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A fragment profiling approach to inhibitors of the orphan M. tuberculosis P450 CYP144A1 Madeline E Kavanagh, Jude Chenge, Azedine Zoufir, Kirsty J. McLean, Anthony G Coyne, Andreas Bender, Andrew William Munro, and Chris Abell Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00954 • Publication Date (Web): 07 Feb 2017 Downloaded from http://pubs.acs.org on February 11, 2017

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A fragment profiling approach to inhibitors of the orphan M. tuberculosis P450 CYP144A1 Madeline E. Kavanagh,a Jude Chenge,b† Azedine Zoufir,a Kirsty J. McLean,b Anthony, G. Coyne,a Andreas Bender,a Andrew W. Munrob and Chris Abella*

a

Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United

Kingdom; bCentre for Synthetic Biology of Fine and Specialty Chemicals (SYNBIOCHEM), Manchester Institute of Biotechnology, School of Chemistry, The University of Manchester, Manchester M1 7DN, United Kingdom. *Corresponding author: Chris Abell, [email protected]

Keywords: fragment screening, ligand profile, cytochrome P450 enzyme, Mycobacterium tuberculosis, CYP144A1

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ABSTRACT

Similarity in the ligand binding profile of two enzymes may provide insight for functional characterization and be of greater relevance to inhibitor development than sequence similarity or structural homology. Fragment screening is an efficient approach to characterizing the ligand profile of an enzyme and has been applied here to study the family of cytochrome P450 enzymes (P450s) expressed by Mycobacterium tuberculosis (Mtb). The Mtb P450s have important roles in bacterial virulence, survival and pathogenicity. Comparing the fragment profiles of seven of these enzymes revealed that P450s which share a similar biological function have significantly similar fragment profiles, while functionally unrelated or orphan P450s exhibit distinct ligand binding properties, despite overall high structural homology. Chemical structures that exhibit promiscuous binding between enzymes have been identified, as have selective fragments that could provide leads for inhibitor development. The similarity in the fragment binding profile of the orphan enzyme CYP144A1 to CYP121A1, an enzyme important for Mtb viability, provides a case study illustrating the subsequent identification of novel CYP144A1 ligands. The different binding modes of these compounds to CYP144A1 provide insight into structural and dynamic aspects of the enzyme, suggest a hypothesis into biological function and provide opportunity for inhibitor development. Expanding this fragment profiling approach to include a greater number of functionally characterized and orphan proteins may provide a valuable resource for understanding

enzyme-ligand

interactions.


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Enzymes that have similar biological functions often bind to sets of ligands that are chemically and structurally similar.1–3 Thus, establishing and comparing the ligand profiles of enzymes can provide insight into their biological role and guide the development of small molecule inhibitors or tool compounds. The value and efficiency of ligand profiling can be improved by screening lower molecular weight compounds called fragments. Fragment screening is an established approach for identifying small molecule leads for drug development, to assess the inherent ligandability of protein targets and to identify binding “hotspots”.4–7 The small size and low structural complexity of fragments means that they represent a greater proportion of their available chemical space in fewer compounds than larger molecules,4 and that they are more likely to find a complementary binding site on a macromolecular target.5,8 Together, these properties allow fragment screening libraries to be small (~100–1000 compounds) facilitating rapid ligand profiling of enzymes. The biochemical mechanisms that are responsible for the virulence and persistence of the pathogen Mycobacterium tuberculosis (Mtb) are poorly understood. Elucidation of the genome sequence of the H37Rv strain of Mtb in 1998 shed light on some of the unique properties of the bacterium.9 One feature of note, was the large number of genes encoding cytochrome P450 enzymes (P450s or CYPs). As many bacterial species produce few or no P450s, the presence of 20 P450 genes in the Mtb genome and evidence for their evolutionary conservation suggested that these enzymes are likely to have important roles for Mtb survival.10–12 The genes encoding several P450s are essential for the viability of Mtb or for the bacterium to establish a chronic intracellular infection. Roles have been demonstrated for the P450s in cell wall synthesis, fatty acid and cholesterol metabolism, the production of secondary metabolites, drug resistance and stress response mechanisms.13–21 Despite their apparent importance, the substrate and biological role of only 5 of the 20 Mtb P450 enzymes (CYP51B1, CYP121A1, CYP124A1, CYP125A1, CYP142A1) have been

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identified.13–15,17,19,20,22 Putative functions have been assigned to a further 2 enzymes (CYP128A1 and CYP136A1) based on their genetic context,11,23 but the remaining 13 P450s are considered orphan enzymes. While several these orphan P450s are widely conserved across mycobacterial species, they share low amino acid sequence similarity (typically 80% relative to protein-free samples was used to identify fragments as hits for the calculation of ligand binding profiles. Additional, weakly binding fragments (50-80% reduction in 1H signal intensity), which were identified when screening a wider fragment library in singleton (1 fragment per tube), were employed in competition NMR experiments in order to observe cooperative binding interactions. Competition NMR experiments


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Competition NMR experiments were performed under identical conditions to fragment screening experiments. Compounds 1−3 were analyzed in singleton (each 200 µM) or in competition with each other or econazole (200 µM), in the presence of CYP144A1 (20 µM), or in buffer alone. Competition NMR experiments for fragments (1 mM) were conducted using clotrimazole or econazole (500 µM) as a displacer and compared to the spectra of the fragment analyzed in singleton. Fragment samples were prepared in MES-NaOH (100 mM) buffer (pH 6.0), containing NaCl (50 mM), d4-3-(trimethylsilyl)propionic acid (20 µM) in D2O (10 % v/v) and a final concentration of d6-DMSO of 2% v/v and d4-MeOD of 2% v/v to aid solubility. All experiments employed a CPMG pulse sequence,31,32 and data acquisition and processing was performed as described above. UV-visible spectroscopy ligand screening Heme-focused library fragments (1 mM) or compounds in the CYP121A1 inhibitor library (100 µM) were prepared as stock solutions (1–100 mM) in d6-DMSO and added to solutions of P450 proteins (4–6 µM) in the appropriate buffer, or to buffer alone to achieve a final concentration of 1% v/v d6-DMSO. Samples were either analyzed in quartz cuvettes with a 1 cm path length using a CARY400 UV-vis spectrophotometer (Varian, UK), or in UV-star® 96-well microplates (Greiner Bio-one, UK) using a CLARIOstar microplate reader (BMG Labtech, Germany) in absorbance mode. Spectra were recorded continuously between 800– 250 nm at 25 oC. Any interference from the inherent absorbance of small molecules was removed by subtracting spectra from buffer control cuvettes from samples containing protein, or from buffer alone, to achieve a final concentration of 1% v/v d6-DMSO. Samples were analyzed in quartz cuvettes with a 1 cm path length. The difference in maximum wavelength of the P450 Soret band (∆λmax) observed in the presence of fragments/compounds compared to that of the ligand-free control (1% v/v d6-DMSO) was used to identify compounds as type


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I or type II heme-binding ligands. Perturbations of ∆λmax < ± 1 nm using the CARY400 spectrophotometer, or < ± 1.5 nm using the CLARIOstar microplate reader were considered within experimental error. All UV-vis spectra were generated using Origin software (OriginLab, Northampton, MA) or MARS Data Analysis Software (BMG Labtech). Data were processed using Microsoft Excel (Microsoft Office, 2010). UV-vis competition assays Competition assays were performed in quartz cuvettes with a 1 cm path length using a CARY400 UV-vis spectrophotometer (Varian, UK). Samples were prepared as for fragment screening except that a type II indicator compound (econazole, 25-50 µM, miconazole, 12.5 µM or compound 2, 50 µM) was also added to fragment/ligand-CYP144A1 solutions. The concentration of d6-DMSO did not exceed 1% v/v of the total assay volume. A decrease or increase in the ∆λmax relative to that produced by the type II compound binding to CYP144A1 alone was used to assess competitive or cooperative ligand binding interactions. Optical titrations Optical titrations to determine the binding affinity (KD values) of ligands were carried out on a Cary 60 UV-visible spectrophotometer (Varian, UK) according to a previously described procedure.33 Ligands were prepared as stock solutions (typically 0.1–100 mM) in DMSO and added as 0.2 µL aliquots to cuvettes containing either a solution of CYP144A1 (~4–8 µM) in 100 mM KPi (pH 7.0), containing 10 mM KCl; or to buffer alone. DMSO concentrations did not exceed 1% v/v of the final titration volume (1 mL) and the absorbance spectrum of CYP144A1 was not affected within this range. Spectra were recorded continuously between 800–250 nm at 25 °C. Spectra collected from the buffer control cuvette were subtracted from protein spectra to remove any optical interference from small-molecule absorbance. Difference spectra were generated by subtraction of the initial ligand-free protein spectrum from each successive ligand-bound protein spectrum. The maximum change in absorbance


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for each difference spectrum was then plotted against ligand concentration. Data were fitted using the Hill function for cooperative binding (Eq. 1) or the Michaelis-Menten equation (Eq. 2), depending on the affinity of the ligand and type of binding curve observed. Equation 1 KD = [L]n ([Eto] – 1)/ [LnEt] Equation 2 Aobs = Amax [L]/ KD + [L] In Equation (1 and 2), Aobs represents the observed difference in Soret absorption of the P450 at each addition of ligand, Amax is maximal absorption difference at ligand saturation, Et is the total amount of enzyme, L is the ligand or substrate concentration, n is the extent of cooperativity, LnEt is the concentration of the ligand-enzyme complex and KD is the dissociation constant for the P450-ligand complex. All data fitting and analysis were performed using Origin software (OriginLab, Northampton, MA). All titrations were repeated in triplicate and data from representative experiments are reported. Isothermal titration calorimetry ITC binding isotherms were recorded on a MicroCal ITC200 instrument (Malvern Instruments). Titrations were conducted at 25 °C by injecting aliquots (2.0 µL) of ligand solutions (1 mM) into protein samples (60.9 µM), both of which had been diluted in 50 mM Tris-HCl buffer (pH 7.5), containing 100 mM KCl, and adjusted to give a final concentration of 10% v/v d6-DMSO. Titrations were typically 20 injections at 90-second intervals. Small background heats from dilution of the ligand were subtracted after performing a second control titration of ligand samples into buffer without protein. Binding isotherms were integrated to give the enthalpy change of each injection and plotted against the molar ratio of ligand added to the sample cell. Titrations were fitted using a one-sites binding model, using Origin Analysis Software provided by the manufacturer. The binding stoichiometry was set at N=1, or to N=2 to reflect the binding stoichiometry observed in nanoESI native mass spectra. Electron paramagnetic resonance spectroscopy


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EPR spectra of both ligand-free CYP144A1 (200 µM), and CYP144A1 (200 µM) bound to ligands (500 µM) were recorded using a Bruker ER-300D series electromagnet and microwave source interfaced with a Bruker EMX control unit. The instrument was 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 a temperature of 10 K, a microwave power of 2.08 mW, and an amplitude of 1 mT. Samples were prepared in 100 mM KPi (pH 7.0), 10 mM KCl, from stock solutions of ligands in DMSO. Data analyses were performed using Origin Software. Nano-ESI native mass spectrometry Protein stock solutions (10–40 µM) were prepared by dilution of purified proteins (500– 1000 µM) in 200 µM ammonium acetate buffer, pH 7.0. Samples were buffer exchanged by size exclusion chromatography using Micro Biospin 6 columns, molecular weight cut-off 6 kDa (BioRad, Hemel Hempstead, UK). Ligands were prepared as stock solutions in d6DMSO at 0.2–2 mM concentrations. Ligand-protein samples were prepared by diluting protein stocks (10 µL) and ligand stocks (1 µL) with ammonium acetate buffer (9 µL) to give final concentrations of 10 µM CYP144A1, 10–50 µM ligand and 5% v/v d6-DMSO. Mass spectra were recorded on a Synapt HDMS instrument (Waters UK Ltd., Manchester, U.K.). Capillaries for nano-ESI were purchased from ThermoFisher, Hemel Hempstead, UK. Capillary tips were cut under a stereo microscope to give inner diameters of 1–5 µm and then loaded with 2.5 µL of sample solutions. Given below are the general instrumental conditions used to acquire the reported spectra. However, parameters were recorded and varied over the course of each experiment to observe the strength of protein-ligand complexes under different ionizing strengths. All measurements were carried out in a positive ion mode with ion source temperature of 20 °C. A capillary voltage of 1.5 kV, cone voltage of 40 V and extraction cone voltage of 4.8 V was applied to perform nanoESI. All reported spectra were collected


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with a trap collision energy 30–60 V, transfer collision energy 12–30 V, IMS pressure 5.02 × 10−1 mbar, TOF analyzer pressure 1.17 × 10−6 mbar. External calibration of the spectra was achieved using cesium iodide at 100 mg mL−1 in water. Mass differences resulting from ligand binding were calculated from the unbound protein peak internal to each spectrum. The unbound protein peak was compared to the relevant 5% v/v d6-DMSO control spectrum for consistency. Mass differences were divided by the molecular weight of the ligand to calculate binding stoichiometry. The percentage of protein-ligand complex was calculated from the sum of the intensities of all peaks corresponding to ligand bound species (species B + C) divided by the sum of the intensities of all protein peaks in the spectrum (A + B + C). Data acquisition and processing were performed using Micromass MassLynx v4.1. Bioinformatics and Chemoinformatics Methods Fragment profile analysis - The similarity in the fragment binding profile of each pair of 7 Mtb P450 enzymes (CYP121A1, CYP124A1, CYP125A1, CYP126A1, CYP142A1, CYP143A1 and CYP144A1) was calculated as previously described in the literature.1,34 The overlapping binding fragments (S) for each pair of enzymes was compared and an E-value was calculated according to equation 3, using a library size of 80 compounds. In equation 36, m and n are the number of compounds that bind to each enzyme, K is the probability search space and λ is a scoring function, which is dependent on the probability that a given ligand binds both enzymes (q) and the probability of finding a hit in the library (p = 1/80). A P-value was computed according to Equation 7 and a threshold of < 10-4 was used to identify statistically significant E-values. Data processing and E-value/P-value computations were performed in Python (v. 2.7.11). Equation 3 E = Kmne-λS Equation 4 K = ((q-p)2)/q Equation 5 q = S/(m x n)


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Equation 6 λ = ln(q/p) Equation 7 P = 1- e-E

Fragment profile phylogeny and fragment heat map - A dendrogram showing the fragment binding similarities between the 7 Mtb P450 enzymes was produced using the pdist, linkage (using the default aggregation method = ’single’) and dendrogram functions of the Python library Scipy35 on pairwise Jaccard distances of the fragment binding profiles. The Jaccard metric was preferred in this case as the fragment binding profiles are binary vectors. A heatmap was produced alongside the dendrogram, in which rows/enzymes were ordered according to the above dendrogram and columns/fragments were ordered by promiscuity (i.e. by number of enzymes bound). The dendrogram and heatmap visualization was produced using the Python library Matplotlib36. Sequence and structural comparisons – Multiple sequence alignment of the amino acid sequences of Mtb P450s was performed using ClustalW2.37,38 BLAST analysis of the fulllength CYP144A1 (CYP144A1-FLV) enzyme amino acid sequence was performed using the online NCBI tool and restricting alignments to proteins in the Swiss-protKB/UniProt databases.39,40 https://blast.ncbi.nlm.nih.gov/. Structure overlays of Mtb CYP144A1-TRV (PDB 5HDI) and CYP121A1 (PDB 3G5F) were performed using the protein structure comparison service PDBeFold at European Bioinformatics Institute (http://www.ebi.ac.uk/msd-srv/ssm), authored by E. Krissinel and K. Henrick.41 Active site properties of enzymes were calculated using the internal SiteMap42–44 function of Schrödinger suite software (Schrödinger LLC, NY). Proteins were prepared prior to analysis using the Maestro v10.0 Protein Preparation Wizard. All ligands and active site water molecules were removed, including the axial heme ligand, and the heme iron was manually adjusted to the ferric oxidation state.


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Ligand docking - Ligands were prepared for docking using the LigPrep, v3.2 and Epik v3.0 functions of Schrödinger suite software (Schrödinger LLC, NY). Duplicate energy minimized (OPLS 2005) protein structures were prepared from the X-ray crystal structure of ligand-free CYP144A1-TRV (PDB 5HDI) 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 coordinates of the axial heme water ligand as a central point. 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. Docking images were generated using the PyMOL Molecular Graphics System, Version 1.3, 2010, Schrödinger, LLC.


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RESULTS Fragment screening A library of 80 randomly selected, rule-of-3 compliant fragments45 was screened against 7 Mtb P450s by relaxation-edited ligand-observed NMR. A Carl-Purcell-Meiboom Gill (CPMG) pulse sequence was employed for the experiments. A reduction of greater than 80% in the intensity of ligand 1H signals in the presence of enzyme compared to enzyme-free samples was used to classify fragments as hits.31,32 The percentage of the fragment library that was identified to bind to a given P450 ranged between 11% for CYP143 to 64% for CYP142 (Figure 1a). Eight percent of the fragments were identified to bind to all 7 P450s, with a greater proportion of shared hits (35%) observed for enzymes that have a common catalytic function, such as CYP125 and CYP142.17,22 No correlation was observed been the screening hit rate and the size or hydrophobicity of the enzyme active site. A second “heme-focused” library of 80 fragments was screened against the 7 Mtb P450s and 4 P450s from unrelated bacteria. Each fragment in this library contained a known heme binding functional group, such as a heteroaromatic nitrogen, aniline, aliphatic amine, nitrile or carboxylic acid group. The library was screened using a UV-vis absorbance assay that enables the detection of ligand binding interactions that perturb the coordination network of the P450 heme iron, from a change in the maximum wavelength of the Soret band (λmax) of the enzyme’s optical spectrum. Ligands that form a stronger coordinate network to the heme than is present in the water-ligated resting state enzyme cause a red shift (type II) in the Soret λmax and difference spectra with λmin ≈ 390 nm and λmax ≈ 430 nm. Type II spectra are most commonly associated with P450 inhibitors and have historically been considered to indicate stabilization of the inactive, low spin state of the enzyme. In contrast, P450 substrates tend to cause a blue shift (type I) in the Soret λmax (and difference spectra with λmin ≈ 420 nm and λmax ≈ 390 nm), as they displace or weaken the coordination of the axial heme water ligand, but do


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not coordinate to the iron themselves.46 Here, only fragments that produced type II, “inhibitor-like” changes in the optical spectrum were classified as hits. However, it should be noted that many exceptions to this classification system have been reported, including competitive inhibitors that produce type I spectra, potent P450 inhibitors that do not perturb the λmax and type II ligands that are oxidized by P450s.13,47–51 The proportion of fragments identified as hits from the heme-focused library was lower (1−19%) than that observed for the unbiased fragment library (Figure 1b). This can largely be attributed to the more restrictive criteria used to classify fragment as hits. That is, fragments must both bind in proximity to the heme group and stabilize the low spin state of the P450. In contrast, NMR hits might bind throughout the enzyme active site. Higher hit rates were observed for other bacterial enzymes than for the Mtb P450s. The fatty acid hydroxylase P450 BM3 (CYP102A1) from Bacillus megaterium had the highest hit rate, with both the wild-type (Wt) and A82F mutant BM3 heme domain proteins binding to 36% and 40% of fragments in library, respectively.28–30,52–54 The fatty acid decarboxylase CYP152L1 (OleT) from a Jeotgalicoccus sp. and the compactin hydroxylase CYP105AS1 (P450 Prava) from Amycolatopsis orientalis had comparable hit rates to the Mtb enzyme with the highest hit rate, CYP142A1. Lower screening hit rates of 1–10% were observed for the orphan Mtb P450s CYP126A1, CYP143A1 and CYP144A1, which reflects the challenge of identifying potent ligands for these particular enzymes. Fragment profile analyses The similarity in the ligand profile or set of fragments that are identified to bind to a pair of enzymes can be calculated as an E-value, using an adaption of the scoring function that is commonly used to assess amino acid sequence similarity (Eq. 3).34 Shortridge and coworkers previously detailed this methodology and found that similar ligand profiles were only observed between pairs of functionally similar proteins (as defined by a FunSimMat


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functional similarity score).1,55 Here, fragment profiling allows relationships to be drawn between enzymes in the absence of functional or structural data. Using Equation 3, the Evalue was calculated between each pair of Mtb P450s, treating fragment profiles established from screening the unbiased and heme-focused fragment libraries independently. The E-value describes the probability that two enzymes bind to the same set of fragments by chance, and thus, a smaller E-value is more significant. Here, only profiles that were calculated to have a P-value (P = 1 – e-E) of less than 10-4 were considered significantly similar. The complete set of pairwise E-values are provided in Tables S1 and S2 of the Supporting Information. Unbiased fragment library - The fragment profiles of the Mtb P450s that were established by screening the unbiased fragment library by NMR revealed statistically significant similarity between each pair of the functionally similar enzymes CYP124A1, CYP125A1 and CYP142A1 (E = 3.1 x 10-5–3.7 x 10-6), all of which catalyze the oxidation of cholesterol/cholestenone in vitro.14,15,17,20,22 Of note, significant similarity was also identified between the fragment profiles of the orphan enzyme CYP144A1 and CYP121A1 (E = 3.0 x 10-7). CYP121A1 is an essential enzyme for Mtb viability that catalyses the cyclisation of the cyclo-dipeptide cyclo-L-Tyr-L-Tyr to form the secondary metabolite mycocyclosin.13,56 CYP121A1 also shared a lower level of similarity with CYP125A1 (E = 7.6 x 10-5). In contrast, the fragment profiles of the other two orphan Mtb P450s that were screened, CYP126A1 and CYP143A1, were not significantly similar to any other enzyme. Heme-focused fragment library - The fragment profiles that were determined from screening the heme-focused fragment library were only significantly similar between 3 enzyme pairs: the Wt and A82F mutant BM3 enzymes from B. megaterium (E = 8.2 x 10-7), both of which catalyze the oxidation of a variety of fatty acid substrates,54 the previously noted cholesterol/fatty acid oxidases CYP124A1 and CYP142A1 (E = 8.6 x 10-5), and also between CYP142A1 and BM3 A82F (E = 9.3 x 10-6). The smaller number of hits per enzyme


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that was obtained from UV-vis screening compared to ligand-observed NMR, meant that any similarity in fragment profile rapidly tended towards insignificance (P ≥ 10-4). Consequently, no statistically robust comparison could be obtained for any of the orphan enzymes CYP126A1, CYP143A1 or CYP144A1. Despite this, detailed information about the preferred heme binding pharmacophore of each P450 was apparent from the chemical scaffolds that were overrepresented within each set of hits. For example, CYP125A1 exhibited a narrow profile in which 80% of the fragment hits had biaryl-pyridine scaffolds. In contrast, CYP121A1 bound to a relatively diverse set of pyridine, aniline and aliphatic amines. Differences in the heme binding pharmacophore of each enzyme reflect variation in the structure of the enzyme active site and access to the heme cofactor. These factors that could be exploited to develop more selective ligands for individual P450 enzymes. Fragment profiling phylogeny - Ligand profile trees can be built up in the same way as phylogenies constructed from amino acid or nucleotide sequence similarity, and can be likened to a focused sequence alignment of the residues comprising an enzyme binding site. A phylogenetic tree of the Mtb P450s was constructed using the E-values that were calculated from all pairwise comparisons of the fragment profiles obtained from screening the unbiased fragment library. A phylogenetic tree of the Mtb P450s was constructed using the E-values that were calculated from all pairwise comparisons of the fragment profiles obtained from screening the unbiased fragment library (Figure 1c). This phylogeny enables the relationships between the Mtb P450s to be visualized with respect to the similarity in their ligand binding profile and could provide insight into convergent or divergent evolution in enzyme function. For example, the orphan enzyme CYP126A1 shares 35−38% amino acid sequence identity with the fatty acid and cholesterol oxidases CYP124A1, CYP125A1 and CYP142A1. However, the fragment profile of CYP126A1 places the enzyme in distinct ligand space, which is consistent with a lack of evidence for CYP126A1 binding to steroid or


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fatty acid substrates.27 Secondly, while the sequence similarity of CYP144A1 is closer to CYP142A1 and CYP126A1, the fragment profile of CYP144A1 clusters the enzyme in closer proximity to CYP121A1, a relationship that subsequently yields the identification of novel ligands for CYP144A1. Fragment selectivity - The relative selectivity of fragments from the unbiased fragment library was analyzed by clustering fragments according to their hit rates against all enzymes. Unsurprisingly, lipophilic biaryl and halogenated fragments were most frequently identified as hits, while smaller more polar fragments showed greater selectivity between enzymes. Identifying promiscuous chemotypes provides guidance for the development more selective ligands, while unique fragment hits provide novel leads for ligand development or may indicate substrate chemotype by highlighting distinguishing properties of an enzyme active site. For example, the orphan enzyme CYP126A1 was identified to disproportionately bind to acidic fragments. This profile suggests that CYP126A1 might have a polar or acidic substrate (opposed to a steroid) and correlates with the recently reported X-ray crystal structure of the enzyme.27 Identification of novel ligands for orphan CYP144A1 CYP144A1 is an orphan Mtb P450 that is required for normal bacterial growth and is postulated to have a role in bacterial stress response or drug resistance mechanisms.18,57,58 Prior to the present study, the only known ligands of CYP144A1 were the imidazolecontaining drugs clotrimazole, econazole, miconazole and ketoconazole, and the small molecule 4-phenylimidazole (4-PIM).18,24 Our attempts to identify inhibitors with improved selectivity or physicochemical properties to these drugs from among other commercially available P450 inhibitors, or to identify CYP144A1 substrates from a diverse collection of biologically relevant lipids and carbohydrates were unsuccessful (Table S3, Supporting Information).


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Here, the pairwise comparison of fragment hits that were identified for the Mtb P450s from NMR screening indicated significant similarity between CYP144A1 and the essential Mtb enzyme CYP121A1.56 A library of CYP121A1 ligands was recently synthesized in-house as part of a fragment optimization campaign that yielded inhibitors with low nanomolar binding affinity.25 The core fragment from which this library of inhibitors was developed, 4-(1H1,2,4-triazol-1-yl)phenol, was also identified to bind to CYP144A1 during fragment profiling, producing a greater than 50% reduction in CPMG 1H signal intensity.25,59,60 While the lead CYP121A1 inhibitors developed from this fragment showed good selectivity over other Mtb and human P450 enzymes, it was postulated that some of the intermediate compounds might yield novel CYP144A1 ligands. Eight of the 21 CYP121A1 inhibitors were identified as hits for CYP144A1 when they were screened against the enzyme by UV-vis spectroscopy (Figure S1, Supporting Information). Indazole 1, imidazole 2 and aniline 3 were the most tightly binding compounds and were calculated to have KD values of 82 ± 4 µM (UV-vis), 200 ± 6 µM (UVvis) and 140 ± 27 µM (ITC), respectively (Table 1 and Figure 3a). Compounds 1, 2, and 3 each exhibit a different mode of binding to CYP144A1, as characterized from their effect on the enzyme’s optical spectrum (Figure S1, Supporting Information). Indazole 1 produced a small blue-shift in the Soret λmax of –1.5 nm and is the first type I, “substrate-like” ligand that has been identified for CYP144A1. Imidazole 2 caused an “inhibitor-like” red-shift in the Soret λmax of +1.5 nm, while aniline 3, and the remaining 5 ligands did not directly perturb the enzyme’s optical spectrum. These compounds were characterized to bind in the distal portion of the active site based on their competitive displacement of econazole in UV-vis competition assays (Figure S3, Supporting Information). In each case, the binding mode of 1−3 differed from their interactions with CYP121A1. For example, although indazole 1 and imidazole 2 have similar KD values for CYP121A1 as CYP144A1, both compounds


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predominantly bind distal to the CYP121A1 heme cofactor. The weak type II shift produced by imidazole 2 in the CYP121A1 optical spectrum was insufficient to determine a KD value by optical titration and X-ray crystallography revealed the compound in a non-heme binding orientation (PDB 5IBH). In contrast, aniline 3 binds to CYP121A1 with nanomolar affinity (KD = 0.29 µM) and coordinates directly to the heme iron using the aniline nitrogen (PDB 5IBG).25 The contrasting binding affinity and binding mode of compounds 1−3 to CYP144A1 and CYP121A1 demonstrate that although the enzymes share a broad similar fragment profile, the proximal active site and heme coordinating properties of the two enzymes differ.

Table 1. The binding mode and affinity of ligands for CYP144A1 and CYP121A1. CYP144A1

CYP121A1

Compound

Binding Modea

KD (µM)b

Methodc

Binding Mode

1

I

82 ± 4

UV

2

II

200 ± 6

UV

II/Distal

3

Distal

140 ± 27

ITC

II

4-PIM

II

2980 ± 300

UV

II

32 ± 256

UV

1-PIM

II

290 ± 74

UV

ND

ND

ND

4

II

230 ± 3

UV

ND

ND

ND

KD (µM b

Method

45 ± 525

ITC

270 ± 1825

ITC

0.29 ± 0.04 /4.5 ± 0.625

UV/ITC

a

Binding mode is defined as type I, type II, distal/non-heme binding, or not determined (ND). bDissociation constant. cITC - isothermal titration calorimetry; UV – UV-vis spectrophotometry/optical titration.

Characterizing CYP144A1 using small molecule ligands and fragment screening. The identification of a series of novel ligands for CYP144A1, which exhibited different modes of binding to the enzyme and have improved physicochemical properties compared to 20 ACS Paragon Plus Environment

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previously studied imidazole antifungal drugs, provided new tools with which to further characterize the properties of the orphan enzyme. Heme binding pharmacophore – Imidazole 2 was the only CYP121A1 inhibitor to produce a type II shift in the optical spectrum of CYP144A1, despite multiple compounds containing functional groups that are commonly identified to bind heme, such as pyridine, pyrazole and aniline groups. This preference of CYP144A1 for imidazole as a heme binding pharmacophore is consistent with the fragment profile obtained for the enzyme when the heme-focused fragment library was screened by UV-vis spectroscopy. CYP144A1 only bound to a single fragment from the heme-focused library, the internal positive control 4PIM. A series of fragments structurally related to 4-PIM were screened against CYP144A1, but only resulted in two additional hits, 1-phenylimidazole (1-PIM) and 4-(4bromophenyl)imidazole 4, despite the presence of numerous other substituted imidazole and biaryl-heteroaromatic fragments. The selectivity of CYP144A1 for imidazole as a heme binding pharmacophore was rationalized by comparing the enzymes’ active site structure with that of CYP121A1. Alignment of the X-ray crystal structures of ligand–free CYP144A1 and CYP121A1 (PDB 5HDI and PDB 3G5F) yielded a root-means-squared deviation of 2.3 Å (over 332 αC),39–41 which is consistent with the low (23%) amino acid sequence identity and distinct evolutionary lineage of the two enzymes (Figure 2a).61,62 The most significant differences in the tertiary structure of CYP144A1 and CYP121A1 occur in regions known to be important for substrate specificity, including the β2-sheets and at the termini of the F- and G-helices.46 In addition, the B-helix is replaced by a long loop region in CYP144, giving the enzyme a more “open” active site than that of CYP121A1. This might enable greater conformational flexibility or allow CYP144A1 to bind to larger substrates than CYP121A1. CYP144A1 and CYP121A1 both have a large active site volume (766 Å3 and 513 Å3, respectively) and wide,


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shallow substrate entry channel relative to the other structurally characterized Mtb P450s.42,43 However, the surface area that is available for metal binding interactions was calculated to be significantly smaller for CYP144A1 (~6 Å2) than CYP121A1 (~12 Å2).

42–44

Amino acid

residues forming the I-helix are highly conserved, including the catalytic alcohol residue serine (S246/247), which is replaced by threonine in the other structurally characterized Mtb P450s (Figure 2b-c).15,20,22,63 However, the CYP144A1 active site is more hydrophobic than that of CYP121A1. The glutamine (Q385) and arginine (R386) residues of CYP121A1 that are important for substrate binding and catalysis are replaced by phenylalanine (F289) and histidine (H292) in CYP144A1. The proximity of His292 and Phe289 to the CYP144A1 heme cofactor likely favors the small size and orthogonal coordination angle of imidazole to the heme iron, or might form T-shaped aromatic stacking interactions with the heterocycle.13,56,64 The KD values of the imidazole compounds for CYP144A1 provides insight into factors contributing to ligand binding affinity (Table 1). The 10-fold difference in the affinity for 1PIM (KD = 280 µM) and 4-PIM (KD = 2980 µM) correlates with the basicity of the respective isomers and the additional desolvation penalty associated with 4-PIM binding.47,48 However, Cryle et al. previously reported a different rank order of affinity between the 1PIM, 2-PIM, and 4-PIM isomers and the Bacillus subtilis enzyme CYP134A1.65 CYP134A1 has comparable KD values of 11 µM and 57 µM for 1-PIM and 4-PIM, respectively, but binds significantly more weakly to 2-PIM (KD = 230 µM). This contrast between CYP134A1 and CYP144A1 highlights the influence of the protein microenvironment on the potency of even structurally simple heme binding ligands. The 13-fold improvement in binding affinity of fragment 4 (KD = 227 µM) compared to 4PIM, and the 50-fold tighter binding affinity of econazole (KD = 0.72 µM) compared to miconazole (KD = 15 µM), amounts to a 1.5−3.0 kJ mol-1 change in the free energy of binding


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Biochemistry

(∆Gbinding) from a single heavy atom substitution (Figure 3a). These values suggest that lipophilic interactions in the distal pocket of CYP144A1 contribute significantly to the affinity of heme binding ligands. Targeting binding hotspots comprised of variable active site residues should enable the selectivity of ligands that contain common metal binding pharmacophores, such as imidazole, to be tailored specifically for CYP144A1. Furthermore, the comparable affinity of aniline 3 to heme binding ligands of a similar molecular weight, such as imidazole 2, suggests that it might be feasible to develop potent, non-heme binding CYP144A1 inhibitors. Electron paramagnetic resonance spectroscopy– X-band EPR spectra of CYP144A1 in complex with the compounds 1−3 and a selection of fragments were collected to provide insight into the effect of ligand binding on the heme coordination sphere and spin-state of the enzyme (Figure 3b). Compounds 1, 2, and 3 each generated new sets of low spin g-values that are consistent with the ligands interacting with the heme iron of CYP144A1 using a strong-field ligand, such as an aromatic nitrogen atom.66–68 Specifically, each compound caused an increase in gz-value and decrease in the gx-value, relative to those of the ligand free protein (gz 2.41, gy 2.24, gx 1.92). The magnitude of the shift in the g-values was small compared to that previously reported for P450s in complex with imidazole ligands.67 This could indicate indirect coordination of the heme iron by an aromatic nitrogen atom via a retained axial water ligand. However, the EPR spectra of previously reported water-ligated complexes, for example human CYP2C9−triazole, Mtb CYP125A1−pyridine and Mtb CYP121A1−fluconazole complexes, show a narrowing of the g-values, which is also inconsistent with CYP144A1 spectra.48,49,66,69 In addition, clotrimazole, which is a potent imidazole-class, CYP144A1 ligand (KD = 0.37 µM) also produces only small shifts in the gvalues of the ligand free protein.18 As such, it is likely that small spectral shifts are characteristic of the CYP144A1 heme.


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The relative intensity of the new low spin species generated by compound 1, 2 and 3 reflects the strength of their interactions with the CYP144A1 heme coordination sphere and supports the binding mode determined for each compound by UV-vis spectroscopy. For example, imidazole 2 produced largest proportion of new low spin CYP144A1 and is expected to directly coordinate to the CYP144A1 heme iron. In contrast, the “non-heme binding” aniline 3 produced the smallest proportion of new low spin CYP144A1. All compounds except imidazole 4, clotrimazole and econazole,18,24 produced heterogeneous sets of low spin g-values that indicate a minimum of two different coordination states of the heme iron. The implication that ligands might acquire different binding modes with respect to the CYP144A1 heme, and that there remains a proportion of enzyme in the water-coordinated resting state when ligand bound, prompted further investigation of the enzyme’s ligand binding properties using competition NMR and UV-vis experiments. Competition experiments – Compounds 1 and 2 mutually displaced each other in competition UV-vis experiments, consistent with both ligands binding to CYP144A1 by interacting with the heme iron (Figure 4a). In contrast, competition ligand-observed NMR experiments indicated that compounds 1 and 2 do not completely displace each other from the CYP144A1 active site. This was evidenced from the failure of the 1H signals of either compound to regain intensity when they were analyzed together by CPMG NMR in the presence of CYP144A1 (Figure S4, Supporting Information). This could indicate a secondary binding site for the compounds in the CYP144A1 active site, or that both compounds are bound simultaneously in significant amounts on the time scale of the NMR experiment. Aniline 3 did not directly perturb the optical spectrum of CYP144A1 (λmax 421.5 nm), but competitively reduced the red-shift in the Soret λmax (∆∆λmax –1.5 nm) that is induced by a type II indicator compound (either imidazole 2 or econazole, λmax 423.5 nm)


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(Figure S3, Supporting Information). This suggests that aniline 3 binds in the distal part of the active site and that binding is mutually exclusive with coordination of imidazole 2/econzole to the heme iron. Competition experiments using selected fragments (e.g. benzothiophene 5) and econazole or imidazole 2 as indicator ligands, resulted in both competitive and cooperative binding effects on the absorbance spectrum (Figure 4b). The ∆∆λmax observed differed depending on the specific combination of ligands and likely reflects the steric properties, affinity and binding location of the respective fragment and indicator. A series of fragments were also assessed in competition NMR experiments using clotrimazole as a displacer ligand. Competitive, non-competitive and cooperative interactions were all observed (Figure 4c-d). For example, the 1H signal intensity of the weakly binding benzothiophene 6 further decreased in the presence of clotrimazole, suggesting that both compounds bind simultaneously to the CYP144A1 active site. However, non-specific aggregation of the ligands cannot be excluded. In contrast, clotrimazole competitively displaced 4-(4bromophenyl)imidazole 4. This result was consistent with the homogeneous g-values generated by clotrimazole and 4 in X-band EPR spectra and suggests that the compounds share a single, common mode of binding to CYP144A1. The results obtained for compounds 1−3 and other fragments 4−6 imply that multiple ligands can bind to the CYP144A1 active site simultaneously and that most ligands have heterogeneous binding modes. Native mass spectrometry – The potential for multiple ligands to simultaneously occupy the CYP144A1 active site was subsequently probed using nanoESI native mass spectrometry. Spectra were collected for compounds 1, 2, 3 and clotrimazole, econazole, and miconazole at a 1:1 and 5:1 ratios of ligand-to-protein (Figure 5). The binding stoichiometry observed in each spectrum was calculated from difference in mass between the ligand-free (A) and ligand-bound (B and C) protein species, divided by the molecular weight of the respective


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ligands (Table 2). All compounds except econazole and miconazole were observed to bind twice (species C) per CYP144A1 monomer at a 5:1 ratio of ligand-to-protein. The proportion of ligand-bound CYP144A1 correlated with the order of binding affinities of compounds that shared a similar chemotype. The apparent weaker affinity of the azole drugs compared to compounds 1−3 is likely a consequence of the contrasting effect that the gas phase of the mass spectrometer has on hydrophobic versus polar intermolecular interactions.70,71 Although multiply bound protein species could arise from non-specific interactions, the combination of these data with EPR and competition UV-vis and NMR experiments strongly suggest that multiple ligands can simultaneously occupy the CYP144A1 active site. Docking – Co-crystallization of compounds 1−3 with CYP144A1 failed to produce diffraction data sets with resolvable ligand density. Consequently, ligand docking studies were performed to gain insight into possible binding modes of the compounds. The accumulated evidence from spectroscopic characterization was used to guide docking simulations (Figure 6a-c). Indazole 1 was modelled to bind CYP144A1 by water-bridged hydrogen bonding interactions between the heme iron or displacement of the axial heme water ligand. Imidazole 2 was predicted to coordinate directly to the heme iron using the lone pair electrons of an imidazole nitrogen. Aniline 3 was docked without constraints and posed in a non-heme binding orientation that is consistent with spectroscopic data. Analysis of the binding poses generated from docking studies suggest that H292 and F289 constrain the access of ligands to the heme iron and indicate interactions in the distil pocket that are likely to form an initial ligand-protein encounter complex. These models allow the SAR observed in the current study to be rationalized and will be used to guide the subsequent optimization of more potent CYP144A1 ligands.

DISCUSSION


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Biochemistry

Characterizing novel gene products continues to prove challenging, particularly when the proteins in question share low sequence or structural similarity to characterized proteins or when systems for assessing gene essentiality are complex, such as in the case of Mtb.72,73 Here, fragment profiling has been shown to provide a rapid approach to draw relationships between enzymes and to identify small molecule compounds that can be used to aid enzyme characterization. Members of the family of P450 enzymes expressed by Mtb have essential roles for bacterial pathogenicity and survival. The enzymes share a broadly conserved heme cofactor, tertiary structure and catalytic mechanism, but have low sequence similarity (< 35% amino acids sequence identity) to each other and to other characterized enzymes. Thus, predicting the biological function of these enzymes from bioinformatics methods alone has proved unfruitful. Biochemical approaches of characterizing the Mtb P450s have met with little success, as there is limited understanding of the endogenous electron transport partner proteins employed by Mtb and slow substrate turnover using either heterologous or Mtb derived systems.13–15,56,74 As such, developing chemical tools with which to study these enzymes is an important strategy to deconvolute their biological importance. The sets of fragments that bind to 7 of the Mtb P450s and 4 P450s from other bacteria were identified and compared, revealing a similar profile between characterized enzymes that share similar functional roles. Chemical scaffolds that bound ubiquitously to all enzymes were highlighted, while more selective fragments, which could provide leads for the development of selective P450 inhibitors, were also identified. The low proportion of enzyme pairs that were identified to have significant similarity in their fragment profiles (24% and 5% of pairs from unbiased and focused fragment libraries, respectively), highlights that, despite sharing a broadly conserved catalytic mechanism and tertiary protein fold, the Mtb P450s are each likely to fulfil a unique biochemical niche.


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CYP144A1 is an orphan Mtb P450 whose expression is upregulated when the bacterium is cultured in the dendritic cell phagosome and in response to challenge with vancomycin.

57,58

The enzyme binds tightly to antifungal drugs that have anti-mycobacterial activity in vivo,18,24 and knockout of CYP144A1 increases the sensitivity of Mtb to killing with these agents.18,75– 77

By establishing that a similar profile of fragments bind to CYP144A1 and the characterized

enzyme CYP121A1, a series of novel ligands for CYP144A1 were identified from a library of CYP121A1 inhibitors. The different binding modes of compounds 1−3 allowed the properties of CYP144A1 to be probed using a collection of biophysical techniques. The enzyme was found to be highly selective for imidazole as a heme binding pharmacophore and to bind multiple large (Mw >300 Da) ligands simultaneously. The distinct binding modes of compounds 1−3 to CYP144A1 and CYP121A1 was rationalized based on the chemical properties and steric constraints imposed by the proximal active site residues of CYP144A1 and the hydrophobicity of the CYP144A1 distal active site.24 The ligand profile and the biophysical characteristics of CYP144A1, elucidated using these novel ligands, provide insight into the potential biological function of the enzyme. The predicted conformational flexibility and the open structure of the CYP144A1 active site, in addition to the low susceptibility of the protein to inhibition with a range of common heme binding functional groups, could indicate a role for CYP144 in xenobiotic detoxification. This would be consistent with induction of CYP144A1 under conditions of cellular stress and in response to antibiotic challenge.57,58 Alternatively, these data could support a role for CYP144A1 in the oxidative tailoring of large substrates, and notably the set of functionally characterized enzymes with the greatest amino acid sequence similarity to CYP144A1 (31– 33% identity) are all macrolide oxidases that synthesize antibiotics.78–80 Indazole 1 is the first ligand to be identified that produces substrate-like shifts in the optical spectrum of CYP144A1. Although turnover of indazole 1 was not detected in biochemical


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assays and X-band EPR spectra did not indicate formation of the catalytically-poised highspin enzyme, this could be a consequence of the non-native conditions employed for these experiments. For example, the low temperature (ca. 10 K) required for X-band EPR of P450 enzymes stabilizes the low spin state of the heme,13,81 and the heterologous redox partner systems that are required to reconstitute biochemical activity in vitro can result in alternative oxidation products to those observed under native conditions.16,46 Alternatively, as suggested from spectroscopic data, indazole 1 may bind CYP144A1 in a water-bridged conformation. X-ray crystal structures of both P450 substrates and inhibitors have been reported where ligands bind indirectly to the heme via a bridging axial heme water ligand. The ∆λmax induced by indirect heme ligation is typically small in magnitude and does not necessarily comply with a traditional type I/type II classification system for P450 substrates and inhibitors.13,49,50,66 In summary, an efficient fragment-based approach has been illustrated which enables relationships between enzymes to be built. Fragment profiling highlights that enzymes with conserved structural and catalytic features, such as the family of P450s expressed by Mtb, have distinct binding properties, and that functionally similar enzymes share similar fragment profiles. Similarity in the binding profile of CYP144A1 to that of CYP121A1 led to the identification of a novel set of ligands for CYP144A1 that have diverse binding modes, good binding affinity and good physicochemical properties. Characterization of CYP144A1 using these compounds highlights properties of the enzyme that distinguish it from other Mtb P450s and subsequent optimization of compounds 1−3 may lead to further insight into the role of this orphan enzyme. Expansion of the fragment profiling campaign reported here to include the remaining 11 orphan Mtb P450s and a series of characterized P450s with diverse functions is in progress, and will provide a unique database of information about that factors


Biochemistry

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contributing to the interactions of cytochrome P450 enzymes with small molecule substrates and inhibitors.


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ASSOCIATED CONTENT Supporting Information Available Tabulated E-values from fragment profile analysis, example data from competition NMR and UV-vis spectroscopy experiments, optical titration and ITC data are provided as Supporting Information. This material is available free of charge via the Internet at: http://pubs.acs.org Open data Additional data relating to this publication are available at the University of Cambridge data repository: https://doi.org/10.17863/CAM.7283 AUTHOR INFORMATION Corresponding Author: *Chris Abell Address: Department of Chemistry, The University of Cambridge, UK Phone: +44 (0) 1223 336405 Email: [email protected] Present address: †

Jude Chenge, Department of Pharmaceutical and Biomedical Sciences, The University of

Georgia, Athens, GA 30602 Author Contributions


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M.E.K. and A.G.C. performed all fragment screening. M.E.K., J.C. and K.J.M. performed all biophysical experiments and protein preparation. M.E.K. and A.Z. performed bioinformatic and chemoinformatic analyses. A.B., A.W.M. and C.A. analyzed data, supervised this research and revised the manuscript. Funding M.E.K. was supported by a Commonwealth (University of Cambridge) Scholarship awarded in conjunction with the Cambridge Commonwealth Trust and Cambridge Overseas Trust. J.C. was supported by funding from the William D Ford program from the US Department of Education. K.J.M. and A.G.C. were supported by grants from the BBSRC (Grant No: BB/K001884/1 and BB/I019227/1). A.Z. was funded by the European Research Commission (ERC Starting Grant 2012 MIXTURE). Notes The authors declare no competing financial interest. Acknowledgements The authors wish to acknowledge Dr Dijana Matak-Vinkovic for assistance with the native mass spectrometry experiments and Dr Alicia Higueruelo for assistance setting up ligands docking models. ABBREVIATIONS CPMG, Carr-Purcell-Meiboom-Gill pulse sequence, relaxation-edited NMR experiment; EPR, electron paramagnetic resonance spectroscopy; ITC, isothermal titration calorimetry; KD, dissociation constant; Mtb, Mycobacterium tuberculosis; m/z, mass-to-charge ratio;


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nanoESI, nano-electrospray ionization mass spectrometry; P450/CYP, cytochrome P450 enzyme, SAR, structure-activity relationship


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45

Biochemistry

(a)

10 5

CYP105AS1

CYP152L1

CYP102A1 HD (A82F)

0

CYP102A1 HD (Wt)

CYP144A1

CYP143A1

CYP142A1

CYP126A1

CYP125A1

CYP124A1

CYP121A1

0

15

CYP144A1

10

20

CYP143A1

20

25

CYP142A1

30

30

CYP126A1

40

35

CYP125A1

50

40

CYP124A1

60

45

CYP121A1

70

Fragment Hits (% of Library)

FIGURES

Fragment Hits (% of Library)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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(b)

(c)

Figure 1. Fragment screening hit rates and fragment profile phylogeny of the Mtb P450s Percentage of fragment library identified to bind each P450 from (a) an unbiased fragment library screened by CPMG ligand-observed NMR; (b) a focused fragment library of heme binding compounds screened by UV-vis spectroscopy. Mtb P450 enzymes are grouped on the left and P450 enzymes from other bacteria (CYP102A1 BM3 wild-type (Wt) and mutant (A82F) heme domains, Bacillus megaterium; CYP152L1, Jeotgalicoccus sp. OleT; CYP105AS1, Amycolatopsis orientalis) are clustered to the right. (c) Phylogenetic tree relating the Mtb P450 enzymes according to the similarity in their fragment binding profile (as determined from screening the unbiased fragment library by ligand-observed NMR). Fragments have been clustered according to their hit rates against the Mtb P450s.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(a)

Biochemistry

(b)

(c)

Figure 2. Structural comparison of Mtb CYP144A1 and CYP121A1 (a) Cartoon representation of the aligned structures of CYP144A1-TRV (PDB 5HDI) (green) and CYP121A1 (PDB 3G5F) (orange). (b) Active site residues of (b) CYP144A1 (green sticks), and (c) CYP121A1 (orange and yellow). CYP121A1 residues that differ from those in CYP144A1 are colored yellow. The heme cofactor (grey sticks), axial water ligand and Ihelix (grey cartoon) are indicated. All figures were prepared using PyMOL v1.7.4 (Schrödinger, LLC).


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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2.43 clotrimazole

2.46

2.25 1.91 1.89

(4)

2.26 2.43 2.41

1.89

(3)

2.24 2.44 2.41

1.92 1.90

(2)

2.26

2.44 2.41

1.92 1.89

(1)

2.24

2.41

1.92 1.89

DMSO

2.24 1.92

2400

2600

2800

3000

3200

3400

3600

3800

Magnetic Field (Gauss)

(a)

(b) Figure 3. Chemical structures of CYP144A1 ligands and X-band EPR spectra of CYP144A1-ligand complexes. (a) Chemical structures of imidazole drugs, novel CYP144A1 ligands 1−3 and fragments 4−6 used to characterize the properties of CYP144A1; (b) Bottom to top: X-band EPR spectra of ligand-free CYP144A1 (200 µΜ), and CYP144A1 (200 µΜ) bound to compounds 1, 2, 3, 4 and clotrimazole (each 500 µΜ), respectively. The ligand-free sample contained DMSO at the same concentration as that used in the ligand-bound forms.


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Biochemistry

Figure 4. Competition ligand binding experiments. (a) Compound 1 (100 µM, blue) caused a decrease in the λmax of the CYP144A1 (4–5 µM) absorbance spectrum compared to the ligand-free enzyme (black line), and competitively decreased the ∆λmax caused by compound 2 (50 µM, green), compared to that observed for compound 2 alone (red). (b) Compound 6 (1 mM) (black dashed) does not perturb the λmax of ligand-free CYP144A1 (solid black), but competes for binding with miconazole (12.5 µM) (orange dashed), decreasing the ∆λmax caused by miconazole alone (solid orange). Compound 5 binds cooperatively with compound 2 (50 µM, red dashed), increasing the ∆λmax compared to compound 2 alone (solid red). (c and d) CPMG NMR spectra of (c) imidazole 4 (1 mM) or (d) benzothiophene 5 (1 mM), in buffer alone (bottom panel), in the presence of CYP144A1 (20 µM) protein (middle panels), or in the presence of CYP144A1 and a displacer (clotrimazole (500 µM)) (top panels). Binding interactions are indicated by a decrease in 1H signal intensity (red arrows). Competitive displacement of fragment 4 by clotrimazole restored 1H signal intensity (blue arrows). Cooperative binding interactions of compound 5 with clotrimazole further reduce 1H signal intensity (blue arrows, top panel).


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 5. NanoESI native mass spectra of native CYP144A1 binding to ligands. Bottom to top: Ligand-free CYP144A1 (10 µM), and CYP144A1 (10 µM) bound to clotrimazole, miconazole, econazole and compounds 1-3 (each 50 µM). All samples contained 5% v/v d6DMSO. Major peaks m/z = 4200-5400 correspond to the CYP144A1 monomer. Minor peaks correspond to an unidentified protein impurity. Dashed lines A, B, C mark the m/z corresponding to the ligand-free protein, and CYP144A1 bound to one molecule and two molecules of ligand, respectively.


Page 51 of 53

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Biochemistry

Table 2. Ligand binding stoichiometry and affinity determined from nanoESI native mass spectrometry.

Compound

KD (µM)a

Binding Stoichiometryb

Ligand Bound CYP144A1(%)c

L (10 μM)

L (50 μM)

B+C

Clotrimazole

0.095d

1

2

100

Econazole

0.72d

0

1

76

Miconazole

15

1

1

41

1

82

1

2

60

2

200

1

2

41

3

140

1

2

48

a

Dissociation constant; calculated by optical titration for azoles, compound 1 and 2, or ITC for compound 3, which was determined by ITC. bNumber of ligand molecules bound per CYP144A1 monomer, from samples containing CYP144A1 (10 μM) and ligands (10-50 μM). cProportion of ligand bound CYP144A1 in each sample, calculated from the relative intensity of peaks A (ligand free), B (1 ligand per monomer), and C (2 ligands per monomer), at a 5:1 ratio of ligand (50 μM) to CYP144A1 (10 μM). dPreviously reported: Chenge, J. et al., Sci. Rep., 2016, 6:26628.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(a)

(b)

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(c)

Figure 6. Ligand docking poses of compounds 1-3 with CYP144A1. (a-c) Compounds 1 (cyan), 2 (salmon), and 3 (green), respectively, in complex with CYP144A1. The heme cofactor and active site residues (grey), axial water ligand (red sphere) and proposed intermolecular interactions (yellow dashes) are shown in each figure. Docking was performed using GLIDE v6.5 (Schrödinger, LLC, New York, NY). All figures were prepared using PyMOL v1.7.4 (Schrödinger, LLC).


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