCl. The samples were then dried over anhydrous
magnesium sulphate and evaporated using a vacuum centrifuge. Sterols in the dried
extracts were derivatized with 0.1 ml BSTFA:TMCS (99:1) and 0.3 ml anhydrous
175 pyridine (2 h at 80°C) prior to analysis by GCMS (33). Individual sterols and sterones
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VT-1129 and cryptococcal CYP51s
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177
178
179
180
were identified by reference to relative retention times, mass ions and fragmentation
patterns. Sterol composition was calculated using peak areas.
Inhibition of human liver CYP enzymes. In vitro studies determined the IC50 of
the test compounds for CYP2C9, CYP2C19, and CYP3A4 (with either midazolam or
testosterone as substrates) in intact human liver microsomes. A separate series of
181 incubation mixtures was prepared with each test compound at final concentration in
182 reaction ranging from 0.0128 to 200 µM. Each incubation mixture contained pooled
183 human liver microsomes at an assay concentration of 1 mg ml-1 microsomal protein (Life
184 Technologies, Grand Island, NY) and metabolic substrates of isozymes for CYP2C9,
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197
CYP2C19, and CYP3A4 (diclofenac, omeprazole, and midazolam or testosterone,
respectively) at their experimentally determined Km concentrations. Active control wells
contained microsomes, substrate(s), and the test-compound diluent (i.e.
DMSO:acetonitrile:phosphate buffer, 5:5:190) substituted for test compound solutions.
The reaction was initiated by addition of an enzyme cofactor source (NADPH
regenerating solution; BD Biosciences, San Jose, CA) and the mixtures were incubated
at 37°C. After ten minutes, incubation mixtures were quenched with acetonitrile, mixed,
and centrifuged. The supernatant was analyzed by HPLC-MS/MS for the hydroxy
metabolite of the substrates. Each product peak area was normalized to be represented
as a percentage of the enzyme control average. The IC50 value for each test compound
was determined by fitting a 4-parameter logistical fit to the dose response data and
graphically determining the inhibitor concentration at 50% of the maximal enzymatic
response.
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VT-1129 and cryptococcal CYP51s
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199
Data analysis. All ligand binding experiments were performed in triplicate and
curve-fitting of data performed using the computer program ProFit 6.1.12 (QuantumSoft,
200 Zurich, Switzerland). GC/MS data were analyzed using Thermo Xcalibur 2.2 software.
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207
Chemicals. VT-1129 was provided by Viamet Pharmaceuticals, Inc. (Durham,
USA). All other chemicals were obtained from Sigma Chemical Company (Poole, UK).
Growth media, sodium ampicillin, IPTG and 5-aminolevulenic acid were obtained from
Foremedium Ltd (Hunstanton, UK). Ni2+-NTA agarose affinity chromatography matrix
was obtained from Qiagen (Crawley, UK).
10

VT-1129 and cryptococcal CYP51s
208
RESULTS
209 Expression and purification of cryptococcal CYP51 proteins. Following
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211
heterologous expression in E. coli, CneoCYP51, CgruCYP51 and CgatCYP51 were
extracted by sonication with 2% (wt/vol) sodium cholate (22), which yielded 240 (±90),
212 160 (±50) and 290 (±80) nmoles per liter culture as determined by carbon monoxide
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214
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218
difference spectroscopy (25). Purification by chromatography on Ni2+-NTA agarose
resulted in 70%, 54% and 45% recoveries of native CneoCYP51, CgruCYP51 and
CgatCYP51 proteins, respectively. SDS polyacrylamide gel electrophoresis confirmed
the purity of Ni2+-NTA agarose eluted cryptococcal CYP51 proteins to be greater than
90% when assessed by staining intensity, with apparent molecular weights of 55,000 to
58,000 compared to the predicted values of 62,708 (Cneo), 62,310 (Cgru), and 62,689
219 (Cgat) including N-terminal modifications and 4 His C-terminal extensions.
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221
Spectral properties of cryptococcal CYP51 proteins. The absolute spectra of
the resting oxidized forms of all three CYP51 proteins (Fig. 2A) were typical for a low-
222 spin ferric cytochrome P450 enzyme (23; 35) with α, β, Soret (γ) and δ spectral bands at
223 566, 536, 418 and 360 nm, respectively. Reduced carbon monoxide difference spectra
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225
226
227
228
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231
(Fig. 2B) gave the red-shifted heme Soret peak at 447 nm, characteristic of P450
enzymes, indicating that all three CYP51 proteins were expressed in the native form.
Sterol binding properties of cryptococcal CYP51 proteins. Progressive
titration with lanosterol, eburicol and obtusifoliol gave characteristic type I difference
spectra for all three CYP51 proteins with a peak at 390 nm and a trough at 425 nm (Fig.
3). Type I binding spectra occur when the substrate or another molecule displaces the
water molecule coordinated as the sixth ligand to the low-spin hexa-coordinated heme
prosthetic group causing the heme to adopt the high-spin penta-coordinated
11

VT-1129 and cryptococcal CYP51s
232 conformation (35). The cryptococcal CYP51 proteins had similar affinities for the three
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sterols (Table 1) with Kd values of 16 to 18 μM for lanosterol, 12 to 16 μM for eburicol
and 12 to 21 μM for obtusifoliol. This suggested that all three 14α-methylated sterols
were potential substrates for the cryptococcal CYP51 proteins.
The sterol binding affinities of the three cryptococcal CYP51 proteins (Kd values
12 to 21 μM) were similar to those reported for other CYP51 proteins. For example, Kd
values for lanosterol and eburicol were 11-16 and 25-28 μM with Candida albicans
CYP51 (21), 11 and 13 μM with Mycosphaerella graminicola CYP51 (36) and 0.5 to 18
μM for lanosterol with human CYP51 (37; 38; 24). However, the sterol Kd values
241 obtained were 10- to 20-fold higher than those obtained for lanosterol with
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243
244
Mycobacterium tuberculosis CYP51 (1 μM) (23) and for lanosterol and eburicol with
Trypanosoma cruzei CYP51 (1.9 and 1.2 μM) (32).
CYP51 reconstitution assays. CYP51 assays using 50 μM sterol gave turnover
245 numbers of 1.2 to 1.9 min-1 for lanosterol, 3.7 to 7.6 min-1 for eburicol and 3.5 to 4.5 min-
1
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247
248
249
250
for obtusifoliol (Table 1), confirming that all three cryptococcal CYP51 proteins readily
catalyzed the 14α-demethylation of these three sterols. Both CneoCYP51 and
CgruCYP51 displayed a substrate preference for eburicol over obtusifoliol and lanosterol
whilst CgatCYP51 displayed a substrate preference for obtusifoliol over eburicol and
lanosterol. The ability of CgatCYP51, in particular, to readily demethylate obtusifoliol
251 indicates a preference for C-24 methylated sterol substrate.
252
253
254
Azole binding properties of CYP51 proteins. All five medical azole antifungal
agents and the investigation agent VT-1129 bound tightly to all three cryptococcal
CYP51 proteins producing type II binding spectra. The binding spectra and saturation
12

VT-1129 and cryptococcal CYP51s
255
256
257
258
curves obtained for fluconazole and itraconazole (Fig. 4) and VT-1129 (Fig. 5) are
shown with a peak at ~429 nm and a trough at ~412 nm. Type II binding spectra are
caused by the triazole ring N-4 nitrogen (fluconazole, itraconazole and voriconazole) or
the imadazole ring N-3 nitrogen (clotrimazole, ketoconazole) coordinating as the sixth
259 ligand with the heme iron (39) to form the low-spin CYP51-azole complex resulting in a
260
261
'red-shift' of the heme Soret peak. The interaction of VT-1129 with the heme ferric ion is
through a terminal (N-3 or N-4) tetrazole nitrogen atom. CneoCYP51 bound the azole
262 antifungal agents the strongest with apparent Kd values of 4 to 11 nM (Table 1), followed
263 by CgatCYP51 with apparent Kd values of 5 to 24 nM and CgruCYP51 bound the azole
264 antifungal agents the weakest with apparent Kd values of 14 to 52 nM. None of the
265 cryptococcal CYP51 enzymes appeared to be inherently resistant to azole antifungal
266 agents as the range of Kd values observed (4 to 52 nM) were similar to those observed
267 with C. albicans CYP51 (10 to 56 nM) (24), unlike Aspegillus fumigatus CYP51A which
268
appeared to be inherently resistant to fluconazole with an apparent Kd value of 11.9 μM
269 (40). The binding affinity of VT-1129 to all three cryptococcal CYP51 proteins was strong
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273
(Kd 11 to 25 nM) and similar to the other five clinical azole antifungal agents examined,
suggesting VT-1129 would be effective as a therapeutic agent against Cryptococcus
species infections. The similar azole binding properties of the three cryptococcal CYP51
proteins agrees with their close sequence homology with CneoCYP51 sharing 98% and
274 96% sequence identity with CgruCYP51 and CgatCYP51.
275 In contrast, VT-1129 bound relatively weakly to human CYP51 (Fig. 5) with an
276 apparent Kd of 4.53 μM (Table 1). The interaction of VT-1129 with human CYP51 was
277 atypical as it gave rise to a red-shifted type I difference spectrum (peak at 410 nm and
278 trough at 426 nm) rather than the expected type II difference spectrum normally
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VT-1129 and cryptococcal CYP51s
279 observed for the interaction of azole antifungal agents with CYP51 proteins. This
280 suggests that the mode of interaction of VT-1129 with the human CYP51 was different
281
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283
to that observed with the three cryptococcal CYP51 proteins. VT-1129 still perturbs the
heme environment of human CYP51 as a difference spectrum was observed, though not
through the azole nitrogen directly coordinating with the heme ferric ion. This altered
284 interaction of VT-1129 with human CYP51 resulted in very weak inhibition of CYP51
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287
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activity in the CYP51 reconstitution assay (see below). The Kd values obtained for VT-
1129 with the cryptococcal CYP51 enzymes were 180- to 410-fold lower than that
obtained with the human homolog, confirming the high selectivity of VT-1129 for the
fungal target enzyme. This compared favorably with fluconazole and voriconazole,
which gave Kd values that were 370- to 1300-fold and 120- to 570-fold lower for
cryptococcal CYP51 enzymes than human CYP51. VT-1129 exhibited far greater
selectivity towards cryptococcal CYP51 enzymes over the human homolog than
292 clotrimazole, ketoconazole of itraconazole, which exhibited Kd values that were only 1.3-
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to 15-fold lower for the fungal CYP51 than human CYP51.
Azole IC50 determinations. IC50 determinations (Fig. 6) confirmed that all three
cryptococcal CYP51 proteins bound fluconazole, itraconazole voriconazole and VT-1129
tightly giving rise to strong inhibition of the CYP51 demethylation of lanosterol. IC50
values of 0.14 to 0.20 μM (Table 1) were obtained which were close to half the CYP51
298 concentration present in the assay system. VT-1129 proved equally as effective at
299 inhibiting cryptococcal CYP51 activity as the three other azole antifungal drugs,
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suggesting VT-1129 would be effective at combating Cryptococcus infections. In
contrast, VT-1129 only weakly inhibited human CYP51 activity (IC50 ~600 μM) (Fig. 7) in
agreement with the weak perturbation of the heme environment of human CYP51
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VT-1129 and cryptococcal CYP51s
303
observed with VT-1129 (Fig. 5), whereas clotrimazole severely inhibited human CYP51
304 activity (IC50 1.9 μM). The IC50 values observed for VT-1129 with the cryptococcal
305 CYP51 enzymes were 3300- to 4000-fold lower than that obtained with the human
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314
homolog (Table 1), again confirming high selectivity for the fungal target enzyme. This
was comparable to fluconazole where the IC50 value obtained with the fungal CYP51
enzymes were 6500- to 9000- fold lower than with human CYP51 and significantly better
than the observed selectivity with voriconazole and itraconazole (Table 1). The IC50
values for VT-1129 were more potent than the Kd values for binding to cryptococcal
CYP51 enzymes, suggesting that the Morrison equation calculated Kd values were an
overestimate in part due to the relatively high CYP51 protein concentrations required for
in vitro binding studies.
Cryptococcus sterol content. The treatment of Cryptococcus spp. with 0.2 to
315 0.4 µg ml-1 fluconazole and 0.0039 to 0.0078 µg ml-1 VT-1129 resulted in the
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317
accumulation of eburicol (Table 2), obtusifolione and lanosterol / obtusifoliol.
Accumulation of CYP51 substrates is indicative of direct CYP51 inhibition in treated
318 cells. Both azole treatments resulted in the depletion of the post-CYP51 sterol
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metabolites ergosta-7,22-dienol and ergosta-7-enol and the partial depletion of
ergosterol levels (Table 2) showing CYP51 inhibition. In these cellular experiments, VT-
1129 was significantly more potent than fluconazole, as VT-1129 had caused greater
322 inhibition of cryptococcal CYP51 activity at fifty-fold lower concentrations than
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fluconazole (relative to DMSO control, VT-1129 caused greater reductions in ergosterol
levels compared to fluconazole at 50-fold higher concentrations and in all cases
accumulation of 14-methylated product showing CYP51 was inhibited in cells; Table 2).
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VT-1129 and cryptococcal CYP51s
326
Inhibition of human liver drug metabolizing CYPs. Inhibition of three critical
327 xenobiotic-metabolizing CYPs by the four approved azole drugs and VT-1129 are shown
328 in Table 3. Where available, the literature IC50 values for the marketed agents (41 - 43)
329 agree well with those measured in this study. The imidazole-containing clotrimazole was
330
331
the most potent CYP inhibitor, inhibiting all activities with sub- or low-micromolar
potency. The three triazole-containing agents had variable inhibitory potencies, with
332 itraconazole potently inhibiting CYP3A4 with either substrate (0.08 and 0.13 μM),
333
334
voriconazole inhibiting all activities with a relatively tight range of potencies (4 to 13 μM),
and fluconazole showing a slightly broader range (6 to 34 μM). In contrast, VT-1129
weakly inhibited each of these activities (79 to 178 μM).
335
336
337
16

VT-1129 and cryptococcal CYP51s
338
339
DISCUSSION
Sionov et al (14) demonstrated that C. neoformans strains are heteroresistant to
340 fluconazole with each strain yielding a sub-population that can survive fluconazole
341 concentrations well above the MIC values through disomy of chromosome 1 which
342 duplicates the CYP51 and AFR1 transporter genes. Disomy of chromosome 1 coupled
343 with reported G468S and Y145F CYP51 mutations (12; 13), increased CYP51 and
344
AFR1 expression levels (14) and the genome plasticity post infection (15) may explain
345 the divergent range of MIC values reported for fluconazole with Cryptococcus spp. of 0.5
to 64 μg ml-1 (44 - 47). MIC values reported for voriconazole (0.008 to 0.5 μg ml-1),
346
347 itraconazole (0.015 to 0.5 μg ml-1) and posaconazole (0.008 to 0.5 μg ml-1) were lower
348 and less variable than for fluconazole (44 - 47), indicating therapeutic efficacy of these
349 triazole antifungals should fluconazole-tolerance become problematic. However, as
350
351
352
353
354
355
previously observed with Candida spp. and Aspergillus spp., it can be anticipated that
azole tolerance will emerge against current triazole therapeutics in Cryptococcus spp.
New antifungal drug candidates for the treatment of systemic Cryptococcus
infection which target CYP51 should ideally have high potency against the intended
cryptococcal CYP51 target enzymes and minimal interaction with human CYP51 and
other critical CYP enzymes such as those that metabolize xenobiotics. VT-1129 meets
356 both these criteria by binding tightly to cryptococcal CYP51 enzymes (Kd values 11 to 25
357 nM) with similarly high affinity as other pharmaceutical azole antifungal agents (Kd
358 values 4 to 52 nM) whilst binding weakly to the host human CYP51 in vitro (Kd 4.53 μM).
359 Binding studies (Fig. 4 and 5) provide useful preliminary information on a 'cyclized
360
361
nitrogen-containing' antifungal drug-candidate's likely effectiveness at inhibiting CYP51
activity. However, only IC50 determinations using a CYP51 reconstitution assay system
17

VT-1129 and cryptococcal CYP51s
362
363
364
365
366
367
368
369
370
371
372
can determine the functional activity of each compound as a CYP51 inhibitor. IC50
determinations confirmed that VT-1129 was a strong inhibitor of cryptococcal CYP51
activity consistent with tight-binding inhibition but only weakly inhibited human CYP51
(13% inhibition at 150 μM VT-1129). VT-1129 selectivity for the cryptococcal CYP51
protein over the human homolog was ~3300-fold in terms of inhibiting CYP51 catalysis
and VT-1129 was equally effective as conventional triazole antifungal drugs at inhibiting
cryptococcal CYP51 activity. VT-1129's selectivity for inhibiting cryptococcal CYP51
was similarly high when compared to key human xenobiotic-metabolizing CYPs,
suggesting a low potential for clinical drug-drug interactions.
Sterol profile analysis confirmed that VT-1129 inhibited cryptococcal CYP51
activity in whole cells, resulting in the depletion of ergosterol and ergosta-7-enol from the
373 cell membranes and the accumulation of 14-methylated compounds eburicol and
374
375
lanosterol / obtusifoliol and obtusifolione. In a separate study measuring a large number
of Cryptococcus spp. isolates and using 50% inhibition as the endpoint, the MIC90 for
376 VT-1129 was 0.060 μg ml-1 against 180 isolates of C. neoformans and 0.25 μg ml-1
377
378
against 321 isolates of C. gattii (19) confirming that VT-1129 is a potent inhibitor of
Cryptococcus spp. growth. In both studies, VT-1129 was a more potent inhibitor of
379 Cryptococcus spp. CYP51 than fluconazole. In addition, VT-1129 retains all or most of
380 its antifungal potency against 50 Ugandan clinical isolates of C. neoformans with
381 elevated fluconazole MIC values (48). This potency coupled with its excellent selectivity
382 versus human CYP enzymes shown here supports VT-1129 as a good candidate for the
383
384
treatment of systemic Cryptococcus infections. Given the unmet need for more potent
drugs for this disease especially in sub-Saharan Africa further assessment towards
18

VT-1129 and cryptococcal CYP51s
385
386
387
388
clinical trials are warranted, with VT-1129 Phase 1 studies now underway in healthy
volunteers.
19

VT-1129 and cryptococcal CYP51s
389
390
391
392
393
ACKNOWLEDGMENT
We are grateful to the Engineering and Physical Sciences Research Council National
Mass Spectrometry Service Centre at Swansea University and Mr. Marcus Hull for
assistance in GC/MS analyses.
This work was in part supported by the European Regional Development Fund /
394 Welsh Government funded BEACON research program (Swansea University), the
395 National Science Foundation of the United States grant NSF-MCB-09020212 awarded
396 to W. David Nes (Texas Tech University) and by Viamet Pharmaceuticals Inc (Durham,
397
398
399
NC 27703, USA).
20

VT-1129 and cryptococcal CYP51s
400
401
402
403
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565
566
567
568
569
28

VT-1129 and cryptococcal CYP51s
570
571
572
TABLE 1 Ligand binding affinities, azole IC50 values and turnover numbers for CYP51
proteins.
______________________________________________________________________
Kd (nM)
________________________________________________________
Ligand
CneoCYP51
CgruCYP51
CgatCYP51
HsapCYP51a
______________________________________________________________________
Sterols:
Lanosterol
Eburicol
Obtusifoliol
16300 ±2800
13000 ±1200
16800 ±2100
17300 ±900
11700 ±600
12200 ±3000
17500 ±1900
15800 ±1300
20600 ±1000
18400 ±1500
-
-
Azoles:
Clotrimazole
Fluconazole
Itraconazole
Ketoconazole
Voriconazole
VT-1129
4 ±3
9 ±5
7 ±3
6 ±2
4 ±2
11 ±5
44 ±18
52 ±15
42 ±11
32 ±15
14 ±9
11 ±4
24 ±9
6 ±2
5 ±2
19 ±1
24 ±10
55 ±5
30400 ±4100
92 ±7
42 ±16
2290 ±120
4530 ±300
25 ±4
______________________________________________________________________
IC50 (μM)
________________________________________________________
Azoles
CneoCYP51
CgruCYP51
CgatCYP51
HsapCYP51
______________________________________________________________________
Clotrimazole
Fluconazole
Itraconazole
Voriconazole
VT-1129
-
-
-
1.9
0.17
0.17
0.17
0.16
0.20
0.19
0.20
0.18
0.14
0.16
0.16
0.15
~1300 b
70 b
112
~600 c
______________________________________________________________________
Turnover Number (min-1)
________________________________________________________
Sterol
CneoCYP51
CgruCYP51
CgatCYP51
HsapCYP51a
______________________________________________________________________
Lanosterol
Eburicol
Obtusifoliol
1.4 ±0.2
6.1 ±0.5
3.5 ±0.3
1.9 ±0.3
7.6 ±0.4
3.6 ±0.3
1.2 ±0.2
3.7 ±0.4
4.5 ±0.6
22.7 ±4.8
-
-
______________________________________________________________________
573
574 a values (except for VT-1129) taken from Warrilow et al 2013 (24).
575
b values taken from Warrilow et al 2013 (24).
29

VT-1129 and cryptococcal CYP51s
576
c 13% inhibition observed in the presence of 150 μM VT-1129.
30