Syntheses and Structure–Activity Relationships of N-Phenethyl-Quinazolin-4-yl-Amines as Potent Inhibitors of Cytochrome bd Oxidase in Mycobacterium tuberculosis

The development of cytochrome bd oxidase (cyt-bd) inhibitors are needed for comprehensive termination of energy production in Mycobacterium tuberculosis (Mtb) to treat tuberculosis infections. Herein, we report on the structure-activity-relationships (SAR) of 22 new N-phenethyl-quinazolin-4-yl-amines that target cyt-bd. Our focused set of compounds was synthesized and screened against three mycobacterial strains: Mycobacterium bovis BCG, Mycobacterium tuberculosis H37Rv and the clinical isolate Mycobacterium tuberculosis N0145 with and without the cytochrome bcc:aa3 inhibitor Q203 in an ATP depletion assay. Two compounds, 12a and 19a, were more active against all three strains than the naturally derived cyt-bd inhibitor aurachin D.

Host immunity increases Mycobacterium tuberculosis reliance on cytochrome bd oxidase

In order to sustain a persistent infection, Mycobacterium tuberculosis (Mtb) must adapt to a changing environment that is shaped by the developing immune response. This necessity to adapt is evident in the flexibility of many aspects of Mtb metabolism, including a respiratory chain that consists of two distinct terminal cytochrome oxidase complexes. Under the conditions tested thus far, the bc1/aa3 complex appears to play a dominant role, while the alternative bd oxidase is largely redundant. However, presence of two terminal oxidases in this obligate pathogen implies that respiratory requirements might change during infection. We report that the cytochrome bd oxidase is specifically required for resisting the adaptive immune response. While the bd oxidase was dispensable for growth in resting macrophages and the establishment of infection in mice, this complex was necessary for optimal fitness after the initiation of adaptive immunity. This requirement was dependent on lymphocyte-derived interferon gamma (IFNγ), but did not involve nitrogen and oxygen radicals that are known to inhibit respiration in other contexts. Instead, we found that ΔcydA mutants were hypersusceptible to the low pH encountered in IFNγ-activated macrophages. Unlike wild type Mtb, cytochrome bd-deficient bacteria were unable to sustain a maximal oxygen consumption rate (OCR) at low pH, indicating that the remaining cytochrome bc1/aa3 complex is preferentially inhibited under acidic conditions. Consistent with this model, the potency of the cytochrome bc1/aa3 inhibitor, Q203, is dramatically enhanced at low pH. This work identifies a critical interaction between host immunity and pathogen respiration that influences both the progression of the infection and the efficacy of potential new TB drugs.

The Inhibition Study of Cytochrome bd Oxidase Using the Enzyme-Based Electrochemical Sensor

Membrane proteins that participate in multiple vital functions of every living organism such as transport, signaling and respiration, provide 80 to 90% of the relevant targets for the pharmaceutical industries. The family of cytochrome bd oxidase enzymes is of great interest for the development of future antibiotics as they are found only in the respiratory chain of the prokaryotes and they are believed to be involved in bacterial adaptability mechanisms. They catalyze the reduction of molecular oxygen in water and oxidation of quinols and contribute to the proton motive force required for ATP synthesis. Due to their hydrophobic nature, membrane proteins are more difficult to handle than soluble proteins. Protein film voltammetry is a very convenient technique, because it allows for working at a very low concentration and for optimizing the electrode surface to the nature of the enzyme. Here, we have developed a biosensor for the study of terminal oxidases based on their immobilization on gold nanoparticles modified with a self-assembled monolayer of thiols. The stability of the protein films can be optimized by varying the nature of thiols and amount of lipids. This enzyme-based electrochemical sensor was successfully used for the inhibition screening of a target-focused library of 34 compounds which belong to the families of quinones, naphthoquinones, phenols, quinolones, coumarins and flavonoids against cytochrome bd oxidase. Moreover, the developed device was applied for the study of the catalytic reaction of the enzyme with small gaseous signaling molecules.

Stabilization of the Highly Hydrophobic Membrane Protein, Cytochrome bd Oxidase, on Metallic Surfaces for Direct Electrochemical Studies

The cytochrome bd oxidase catalyzes the reduction of oxygen to water in bacteria and it is thus an interesting target for electrocatalytic studies and biosensor applications. The bd oxidase is completely embedded in the phospholipid membrane. In this study, the variation of the surface charge of thiol-modified gold nanoparticles, the length of the thiols and the other crucial parameters including optimal phospholipid content and type, have been performed, giving insight into the role of these factors for the optimal interaction and direct electron transfer of an integral membrane protein. Importantly, all three tested factors, the lipid type, the electrode surface charge and the thiol length mutually influenced the stability of films of the cytochrome bd oxidase. The best electrocatalytic responses were obtained on the neutral gold surface when the negatively charged phosphatidylglycerol (PG) was used and on the charged gold surface when the zwitterionic phosphatidylethanolamine (PE) was used. The advantages of the covalent binding of the membrane protein to the electrode surface over the non-covalent binding are also discussed.