Substrate Fragmentation for the Design of M. tuberculosis CYP121 Inhibitors

Abstract The cyclo‐dipeptide substrates of the essential M. tuberculosis (Mtb) enzyme CYP121 were deconstructed into their component fragments and screened against the enzyme. A number of hits were identified, one of which exhibited an unexpected inhibitor‐like binding mode. The inhibitory pharmacophore was elucidated, and fragment binding affinity was rapidly improved by synthetic elaboration guided by the structures of CYP121 substrates. The resulting inhibitors have low micromolar affinity, good predicted physicochemical properties and selectivity for CYP121 over other Mtb P450s. Spectroscopic characterisation of the inhibitors′ binding mode provides insight into the effect of weak nitrogen‐donor ligands on the P450 heme, an improved understanding of factors governing CYP121–ligand recognition and speculation into the biological role of the enzyme for Mtb.

Page S2  Table S1. Structures and screening data for select substrate fragments and structurally related analogues. Page S5 Figure S1. UV-vis absorbance spectra of CYP121 and compound 6 in competition with the type II ligand clotrimazole. Page S6  Table S3. The g-values and proportion of high spin enzyme generated by ligands binding to CYP121 in X-band EPR spectroscopy experiments. Page S7 Figure S2. EPR spectra of ligand-free CYP121, and CYP121 bound to cYY (blue) or aniline ligand 25a. Page S8 Table S4. The g-values and proportion of high spin enzyme generated by CYP121 binding to cYY, fluconazole, econazole and compound 25a.

Page S9
Supplementary synthetic schemes -Schemes S1-S6 Page S12 Synthesis Page S13 General synthetic procedures A-G Page S14 Compound characterisation Page S48 Molecular biology, biophysical and biochemical methods Table S1. Structures and screening data select fragments that were deconstructed from CYP121 substrates, and structurally related fragments that were screened to establish the binding mode and minimal pharmacophore of fragment hit 1a.
Compound ΔT m (°C) [a] ∆λ max (nm) [b] .  [c] Fragments that are also reported in Table  1 of the main paper. Table S3. The g-values and proportion of high spin enzyme generated by ligands binding to CYP121 in X-band EPR spectroscopy experiments.

Compound [a] g-values of low-spin CYP121
g-values of high-spin CYP121 HS (%) [b] g z g y g x g z g y g x DMSO [c] 2 [b] The proportion of high-spin enzyme was calculated from the relative peak area of HS to LS g z signals in each spectrum.
[c] The gvalues from 2 different spectra of ligand-free CYP121 (DMSO, 4% v/v) have been provided to account for any possible variation between different batches of the purified protein that was used to collect ligand bound spectra. Figure S2. EPR spectra of ligand-free CYP121 (black) bound to cYY (blue) and aniline ligand 25a (red). [1] (a) The g-values for low-spin CYP121 have been annotated for each species. (b) The g-values of high-spin CYP121 that is generated on binding to cYY have been annotated. The g x -value could not be determined. Table S4. The g-values and proportion of high spin enzyme generated by CYP121 binding to previously reported substrates and inhibitors (cYY, fluconazole, [2] econazole and compound 25a [1] in X-band EPR spectroscopy experiments.

Compound [a] G-values of low-spin CYP121
G-values of highspin CYP121 HS (%) [b] g z g y g x g z g y g  [1] 2.44 2.24 1.90 ---0 [a] Samples contained 100 μM CYP121 and 2 mM compounds, or 4% v/v DMSO. [b] The proportion of high-spin (HS) enzyme was calculated from the relative peak area of HS to LS g z signals in each spectrum. The proportion of HS enzyme was not reported (ND) for fluconazole-bound CYP121. Ar.

Synthesis
All reagents were commercially sourced and used without further purification, unless otherwise specified. All reactions were conducted under the positive pressure of a dry nitrogen atmosphere. Temperatures of 0 o C and -15 o C were obtained by cooling the reaction vessel in a bath of ice, or salt and ice, respectively. Anhydrous solvents were either freshly distilled over the appropriate drying reagent (DCM and MeOH over CaH 2 , THF over CaH 2 and LiAlH 4 using triphenylmethane as an indicator), or purchased directly from commercial sources.
Analytical thin layer chromatography (TLC) was performed using Merck glass-backed silica (Kieselgel 60 F254 0.25 mm) plates. Compounds were visualised using short wave (254 nm) or long wave (365 nm) ultra-violet light. Retention factors (R f ) are quoted with respect to the solvent system used to develop the plate. Flash column chromatography was performed using an Isolera TM Spektra One/Four purification system and the appropriately pre-packed GraceResolv™ LOK flash cartridge containing silica gel (40 μm) (Grace Discovery Sciences, USA). Solvents are reported as volume/volume eluent mixture where applicable. Reactions were monitored by TLC and LCMS to determine consumption of starting materials. Infrared (IR) absorption spectra were recorded on a Bruker ALPHA FT-IR or Spectrum One™ FT-IR (Perkin Elmer) spectrometer by attenuated total reflectance (ATR) using a diamond crystal. Data are reported as vibrational frequency (υ max, cm -1 ) and intensity (strong, medium, weak or broad) for the assigned functional group.
Nuclear magnetic resonance (NMR) spectra were recorded at 300 K unless otherwise stated, using either a Bruker 400 MHz AVANCE III HD Smart Probe, 400 MHz QNP cryoprobe or 500 MHz DCH cryoprobe spectrometer. All spectra were recorded in the deuterated solvent indicated. Data are reported as chemical shift in parts per million (δ ppm) relative to the residual protonated solvent resonance peak. The relative integral, multiplicity, coupling constants (J Hz) has been provided where possible. Assignment of 1 H-NMR and 13 C-NMR spectra was assisted by DEPT, homonuclear (COSY), and heteronuclear (edited 1 H-13 C-HSQC and 1 H-13 C HMBC) 2D-NMR experiments.
General Procedure B: Synthesis of thiol compounds 37h-k Thiourea (2 mmol, 2 equiv.) was added to a solution of the alkyl bromide (1 mmol, 1 equiv.) in absolute EtOH (16 mL) and the reaction was heated under reflux for 3-16 hours. The solution was then cooled to room temperature and a solution of 2 M NaOH (4 mmol, 4 equiv.) was added. The reaction was heated under reflux for 3-4 hours, then cooled to room temperature and neutralised with 1 M HCl. The volatiles were removed under reduced pressure and then the aqueous phase was diluted with DCM (50 mL). The phases were separated and the aqueous phase was extracted with DCM (10 mL). The combined organic fractions were washed with brine (2 x 10 mL), dried over anhydrous Na2SO4 and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography, eluting with the solvent system specified.
General Procedure C: Synthesis of ester compounds 5, 35a-b, 38f-g and thioester compounds 36a, 36c, 36e-h, and 38h, j-k A solution of DCC (0.55 mmol, 1.1 equiv.) in anhydrous DCM (1 mL) was added dropwise at 0 °C to a stirred solution of the acid (0.50 mmol, 1.0 equiv.), alcohol/thiol (0.60 mmol, 1.0-1.2 equiv.) and DMAP (0.05 mmol, 0.1 equiv.) in anhydrous DCM (3 mL). The reaction was allowed to come to come slowly to room temperature and stirred overnight. The reaction was then filtered and the DCC residue was washed with DCM. The filtrate was concentrated under reduced pressure and purified by flash chromatography eluting with the solvent system specified.
General Procedure D: Synthesis of amide compounds 38a-d and 39a EDC.HCl (1.1 mmol, 1.1 equiv.) and HOAt (1.1 mmol, 1.1 equiv.) were added to a stirred solution of N-Boc-D-tryptophan (1 mmol, 1 equiv.) in anhydrous DCM (25 mL) at 0 °C. The reaction was stirred for 10 minutes and then a solution of the amine (1 mmol, 1 equiv.) and Et 3 N (1.2 mmol, 1.2 equiv) in anhydrous DCM (5 ml) was added dropwise. The reaction was allowed to warm to room temperature and stirred overnight. When complete, the reaction was diluted with DCM (25 mL) washed with water (25 ml), brine (10 ml), dried over anhydrous Na 2 SO 4 and the solvent was removed under reduced pressure. Where required, the crude product was purified by flash chromatography, eluting with the solvent system specified.
General Procedure E: Synthesis of primary amide compounds 42a, f, h Isobutylchloroformate (2.4 mmol, 1.2 equiv.) was added dropwise to a stirred solution of the N-Bocprotected amino acid (2 mmol, 1 equiv.) and N-methylmorpholine (2.4 mmol, 1.2 equiv.) in dry DME (9.2 mL) at 0 °C. The reaction was stirred for 2 minutes then 35% aqueous solution of NH 3 (740 μL) was added dropwise. The reaction was stirred vigorously at 0 o C for 1 hour, then allowed to warm to room temperature and stirred for 3 hours. The reaction was then quenched with water (15 mL) and extracted with EtOAc (3 x 15 mL). The combined organic fractions were washed with 1 M HCl (5 mL) and brine (5 mL), dried over anhydrous Na 2 SO 4 and the solvent removed under reduced pressure to yield the desired product which did not require further purification.
General Procedure F: Synthesis of thiazole compounds 44a, f, h, and 45 The primary thioamide (0.5 mmol, 1 equiv.) and α-bromoketone (0.6 mmol, 1.2 equiv.) were combined in absolute EtOH (8 mL) and the reaction was allowed to stir at room temperature for 7-20 hours. When complete, the reaction was diluted with DCM (50 mL), washed with saturated NaHCO 3 (30 mL), water (20 mL), brine (20 mL) and dried over anhydrous Na 2 SO 4 . The solvent was removed under reduced pressure and the crude material was purified by flash chromatography, eluting with the solvent system specified.
General Procedure G: Deprotection of α-amine to yield compounds 2, 4, and 6-32 A solution of anhydrous 4M HCl in dioxane (5 equiv.) was added to the Boc-protected compounds (1 equiv.) and the reaction was stirred for 1-2 hours at room temperature. The solvent was then removed under reduced pressure. The crude product was redissolved in a minimal quantity of DCM and MeOH and redried under reduced pressure to yield compounds as their hydrochloride salts.

1-Diazo-3-(1H-indol-3-yl)propan-2-one (40)
HN N 2 O SOCl 2 (218 μL, 3.00 mmol) and 3 drops of dry DMF were added to a stirred suspension of indole-3acetic acid (350 mg, 2.0 mmol) in dry THF (40 mL) at 0 °C. The reaction was allowed to stir at 0 o C for 5 hours and then additional SOCl 2 (20 μL, 0.28 mmol) and dry DMF (2 drops) was added. The reaction was allowed to stir for a further 20 minutes at room temperature and then the volatiles were removed under reduced pressure. The residue was diluted with dry THF (10 mL) and added immediately to a stirred solution of TMS-diazomethane (3.5 mL, 7 mmol) in dry MeCN (10 mL) at 0 o C, under a positive pressure of N 2 . The reaction was stirred at 0 o C for 3.5 hours, then the solvent was removed under reduced pressure. The crude product was immediately purified by flash chromatography (10-50% EtOAc in hexane). Product containing fractions were collected and concentrated under reduced pressure. The resulting residue was redissolved in EtOAc (20 mL) and washed with a saturated solution of NaHCO 3 (4 x 2 mL). The solvent was then removed under reduced pressure to yield compound 40 as an orange oil (246.

tert-Butyl (R)-(2-(1H-indol-3-yl)-1-(5-phenyl-1H-imidazol-2-yl)ethyl)carbamate (48)
N NHBoc NH NH Cs 2 CO 3 (163 mg, 0.5 mmol) was added to a solution of N-Boc-D-tryptophan (304 mg, 1.0 mmol) in absolute EtOH (2 mL) and the reaction was stirred at room temperature for 30 minutes before being concentrated under reduced pressure. The residue was redissolved in anhydrous DMF (3.6 mL) and 2-bromoacetophenone (199 mg, 1.0 mmol) was added. The reaction was stirred at room temperature for 4 hours and then the solvent was removed under reduced pressure. The residue was suspended with EtOAc (20 mL), filtered and washed with EtOAc (20 mL). The combined filtrates were concentrated under reduced pressure and the resulting yellow oil was redissolved in anhydrous m-xylene (12 mL). NH 4 OAc (1.5 g, 1.95 mmol) was added and the reaction was heated performed in reduced volume (200 μL) quartz cuvettes with a path length of 1 cm (Starna, Essex, UK). Ligands were prepared as d 6 -DMSO stock solutions (2.5 mM-500 mM) and proteins (5 μM) were prepared in the corresponding buffer described in the ligand screening by UV-visible spectroscopy protocol above. Aliquots (0.2 μL) of ligand stock solutions were added directly to cuvettes containing either protein solutions, or buffer alone. The final d 6 -DMSO concentration did not exceed 1% v/v of the assay solution. Spectra were recorded between 800-250 nm at 25 o C after the addition of each aliquot of ligand. Buffer control spectra were subtracted from protein spectra to account for any inherent absorbance of added ligands/solvent and all solutions were inspected and found to be free of precipitate. Difference spectra were generated by subtracting the initial ligand-free protein spectrum from each successive titration spectrum. The maximum change in absorbance for each difference spectrum was then plotted against ligand concentration and fitted using a one-site binding model hyperbolic/Michaelis-Menten equation (Eqn. 1). All spectral analysis and curve fitting was performed using Origin software (OriginLab, Northampton, MA). Data were processed using Microsoft Excel (Microsoft Office, 2013).

Equation 1. A obs = (A max x L)/(K D + L)
In Equation 1, A obs is the observed change in absorbance, A max is the maximum absorbance change at saturation, L is the concentration of ligand and K D is the dissociation constant of the enzyme-ligand complex.

Electron Paramagnetic Resonance Spectroscopy
EPR spectra for CYP121 were recorded on a Bruker ER-300D series electromagnet and microwave source interfaced with a Bruker EMX control unit and fitted with an ESR-9 liquid helium flow cryostat (Oxford Instruments) and a dual mode microwave cavity from Bruker (ER-4116DM). Spectra were recorded at 10 K with a microwave power of 2.08 milliwatts and a modulation amplitude of 10 gauss. Samples were prepared with CYP121 (100 μM), and ligands (2 mM) or DMSO (4% v/v), in 100 mM HEPES (pH 7.6) buffer, containing 100 mM NaCl and 4% glycerol. Samples were incubated for 30 minutes prior to freezing. Spectra were recorded over a wide scan (500-4500 G) and narrow scan (2000-4000 G) under the identical experimental conditions. Data analysis was performed using Origin software (OriginLab, Northampton, MA) and Microsoft Excel (Microsoft Office, 2013).

Molecular Modelling and Docking
Ligands were prepared for docking using the LigPrep, v3.2 and Epik v3.0 functions of Schrödinger suite software (Schrödinger LLC, NY). [9,10] Duplicate energy minimized (OPLS 2005) protein structures were prepared from the X-ray crystal structure of CYP121 in complex with cyclo-L-Tyr-L-Trp (cYW) (PDB 4IQ9) using the internal Protein Preparation Wizard in Maestro v10.0. Ionization states were generated to be compatible with metal-binding interactions and the heme-iron was manually adjusted to the ferric (+3) oxidation state. All water molecules were removed from one structure, while the axial heme water ligand was retained in the second protein structure. Docking grids were prepared using the structure of cYW to center the site. Ligands were allowed to dock into both grids under a range of scenarios; either employing no constraints, enforcing hydrogen bonding interactions with the axial heme water ligand or metal coordination to the ferric iron, and hydrogen bonding interactions with residue arginine 386. Docking was performed using GLIDE v6. 5 (Schrödinger, LLC, NY, 2014-4) and docking figures were prepared using the PyMOL Molecular Graphics System, Version 1.3, 2010, Schrödinger, LLC.