Photocontrol of the ß-Hairpin Polypeptide Structure through an Optimized Azobenzene-Based Amino Acid Analogue.

A family of neurodegenerative diseases, including Huntington's disease (HD) and spinocerebellar ataxias, are associated with an abnormal polyglutamine (polyQ) expansion in mutant proteins that become prone to form amyloid-like aggregates. Prior studies have suggested a key role for ß-hairpin formation as a driver of nucleation and aggregation, but direct experimental studies have been challenging. Toward such research, we set out to enable spatiotemporal control over ß-hairpin formation by the introduction of a photosensitive ß-turn mimic in the polypeptide backbone, consisting of a newly designed azobenzene derivative. The reported derivative overcomes the limitations of prior approaches associated with poor photochemical properties and imperfect structural compatibility with the desired ß-turn structure. A new azobenzene-based ß-turn mimic was designed, synthesized, and found to display improved photochemical properties, both prior and after incorporation into the backbone of a polyQ polypeptide. The two isomers of the azobenzene-polyQ peptide showed different aggregate structures of the polyQ peptide fibrils, as demonstrated by electron microscopy and solid-state NMR (ssNMR). Notably, only peptides in which the ß-turn structure was stabilized (azobenzene in the cis configuration) closely reproduced the spectral fingerprints of toxic, ß-hairpin-containing fibrils formed by mutant huntingtin protein fragments implicated in HD. These approaches and findings will enable better deciphering of the roles of ß-hairpin structures in protein aggregation processes in HD and other amyloid-related neurodegenerative diseases.

Rational Design in Photopharmacology with Molecular Photoswitches.

Photopharmacology is an attractive approach for achieving targeted drug action with the use of light. In photopharmacology, molecular photoswitches are introduced into the structure of biologically active small molecules to allow for the optical control of their potency. Going beyond trial and error, photopharmacology has progressively applied rational drug design methodologies to devise light-controlled bioactive ligands. In this review, we categorize photopharmacological efforts from the standpoint of medicinal chemistry strategies, focusing on diffusible photochromic ligands modified with photoswitches that operate through E-Z bond isomerization. In the vast majority of cases, photoswitchable ligands are designed as analogs of existing compounds, through a variety of approaches. By analyzing in detail a comprehensive list of instructive examples, we describe the state of the art and discuss future opportunities for rational design in photopharmacology.

Hypothesis-Driven, Structure-Based Design in Photopharmacology: The Case of eDHFR Inhibitors.

Photopharmacology uses light to regulate the biological activity of drugs. This precise control is obtained through the incorporation of molecular photoswitches into bioactive molecules. A major challenge for photopharmacology is the rational design of photoswitchable drugs that show light-induced activation. Computer-aided drug design is an attractive approach toward more effective, targeted design. Herein, we critically evaluated different structure-based approaches for photopharmacology with Escherichia coli dihydrofolate reductase (eDHFR) as a case study. Through the iterative examination of our hypotheses, we progressively tuned the design of azobenzene-based, photoswitchable eDHFR inhibitors in five design-make-switch-test-analyze cycles. Targeting a hydrophobic subpocket of the enzyme and a specific salt bridge only with the thermally metastable cis-isomer emerged as the most promising design strategy. We identified three inhibitors that could be activated upon irradiation and reached potencies in the low-nanomolar range. Above all, this systematic study provided valuable insights for future endeavors toward rational photopharmacology.

A light-fuelled nanoratchet shifts a coupled chemical equilibrium.

Biological molecular machines enable chemical transformations, assembly, replication and motility, but most distinctively drive chemical systems out of-equilibrium to sustain life1,2. In such processes, nanometre-sized machines produce molecular energy carriers by driving endergonic equilibrium reactions. However, transforming the work performed by artificial nanomachines3-5 into chemical energy remains highly challenging. Here, we report a light-fuelled small-molecule ratchet capable of driving a coupled chemical equilibrium energetically uphill. By bridging two imine6-9 macrocycles with a molecular motor10,11, the machine forms crossings and consequently adopts several distinct topologies by either a thermal (temporary bond-dissociation) or photochemical (unidirectional rotation) pathway. While the former will relax the machine towards the global energetic minimum, the latter increases the number of crossings in the system above the equilibrium value. Our approach provides a blueprint for coupling continuous mechanical motion performed by a molecular machine with a chemical transformation to reach an out-of-equilibrium state.

Rational design of a photoswitchable DNA glue enabling high regulatory function and supramolecular chirality transfer.

Short, complementary DNA single strands with mismatched base pairs cannot undergo spontaneous formation of duplex DNA (dsDNA). Mismatch binding ligands (MBLs) can compensate this effect, inducing the formation of the double helix and thereby acting as a molecular glue. Here, we present the rational design of photoswitchable MBLs that allow for reversible dsDNA assembly by light. Careful choice of the azobenzene core structure results in excellent band separation of the E and Z isomers of the involved chromophores. This effect allows for efficient use of light as an external control element for duplex DNA formation and for an in-depth study of the DNA-ligand interaction by UV-Vis, SPR, and CD spectroscopy, revealing a tight mutual interaction and complementarity between the photoswitchable ligand and the mismatched DNA. We also show that the configuration of the switch reversibly dictates the conformation of the DNA strands, while the dsDNA serves as a chiral clamp and translates its chiral information onto the ligand inducing a preference in helical chirality of the Z isomer of the MBLs.

Biaryl sulfonamides as cisoid azosteres for photopharmacology.

Biaryl sulfonamides are excellent candidates for the azologization approach that yields photoswitchable drugs more active in their metastable cis state, compared to the stable trans state. Here we present the scope and limitations of this strategy for rational design in photopharmacology.

Mechanism of Resistance Development in E. coli against TCAT, a Trimethoprim-Based Photoswitchable Antibiotic.

During the last decades, a continuous rise of multi-drug resistant pathogens has threatened antibiotic efficacy. To tackle this key challenge, novel antimicrobial therapies are needed with increased specificity for the site of infection. Photopharmacology could enable such specificity by allowing for the control of antibiotic activity with light, as exemplified by trans/cis-tetra-ortho-chloroazobenzene-trimethoprim (TCAT) conjugates. Resistance development against the on (irradiated, TCATa) and off (thermally adapted, TCATd) states of TCAT were compared to that of trimethoprim (TMP) in Escherichia coli mutant strain CS1562. Genomics and transcriptomics were used to explore the acquired resistance. Although TCAT shows TMP-like dihydrofolate reductase (DHFR) inhibition in vitro, transcriptome analyses show different responses in acquired resistance. Resistance against TCATa (on) relies on the production of exopolysaccharides and overexpression of TolC. While resistance against TCATd (off) follows a slightly different gene expression profile, both indicate hampering the entrance of the molecule into the cell. Conversely, resistance against TMP is based on alterations in cell metabolism towards a more persister-like phenotype, as well as alteration of expression levels of enzymes involved in the folate biosynthesis. This study provides a deeper understanding of the development of new therapeutic strategies and the consequences on resistance development against photopharmacological drugs.

Photopharmacological Manipulation of Mammalian CRY1 for Regulation of the Circadian Clock.

CRY1 and CRY2 proteins are highly conserved components of the circadian clock that controls daily physiological rhythms. Disruption of CRY functions are related to many diseases, including circadian sleep phase disorder. Development of isoform-selective and spatiotemporally controllable tools will facilitate the understanding of shared and distinct functions of CRY1 and CRY2. Here, we developed CRY1-selective compounds that enable light-dependent manipulation of the circadian clock. From phenotypic chemical screening in human cells, we identified benzophenone derivatives that lengthened the circadian period. These compounds selectively interacted with the CRY1 photolyase homology region, resulting in activation of CRY1 but not CRY2. The benzophenone moiety rearranged a CRY1 region called the "lid loop" located outside of the compound-binding pocket and formed a unique interaction with Phe409 in the lid loop. Manipulation of this key interaction was achieved by rationally designed replacement of the benzophenone with a switchable azobenzene moiety whose cis-trans isomerization can be controlled by light. The metastable cis form exhibited sufficiently high half-life in aqueous solutions and structurally mimicked the benzophenone unit, enabling reversible period regulation over days by cellular irradiation with visible light. This study revealed an unprecedented role of the lid loop in CRY-compound interaction and paves the way for spatiotemporal regulation of CRY1 activity by photopharmacology for molecular understanding of CRY1-dependent functions in health and disease.

Modular Medical Imaging Agents Based on Azide-Alkyne Huisgen Cycloadditions: Synthesis and Pre-Clinical Evaluation of 18 F-Labeled PSMA-Tracers for Prostate Cancer Imaging.

Since the seminal contribution of Rolf Huisgen to develop the [3+2] cycloaddition of 1,3-dipolar compounds, its azide-alkyne variant has established itself as the key step in numerous organic syntheses and bioorthogonal processes in materials science and chemical biology. In the present study, the copper(I)-catalyzed azide-alkyne cycloaddition was applied for the development of a modular molecular platform for medical imaging of the prostate-specific membrane antigen (PSMA), using positron emission tomography. This process is shown from molecular design, through synthesis automation and in vitro studies, all the way to pre-clinical in vivo evaluation of fluorine-18- labeled PSMA-targeting 'F-PSMA-MIC' radiotracers (t1/2 =109.7 min). Pre-clinical data indicate that the modular PSMA-scaffold has similar binding affinity and imaging properties to the clinically used [68 Ga]PSMA-11. Furthermore, we demonstrated that targeting the arene-binding in PSMA, facilitated through the [3+2]cycloaddition, can improve binding affinity, which was rationalized by molecular modeling. The here presented PSMA-binding scaffold potentially facilitates easy coupling to other medical imaging moieties, enabling future developments of new modular imaging agents.

Light-controlled inhibition of BRAFV600E kinase.

Metastatic melanoma is amongst the most difficult types of cancer to treat, with current therapies mainly relying on the inhibition of the BRAFV600E mutant kinase. However, systemic inhibition of BRAF by small molecule drugs in cancer patients results - paradoxically - in increased wild-type BRAF activity in healthy tissue, causing side-effects and even the formation of new tumors. Here we show the development of BRAFV600E kinase inhibitors of which the activity can be switched on and off reversibly with light, offering the possibility to overcome problems of systemic drug activity by selectively activating the drug at the desired site of action. Based on a known inhibitor, eight photoswitchable effectors containing an azobenzene photoswitch were designed, synthesized and evaluated. The most promising inhibitor showed an approximately 10-fold increase in activity upon light-activation. This research offers inspiration for the development of therapies for metastatic melanoma in which tumor tissue is treated with an active BRAFV600E inhibitor with high spatial and temporal resolution, thus limiting the damage to other tissues.

Photoactivation of MDM2 Inhibitors: Controlling Protein-Protein Interaction with Light.

Selectivity remains a major challenge in anticancer therapy, which potentially can be overcome by local activation of a cytotoxic drug. Such triggered activation can be obtained through modification of a drug with a photoremovable protecting group (PPG), and subsequent irradiation in the chosen place and time. Herein, the design, synthesis and biological evaluation is described of a photoactivatable MDM2 inhibitor, PPG-idasanutlin, which exerts no functional effect on cellular outgrowth, but allows for the selective, noninvasive activation of antitumor properties upon irradiation visible light, demonstrating activation with micrometer, single cell precision. The generality of this method has been demonstrated by growth inhibition of multiple cancer cell lines showing p53 stabilization and subsequent growth inhibition effects upon irradiation. Light activation to regulate protein-protein interactions between MDM2 and p53 offers exciting opportunities to control a multitude of biological processes and has the potential to circumvent common selectivity issues in antitumor drug development.

Structure-activity relationships of strigolactones via a novel, quantitative in planta bioassay.

Strigolactones (SLs) are plant hormones with various functions in development, responses to stress, and interactions with (micro)organisms in the rhizosphere, including with seeds of parasitic plants. Their perception for hormonal functions requires an α,ß-hydrolase belonging to the D14 clade in higher plants; perception of host-produced SLs by parasitic seeds relies on similar but phylogenetically distinct proteins (D14-like). D14 and D14-like proteins are peculiar receptors, because they cleave SLs before undergoing a conformational change that elicits downstream events. Structure-activity relationship data show that the butenolide D-ring is crucial for bioactivity. We applied a bioisosteric approach to the structure of SLs by synthetizing analogues and mimics of natural SLs in which the D-ring was changed from a butenolide to a lactam and then evaluating their bioactivity. This was done by using a novel bioassay based on Arabidopsis transgenic lines expressing AtD14 fused to firefly luciferase, in parallel with the quantification of germination-inducing activity on parasitic seeds. The results obtained showed that the in planta bioassay is robust and quantitative, and thus can be confidently added to the SL-survey toolbox. The results also showed that modification of the butenolide ring into a lactam one significantly hampers the biological activity exhibited by SLs possessing a canonical lactonic D-ring.