Leveraging the histidine kinase-phosphatase duality to sculpt two-component signaling.

Bacteria must constantly probe their environment for rapid adaptation, a crucial need most frequently served by two-component systems (TCS). As one component, sensor histidine kinases (SHK) control the phosphorylation of the second component, the response regulator (RR). Downstream responses hinge on RR phosphorylation and can be highly stringent, acute, and sensitive because SHKs commonly exert both kinase and phosphatase activity. With a bacteriophytochrome TCS as a paradigm, we here interrogate how this catalytic duality underlies signal responses. Derivative systems exhibit tenfold higher red-light sensitivity, owing to an altered kinase-phosphatase balance. Modifications of the linker intervening the SHK sensor and catalytic entities likewise tilt this balance and provide TCSs with inverted output that increases under red light. These TCSs expand synthetic biology and showcase how deliberate perturbations of the kinase-phosphatase duality unlock altered signal-response regimes. Arguably, these aspects equally pertain to the engineering and the natural evolution of TCSs.

Multimodal Control of Bacterial Gene Expression by Red and Blue Light.

By applying sensory photoreceptors, optogenetics realizes the light-dependent control of cellular events and state. Given reversibility, noninvasiveness, and exquisite spatiotemporal precision, optogenetic approaches enable innovative use cases in cell biology, synthetic biology, and biotechnology. In this chapter, we detail the implementation of the pREDusk, pREDawn, pCrepusculo, and pAurora optogenetic circuits for controlling bacterial gene expression by red and blue light, respectively. The protocols provided here guide the practical use and multiplexing of these circuits, thereby enabling graded protein production in bacteria at analytical and semi-preparative scales.

Optogenetic Control of Bacterial Expression by Red Light.

In optogenetics, as in nature, sensory photoreceptors serve to control cellular processes by light. Bacteriophytochrome (BphP) photoreceptors sense red and far-red light via a biliverdin chromophore and, in response, cycle between the spectroscopically, structurally, and functionally distinct Pr and Pfr states. BphPs commonly belong to two-component systems that control the phosphorylation of cognate response regulators and downstream gene expression through histidine kinase modules. We recently demonstrated that the paradigm BphP from Deinococcus radiodurans exclusively acts as a phosphatase but that its photosensory module can control the histidine kinase activity of homologous receptors. Here, we apply this insight to reprogram two widely used setups for bacterial gene expression from blue-light to red-light control. The resultant pREDusk and pREDawn systems allow gene expression to be regulated down and up, respectively, uniformly under red light by 100-fold or more. Both setups are realized as portable, single plasmids that encode all necessary components including the biliverdin-producing machinery. The triggering by red light affords high spatial resolution down to the single-cell level. As pREDusk and pREDawn respond sensitively to red light, they support multiplexing with optogenetic systems sensitive to other light colors. Owing to the superior tissue penetration of red light, the pREDawn system can be triggered at therapeutically safe light intensities through material layers, replicating the optical properties of the skin and skull. Given these advantages, pREDusk and pREDawn enable red-light-regulated expression for diverse use cases in bacteria.

Comparative analysis of two paradigm bacteriophytochromes reveals opposite functionalities in two-component signaling.

Bacterial phytochrome photoreceptors usually belong to two-component signaling systems which transmit environmental stimuli to a response regulator through a histidine kinase domain. Phytochromes switch between red light-absorbing and far-red light-absorbing states. Despite exhibiting extensive structural responses during this transition, the model bacteriophytochrome from Deinococcus radiodurans (DrBphP) lacks detectable kinase activity. Here, we resolve this long-standing conundrum by comparatively analyzing the interactions and output activities of DrBphP and a bacteriophytochrome from Agrobacterium fabrum (Agp1). Whereas Agp1 acts as a conventional histidine kinase, we identify DrBphP as a light-sensitive phosphatase. While Agp1 binds its cognate response regulator only transiently, DrBphP does so strongly, which is rationalized at the structural level. Our data pinpoint two key residues affecting the balance between kinase and phosphatase activities, which immediately bears on photoreception and two-component signaling. The opposing output activities in two highly similar bacteriophytochromes suggest the use of light-controllable histidine kinases and phosphatases for optogenetics.

Discovery of Retinoic Acid-Related Orphan Receptor γt Inverse Agonists via Docking and Negative Image-Based Screening.

Retinoic acid-related orphan receptor γt (RORγt) has a vital role in the differentiation of T-helper 17 (TH17) cells. Potent and specific RORγt inverse agonists are sought for treating TH17-related diseases such as psoriasis, rheumatoid arthritis, and type 1 diabetes. Here, the aim was to discover novel RORγt ligands using both standard molecular docking and negative image-based screening. Interestingly, both of these in silico techniques put forward mostly the same compounds for experimental testing. In total, 11 of the 34 molecules purchased for testing were verified as RORγt inverse agonists, thus making the effective hit rate 32%. The pIC50 values for the compounds varied from 4.9 (11 µM) to 6.2 (590 nM). Importantly, the fact that the verified hits represent four different cores highlights the structural diversity of the RORγt inverse agonism and the ability of the applied screening methodologies to facilitate much-desired scaffold hopping for drug design.

Blocking oestradiol synthesis pathways with potent and selective coumarin derivatives.

A comprehensive set of 3-phenylcoumarin analogues with polar substituents was synthesised for blocking oestradiol synthesis by 17-ß-hydroxysteroid dehydrogenase 1 (HSD1) in the latter part of the sulphatase pathway. Five analogues produced ≥62% HSD1 inhibition at 5 µM and, furthermore, three of them produced ≥68% inhibition at 1 µM. A docking-based structure-activity relationship analysis was done to determine the molecular basis of the inhibition and the cross-reactivity of the analogues was tested against oestrogen receptor, aromatase, cytochrome P450 1A2, and monoamine oxidases. Most of the analogues are only modestly active with 17-ß-hydroxysteroid dehydrogenase 2 - a requirement for lowering effective oestradiol levels in vivo. Moreover, the analysis led to the synthesis and discovery of 3-imidazolecoumarin as a potent aromatase inhibitor. In short, coumarin core can be tailored with specific ring and polar moiety substitutions to block either the sulphatase pathway or the aromatase pathway for treating breast cancer and endometriosis.

Structure-Activity Relationship Analysis of 3-Phenylcoumarin-Based Monoamine Oxidase B Inhibitors.

Monoamine oxidase B (MAO-B) catalyzes deamination of monoamines such as neurotransmitters dopamine and norepinephrine. Accordingly, small-molecule MAO-B inhibitors potentially alleviate the symptoms of dopamine-linked neuropathologies such as depression or Parkinson's disease. Coumarin with a functionalized 3-phenyl ring system is a promising scaffold for building potent MAO-B inhibitors. Here, a vast set of 3-phenylcoumarin derivatives was designed using virtual combinatorial chemistry or rationally de novo and synthesized using microwave chemistry. The derivatives inhibited the MAO-B at 100 nM-1 µM. The IC50 value of the most potent derivative 1 was 56 nM. A docking-based structure-activity relationship analysis summarizes the atom-level determinants of the MAO-B inhibition by the derivatives. Finally, the cross-reactivity of the derivatives was tested against monoamine oxidase A and a specific subset of enzymes linked to estradiol metabolism, known to have coumarin-based inhibitors. Overall, the results indicate that the 3-phenylcoumarins, especially derivative 1, present unique pharmacological features worth considering in future drug development.