Small molecule inactivation of HIV-1 NCp7 by repetitive … · Nature Chemical Biology:...

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Small molecule inactivation of HIV-1 NCp7 by repetitive intracellular acyl

transfer

Lisa M. Miller Jenkins, David E. Ott, Ryo Hayashi, Lori V. Coren, Deyun Wang, Qun

Xu, Marco L. Schito, John K. Inman, Daniel H. Appella, Ettore Appella

Supplementary Information

Supplementary Methods

Supplementary Results

Nature Chemical Biology: doi:10.1038/nchembio.456

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Supplementary Methods

General. All chemicals and solvents were obtained from Sigma-Aldrich (Milwaukee,

WI) and were used without additional purification unless noted. Proton NMR spectra

were obtained on a Bruker 400 (400 MHz) spectrometer. Carbon NMR spectra were

obtained on a Bruker 400 (100.5 MHz) spectrometer. Proton chemical shifts are reported

as values relative to tetramethylsilane (TMS, 0.00 ppm) or to the particular solvent used

in the experiment (CDCl3: 7.26 ppm, DMSO: 2.54 ppm). Carbon chemical shifts are

reported as values relative to TMS (0.00 ppm) or to the particular solvent used in the

experiment (CDCl3: 77.0 ppm, DMSO: 77.6 ppm). Data is reported as follows: chemical

shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd =

doublet of doublets, br = broad), coupling constant, and integration. High-resolution mass

spectra (HRMS) were obtained on a LC/MSD TOF (Agilent Technologies).

I. Synthesis of 14C-labeled N-[2-(acetylthio)benzoyl]--alanine amide (SAMT-247)

The synthesis of SAMT-247 (1) and MT-1 (2) (N-(2-mercaptobenzoyl)--

alaninamide), MT-2 (3) (N-(3-mercaptobenzoyl)--alaninamide), and MT-3 (4) (N-(2-

mercaptobenzoyl)--alanine methyl ester) was performed according to previous reports1,2.

For the synthesis of 14C-labeled SAMT-247, MT-1 (15.6 mol) was incubated with

unlabeled SAMT-247 (9.39 mol) in dimethylacetamide. This mixture was added to 14C-

acetyl chloride and allowed to react for 16 h at 25 °C. The labeled product was then

separated from thiol 2 by preparative TLC (90:10 CHCl3:MeOH). The specific activity of

14C-labeled SAMT-247, as determined by scintillation counting, was 98 Ci/mol.


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II. Synthesis of N-{2-[4-(isopropylcarbonyl-oxy)benzylthio]benzoyl}--alaninamide

(MTE-1)

The synthesis and characterization of 4-(isopropylcarbonyl-oxy)benzyl iodide (9) was

carried out following a published procedure3 starting from 4-hydroxybenzyl alcohol 8 in

65% yield over four steps. Disulfide 10 was synthesized and characterized as previously

reported4.

MTE-1 was synthesized using Zhong’s method5. Sodium hydroxymethanesulfinate

hydrate (4.29 g, 36.3 mmol) and water (ca. 100 L) was added to a solution of disulfide

10 (2.70 g, 6.05 mmol), 4-(isopropylcarbonyl-oxy)benzyl iodide 9 (3.69 g, 12.1 mmol),

and potassium carbonate (3.34 g, 24.2 mmol) in DMF (20 mL) at room temperature.

The reaction mixture was stirred overnight and diluted with DCM (200 mL). The organic

layer was washed with brine, 1 N aq. HCl, dried with anhydrous sodium sulfate, filtered

and concentrated by rotary evaporation. The residue was recrystallized from EtOAc to

give MTE-1 (3.39 g, 70%) as a white solid. mp: 131-133°C; 1H NMR (400 MHz,

OH H O O

I

O

S S

HN

H2N

NH

N H 2

O O

O O

S

HN

H2N

O

O

O

O

58 9

10

a) TBD M S -C l , p y ri d in e; b ) Is o b u ty ry l chloride, pyridine; c) TBAF, THF; d) PPh3, I2, DCM; e) S 3 , K 2 C O3 , HOC H2 S O 2 N a, H2 O (C at. ), D M F

a, b,

c ,

d e

65% ov er

4

s tep s 70%




4

DMSO) δ 8.31 (t, J = 5.6 Hz, 1H), 7.50 – 7.29 (m, 6H), 7.20 (t, J = 7.0 Hz, 1H), 7.05 (d,

J = 8.5 Hz, 2H), 6.85 (s, 1H), 4.19 (s, 2H), 3.39 (dd, J = 7.1, 13.1 Hz, 2H), 2.80 (dt, J =

7.0, 13.9 Hz, 1H), 2.35 (t, J = 7.3 Hz, 2H), 1.22 (d, J = 7.0 Hz, 6H); 13C NMR (100 MHz,

DMSO) δ 175.51, 172.92, 168.00, 149.99, 136.95, 135.95, 135.18, 130.49, 130.36,

128.30, 128.14, 125.50, 122.12, 36.39, 36.27, 35.37, 33.76, 19.13; HRMS m/z for

C21H25N2O4S [M+H]+, calcd. 401.1457, found 401.1523.

III. Synthesis of N-(3-amino-3-oxopropyl)-2-(benzylthio)benzamide (MTE-2)

N-(3-amino-3-oxopropyl)-2-(benzylthio)benzamide (MTE-2, 6) was synthesized

similarly to MTE-1 by coupling disulfide 10 with benzyl bromide in 75% yield. 1H NMR

(400 MHz, DMSO) δ 8.30 (t, J = 5.5 Hz, 1H), 7.49 – 7.29 (m, 8H), 7.25 (d, J = 7.2 Hz,

1H), 7.20 (t, J = 5.4 Hz, 1H), 6.85 (s, 1H), 4.18 (s, 2H), 3.38 (dd, J = 7.1, 13.2 Hz, 2H),

2.34 (t, J = 7.4 Hz, 2H); 13C NMR (100 MHz, CD3OD) δ 176.62, 171.62, 139.26, 138.82,

135.98, 131.94, 131.43, 130.19, 129.58, 128.90, 128.33, 127.43, 39.97, 37.42, 36.01.;

HRMS m/z for C17H19N2O2S [M+H]+, calcd. 315.1089, found 315.1177.

IV. Synthesis of 2-((3-amino-3-oxopropyl)carbamoyl)-3,4,5,6-tetrafluorophenyl acetate

(7)


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A solution of 3,4,5,6-tetrafluorosalicylic acid (11, 904 mg, 4.3 mmol), benzyl bromide

(1.84 g, 10.76 mmol), and K2CO3 (1.78 g, 12.9 mmol) in acetone (40 mL) was refluxed

under nitrogen for 16 h. After filtration, the solution was concentrated in vacuo to give

the crude product (12) as a clear oil. The crude product was dissolved in THF (60 mL),

and a solution of 5 M NaOH (20 mL) was added. The mixture was reflux at 80 oC for 6 h

and the solution concentrated in vacuo to remove the organic solvents. The residue was

dissolved in water and extracted twice with hexane. The water phase was then acidified

with 3 M HCl to pH 2 and filtered to give the product as a white solid (1.01 g, 97%). Rf =

0.11 (CH2Cl2/MeOH 10:1).1H NMR (400 MHz, CDCl3) δ 7.38-7.35 (m, 5H), 4.70 (s, 2H).

To a solution of 12 (980 mg, 3.27 mmol) in DMF was added HATU (1.49 g, 3.92 mmol).

The resulting mixture was stirred at room temperature for 5 min. -Alanine amide

hydrochloride (488 mg, 3.92 mmol) was added followed by the addition of DIEPA (N,N-

diisopropylethylamine) (1.27 g, 1.71 mL, 9.81 mmol). The mixture was stirred at room

temperature overnight. Water was added and the reaction solution was extracted with

EtOAc (3 × 10 mL), dried over Na2SO4, filtered, concentrated, and purified by column

OH

OH

F

F

F F

O

O Bn

OH

F

F

F F

O

OBn

NH

F

F

F

F

O

NH2

O

O Ac

N H

F F

F F

O

NH2

O

a b

a : ( 1) BnBr , K2CO3, acetone , 75 oC , 16 h, 80% (2) NaOH, H2O/THF, 80

oC, 6 h, 97%

b : H 2N CH2C H2C ON H 2 ( H Cl) , H AT U, DIEPA, DMF, 88%

c : 10 % Pd/C , EtOH/C H 2C l2 d : Pyr idine, Ac 2 O, CH 2 C l2 , 4 h , 76% for two steps

OH

NH

F

F

F

F

O

NH2

O

c

d

147

11 12 13


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chromatography (CH2Cl2/MeOH 10:1) to afford 1.06 g (88%) of 13. Rf = 0.26

(CH2Cl2/MeOH 10:1). 1H NMR (400 MHz, CDCl3) δ 7.54-7.43 (m, 5H), 6.71(br, 1H),

5.41 (br, 1H), 5.26 (s, 2H), 3.75 (dd, 2H), 2.53 (dd, 2H). 13C NMR (100 MHz, CDCl3) δ

174.5, 160.8, 143.5, 142.3, 140.1, 135.6, 128.9, 128.6, 116.5, 116.3, 77.7, 36.0, 34.4.

HRMS (ESI+) Calcd. for C17H14F4N2O3 +Na: 393.0838. Found: 393.0842.

To a hydrogenation jar was added 10% Pd on carbon (74 mg), followed by the addition

of a solution of 13 (370 mg, 1.0 mmol) in (CH2Cl2/MeOH 4 /6 mL), the solution was

hydrogenated on a Parr hydrogenation apparatus under hydrogen (30 psi) overnight. After

filtration, solvents were evaporated in vacuo and the crude product was purified by

column chromatography (CH2Cl2/MeOH 10:1) to afford 263 mg (94%) of 14. Rf = 0.26

(CH2Cl2/MeOH 10:1, UV light: blue). 1H NMR (400 MHz, CDCl3) δ 8.08 (br, 1H), 5.58

(br, 1H), 3.88 (m, 2H), 2.70 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 176.5, 166.0, 149.3,

147.2, 145.5, 143.1, 138.0, 107.5, 37.2, 35.3. HRMS (ESI-) Calcd. for C10H8F4N2O3 :

280.0471. Found: 279.0463.

To a stirred solution of 14 (263 mg, 0.94 mmol) and pyridine (632 mg, 0.65 mL, 8.0

mmol) in CH2Cl2 (30 mL) at 0 oC was added acetic anhydride (612 mg, 0.57 mL, 6.0

mmol). The reaction was warmed to room temperature and allowed to stir for 4 h. The

mixture was poured into 20 mL of water and extracted with CH2Cl2 (3 × 10 mL). The

organic phase was then washed with 10% HCl (3 × 3 mL), dried over Na2SO4, filtered,

and concentrated. The crude product was purified by column chromatography

(CH2Cl2/MeOH 10:1) to afford 245 mg (81%) of the acetate ester-7 (7). Rf = 0.19

(CH2Cl2/MeOH 10:1, UV light: dark) 1H NMR (400 MHz, CDCl3) δ 6.81 (br, 1H), 5.56

(br, 1H), 5.5.38 (br, 1H), 3.70 (dd, 2H), 2.53 (t, 2H), 2.35 (s, 3H), 13C NMR (100 MHz,


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CDCl3) δ 174.4, 167.8, 159.5, 145.9, 143.6, 142.5, 137.1, 132.5, 106.7, 36.2, 34.4, 19.9.

HRMS (ESI-) Calcd. for C12H10F4N2O4 : 322.0577. Found: 323.0658.

Cell culture. The chronically-infected HIV-1MN and cloned H9 human T-lymphoid cell

line (Clone 46) and uninfected H9 cells used for drug treatments were cultured in RPMI

1640 with supplements: 10% fetal bovine serum, 2 mM glutamine and 100 U of

penicillin/100 g of streptomycin per ml. HEK293T cells used for transfections were

cultured in DMEM with the above supplements. Protease-deficient virions were produced

by transfecting our previously described protease mutant proviral construct PrR57G7 with

the Transit293 reagent (Mirus Bio Corp, Madison, WI).

Compound-treatment and virion analysis. To test compounds for activity against the zinc

fingers of NCp7, 5 x 106 Clone 4 cells were washed to remove virions and resuspended in

2 ml of 100 μM compound-containing medium for 1 hour at 37oC. To eliminate those

virions that were formed before treatment, this medium was then removed by

centrifugation and the cells resuspended in 2 ml of compound-containing medium.

Virions were prepared from clarified supernatants (centrifuged >1,000 X g for 10 min) by

centrifugation through a 20% wt/vol sucrose in PBS pad at >120,000 X g for 1 h at 4 oC.

Semi-dry immunoblot analysis was performed as previously described8. Primary goat

antisera against CA (#81), MA (#83), or NCp7 (#77) were obtained from the AIDS and

Cancer Virus Program, NCI-Frederick, Frederick, MD. Proteins were detected by

developing blots with HRP-conjugated anti-goat or the Immun-star HRP substrate kit

(Bio-Rad, Hercules, CA) on LumiFilm, (Roche Applied Science, Indianapolis, IN).


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Radiolabeling experiments. For experiments using radiolabeled SAMT-247, 5 Ci 14C-

labeled compound was used in the standard compound treatment assay. The synthesis of

14C-labeled SAMT-247 is described in the Supplementary Methods online. For 14C-

labeled acetate and 14C-labeled pyruvate experiments, cells were washed twice to remove

medium components and then placed in RPMI 1640, which has no pyruvate,

supplemented with 10% vol/vol dialyzed fetal bovine serum. Then, 10 mCi of 14C-acetate

or 2-14C-pyruvate (PerkinElmer, Waltham, MA) was added per sample for the respective

labelings. Labeling was carried out overnight before preparing virions and separating on

SDS-PAGE gels. Gels were then dried and analyzed with a PMI phosphorimager and

Quantity One software (Bio-Rad Laboratories, Hercules, CA).

In vitro protease digestion. Virions were lysed in 0.1% Triton X-100 and then digested

with 0.5 g recombinant HIV-1 protease (Sigma Aldrich, St. Louis, MO) in the

provider’s recommended buffer at room temperature for 4 hours. Samples were then

analyzed by immunoblot as described above.

Antiviral and virucidal assays. The HIV cytoprotection assay has been previously

described9-11. CEM-SS cells infected with HIV-1RF or HIV-1IIIB (as indicated), monocyte-

macrophoages infected with HIV-1BaL, or peripheral blood mononuclear cells (PBMCs)

infected with HIV-1 HT/92/599 were incubated with the compound for 6 days and

antiviral activity determined by measurement of cell survival (cytoprotection) using XTT

(2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium

hydroxide) dye reduction. AZT was used as a positive control for all assays. Compound

toxicity was measured the same way in uninfected cells.


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Metal ejection and mass spectrometry. Metal ejection was measured using cobalt-

refolded NCp732-55 as previously described12. Briefly, 300 M NCp732-55 was incubated

with equimolar compound in 20 mM sodium phosphate buffer, pH 7.0 at 25 ºC. The

UV/visible spectrum from 400-800 nm was collected every 0.5 h for 2.5 h. Tetrahedrally-

coordinated cobalt in NCp732-55 has maximum absorbance at 698 and 642 nm. The

absorbance at these two wavelengths was specifically monitored and compared against a

control sample to which no compound was added; experiments were repeated a minimum

of three times. Analysis of the covalent modification of zinc-coordinated NCp732-55 was

performed as previously described12. Briefly, protein was incubated with equimolar

compound at 30 ºC for 0-6 h. Samples were taken at the indicated time points and

analyzed by MALDI-TOF MS. Analysis of the covalent modification of Gag in cells was

performed by HPLC purification of Gag from density centrifugation-purified virions

(performed as previously described8) produced by infected cells either untreated or

treated with thioester 1. Quantitation of the amount of modified Gag was determined

from peak areas following this HPLC purification. Part of the purified protein was then

digested with endoproteinase GluC for 16 h at 25 °C. The remainder was reduced with

DTT for 45 min, 56 °C, then alkylated with iodoacetamide for 30 min, 25 °C, before

being digested with endoproteinase GluC for 16 h at 25 °C. The resultant peptides were

desalted and analyzed by MALDI-TOF MS. For MS/MS analysis, the peptides were

dried by vacuum-evaporation using a Vacufuge (Eppendorf) and then resuspended in

water containing 2% acetonitrile, 0.5% acetic acid. They were then injected onto a 0.2 ×

50 mm Magic C18AQ reverse phase column (Michrom Bioresources, Inc.) using the

Paradigm MS4 HPLC (Michrom Bioresources, Inc.) and separated at a flow rate of 2


10

nL/min followed by online analysis by tandem mass spectrometry using an LTQ ion trap

mass spectrometer (Thermo Scientific) equipped with an ADVANCE CaptiveSpray ion

source (Michrom Bioresources, Inc.). Peptides were eluted into the mass spectrometer

using a linear gradient from 95% mobile phase A (2% acetonitrile, 0.5% acetic acid,

97.5% water) to 65% mobile phase B (10% water, 0.5% formic acid, 89.5% acetonitrile)

over 20 minutes followed by 95% mobile phase B over 5 minutes. Peptides were

detected in positive ion mode using a data-dependent method in which the nine most

abundant ions detected in an initial survey scan were selected for MS/MS analysis. The

MS/MS spectra were searched against the HIV proteome using TurboSEQUEST in

BioWorks v. 3.3.1 SP1 (ThermoElectron, San Jose, CA). The search parameters

included: precursor mass tolerance: ±1.5 amu; fragment mass tolerance: ±0.8 amu; a

static modification of +57.02 on cysteine; a variable modification of +15.99 for

methionine oxidation; and a variable modification of +42.01 on lysine.

References 1. Song, Y. et al. Bioorg. Med. Chem. 10, 1263-73 (2002). 2. Srivastava, P. et al. Bioorg. Med. Chem. 12, 6437-50 (2004). 3. Iyer, R.P., Yu, D., Devlin, T., Ho, N.-H. & Agrawal, S. Bioorg. Med. Chem. Lett.

6, 1917-22 (1996). 4. Srivastava, P. et al. Bioorgan. Med. Chem. 12, 6437-50 (2004). 5. Tang, R.Y., Zhong, P. & Lin, Q.L. Synthesis-Stuttgart, 85-91 (2007). 6. Ott, D.E., Nigida, S.M., Jr., Henderson, L.E. & Arthur, L.O. J. Virol. 69, 2443-50

(1995). 7. Ott, D.E., Coren, L.V., Chertova, E.N., Gagliardi, T.D. & Schubert, U. Virology

278, 111-21 (2000). 8. Ott, D.E. et al. J. Virol. 73, 19-28 (1999). 9. Rice, W.G. et al. Antimicrob. Agents Chemother. 41, 419-26 (1997). 10. Rice, W.G. et al. Proc. Natl. Acad. Sci. USA 90, 9721-4 (1993). 11. Weislow, O.S. et al. J. Natl. Cancer Inst. 81, 577-86 (1989). 12. Jenkins, L.M.M. et al. J. Am. Chem. Soc. 129, 11067-78 (2007).


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Supplementary Results Supplementary Table 1. Antiviral activity of SAMT-247 in primary cells as measured by the XTT cytoprotection assay.

Cell Type Virus EC50 (M)

TC50 (M)

PBMC HIV-1

HT/92/599 5.74 >100

monocyte-macrophage

HIV-1BaL 1.97 >100


12

Supplementary Figure 1. Treatment of infected cells with SAMT-247 results in accumulation of aggregated, unprocessed Gag polyprotein. (a) Virions from infected H9 cells treated with 100 M compound were separated and analyzed on a non-reducing gel. Proteins were immunoblotted with an antibody against NCp7. (b) Virions from infected H9 cells treated with 100 M compound were separated and analyzed on a reducing gel. Additionally, samples were treated with exogenous protease (+) before electrophoresis. PR- denotes samples containing protease-deficient virions isolated from HEK293T cell transfections. Proteins were immunoblotted with an antibody against MA. (c) Virions from infected H9 cells treated with 10 or 100 M SAMT-247 were separated and analyzed on a non-reducing gel. Proteins were immunoblotted with an antibody against CA.


13

Supplementary Figure 2. Covalent modification of Gag by SAMT-247 in HIV-infected H9 cells. (a) MALDI-TOF mass spectrum showing a peptide from Gag with an altered mass following treatment with SAMT-247. Untreated protein: lower spectrum; treated protein: middle spectrum; treated protein + cysteine alkylation: upper spectrum. (b) MS/MS spectrum showing two peptides from Gag (chimeric spectrum) modified at the specified Lys residues.


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Supplementary Figure 3. MT-1 can be acetylated and is active against HIV-1 in cells. (a) Formation of SAMT-247 by reaction of MT-1 with acetyl-CoA as monitored by analytical HPLC. (b) Autoradiogram of virions from infected H9 cells treated with 14C-labeled pyruvate and either SAMT-247 or a thiol compound (100 μM). Un. denotes an uninfected control and – denotes infected cells not given a compound. (c) Virions from infected H9 cells treated with compounds (10 or 100 μM) were separated and analyzed on a reducing gel. Proteins were immunoblotted with an antibody against NCp7. (d) Virions from infected H9 cells treated with compounds (100 μM) were digested with exogenous HIV-1 protease (+), then separated on a reducing gel. Proteins were immunoblotted with an antibody against MA. An uninfected control is denoted with un.


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Supplementary Figure 4. MTE-1 treatment results in aggregation of unprocessed Gag. (a) Virions from HIV-infected H9 cells treated with the thioether compounds (100 μM) were separated and analyzed on a non-reducing gel. Proteins were immunoblotted with an antibody against NCp7. (b) Autoradiogram of virions from infected H9 cells treated with 14C-labeled pyruvate and SAMT-247 or a thioether compound (100 μM).


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Supplementary Figure 5. A thioester linkage is required for Gag inactivation in vivo. (a) Chemical structure of ester-7. (b) MALDI-TOF mass spectra collected of NCp732-55 incubated with ester-7 for 0-6 hours. The mass of unmodified NCp732-55 is indicated with an asterisk. (c) Virions from infected H9 cells treated with compounds (100 μM) were separated and analyzed on a non-reducing gel. Proteins were immunoblotted with an antibody against NCp7. (d) Virions from infected H9 cells treated with compounds (100 μM) were separated and analyzed on a reducing gel. Proteins were immunoblotted with an antibody against CA.


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