Multiple drug binding modes in Mycobacterium tuberculosis CYP51B1

Highlights

• There is a mixture of binding modes for nitrogenous drug fragments.

• Water-bridged complexes are present in each cytochrome P450 (CYP)-drug combination.

• A mixture of binding modes impacts inhibitor design for Mycobacterium tuberculosis CYPs.

Abstract

The Mycobacterium tuberculosis (Mtb) genome encodes 20 different cytochrome P450 enzymes (CYPs), many of which serve essential biosynthetic roles. CYP51B1, the Mtb version of eukaryotic sterol demethylase, remains a potential therapeutic target. The binding of three drug fragments containing nitrogen heterocycles to CYP51B1 is studied here by continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) techniques to determine how each drug fragment binds to the heme active-site. All three drug fragments form a mixture of complexes, some of which retain the axial water ligand from the resting state. Hyperfine sublevel correlation spectroscopy (HYSCORE) and electron-nuclear double resonance spectroscopy (ENDOR) observe protons of the axial water and on the drug fragments that reveal drug binding modes. Binding in CYP51B1 is complicated by the presence of multiple binding modes that coexist in the same solution. These results aid our understanding of CYP-inhibitor interactions and will help guide future inhibitor design.

Introduction

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), an infectious disease that has afflicted humans for centuries. TB remains a threat and a major cause of death today. Current treatments are complicated by the rise of resistant strains of Mtb, which has caused renewed urgency in TB research and drug development. A major recent advance in TB research was the sequencing of the Mtb genome, which revealed new potential drug targets. One of the most noteworthy aspects of the genome is that Mtb encodes 20 different cytochrome P450 enzymes (CYPs) [1]. CYPs are heme-containing monooxygenase enzymes responsible for essential biosynthetic and detoxification tasks in nearly all living organisms. CYP51B1 in Mtb was the first member of the CYP51 sterol demethylase family to be found in prokaryotes [1]. CYP51B1 catalyzes the oxidative demethylation of dihydrolanosterol and obtusifoliol, which is similar to the role and function of eukaryotic CYP51 [2,3]. While a complete sterol biosynthesis pathway is not known in Mtb, CYP51B1 remains a potential drug target. The first effective drugs against Mtb, isoniazid, pyrazinamide, and rifampicin, came out in the 1950s [4]. These remained the most effective drugs for decades and are still among the few treatment options available. A robust understanding of how drugs bind to and inhibit CYPs will help to target Mtb CYPs in the development of new and effective treatments.

CYPs contain an active site comprised of a ferric, low-spin heme coordinated to a conserved cysteine residue (Fig. 1, left). The sixth axial ligand present in the resting state is a water molecule, but it is assumed to be either displaced or replaced upon drug binding. In the traditional drug-binding paradigm, nitrogen-containing heterocycles are considered to act as CYP inhibitors because they replace the axial water by direct coordination to the heme (Fig. 1, middle). These complexes are typically thought to “trap” the heme in the low-spin state, thus preventing the one electron reduction that begins the catalytic cycle. Substrates, on the other hand, displace the axial water without replacing it, which leaves the heme in a high-spin state that is more easily reduced and serves as the entry point to the catalytic cycle [5]. In some cases, the drug does not fit into the traditional substrate/inhibitor paradigm, and instead of displacing or replacing the axial water, it interacts with the heme via a hydrogen bonding network (Fig. 1, right). This complex, which we refer to as water-bridged, is only observed in a few crystal structures and is considered rare [[6], [7], [8], [9], [10]]. The water-bridged binding mode is not necessarily associated with substrates or inhibitors; in fact, it has been observed with both in a variety of CYP isoforms [11]. While the water-bridged complex is still low-spin, it is not necessarily catalytically inactive. For example, in CYP3A4, the major human isoform responsible for drug metabolism, we found that a potential estradiol-based drug, 17α-(2H-2,3,4-triazolyl)-estradiol (17-click), formed a water-bridged complex that was still catalytically active, producing a library of altered drug products [12].

In the case of Mtb CYPs, a few crystal structures show drugs, substrates, and substrate analogs interacting with the heme through active site waters [[7], [8], [9], [10],[13], [14], [15]]. Recently, a water-bridged complex was identified in the Mtb CYP121 with the substrate cyclodityrosine (cYY) [9]. The H-bond network involves two ordered water molecules which are present in other CYP121 ligand bound crystal structures, and the authors suggest that such H-bond networks could position substrates or drugs in the active site for metabolism [9]. The drug fluconazole does not always displace the axial water in Mtb CYP121; sometimes, it forms a hydrogen-bonding network with the heme [8]. A water-bridged complex of Mtb CYP125A1 with the pyridine-based inhibitor α-[(4-methylcyclohexyl)carbonyl amino]-N-4-pyridinyl-1H-indole-3-propanamide (LP10; structure in supplementary data section S1) has also been observed and studied using electron paramagnetic resonance (EPR) and visible and near-infrared magnetic circular dichroism (MCD) spectroscopy [5].

A few water-bridged complexes have been characterized in Mtb CYPs, but the extent to which these complexes exist in other isoforms and with other drugs is not known. We have found that water-bridged complexes oftentimes coexist in frozen solution with directly-coordinated complexes for several CYPs, producing a mixture of binding modes for the same drug that are not captured in the crystal structure databases [11]. This mixture of binding modes makes the question of their catalytic competency important because water-bridged complexes have been observed with both inhibitors and substrates. Mixtures of binding modes are common in the human CYPs we have studied, and this study suggests that a similar mixture of binding modes in solution may exist with CYP51B1 with nitrogen heterocycles that would traditionally be expected to replace the axial water and directly coordinate to the heme.

Here, we use EPR to characterize the binding of three nitrogen-containing heterocycles that traditionally would be considered inhibitor-like ligands to CYP51B1. These three compounds will be referred to as “drugs” henceforth for convenience. Continuous wave (CW) EPR and hyperfine sublevel correlation spectroscopy (HYSCORE) are used to directly probe the protons on the axial water and distinguish drugs that replace the axial water and are directly-coordinated from those that form a water-bridged complex [11]. In addition, we use electron-nuclear double resonance (ENDOR) spectroscopy to show that the drugs occupy the active site and are in close proximity to the heme. All three drugs bind as a mixture of water-bridged and directly-coordinated complexes. The EPR and HYSCORE spectra both show that the axial water is perturbed and reoriented with respect to the heme in the water-bridged complexes. This study suggests that water-bridged complexes may not be rare in Mtb CYPs, and supports the suggestion from CYP121 that the axial water can play a structural role in drug binding [9]. These multiple complexes and their possible individual activities have implications for drug design and need to be considered in the design of effective inhibitors for CYP51B1 and the other Mtb CYP isoforms.