Experimental and theoretical comprehension of ESIPT fluorophores based on a 2-(2'-hydroxyphenyl)-3,3'-dimethylindole (HDMI) scaffold.

Excited-State Intramolecular Proton Transfer (ESIPT) emission is associated with intense single or multiple fluorescence in the solid-state, along with enhanced photostability and sensitivity to the close environment. As a result, ESIPT probes are attractive candidates for ratiometric sensing of a variety of substrates. A new family of ESIPT fluorophores is described herein, inspired by the well-known 2-(2'hydroxyphenyl)benzazole (HBX) organic scaffold. The connection of 3,3'-dimethylindole (or 3H-indole) derivatives with phenol rings triggers the formation of novel 2-(2'-hydroxyphenyl)-3,3'-dimethylindole (HDMI) fluorophores, capable of stimuli-responsive ESIPT emission. This brand new family of dyes displays redshifted emission, as compared to HBX, along with an unprecedented acid/base-mediated stabilization of different rotamers, owing to supramolecular interactions with methyl groups. These compounds are therefore highly sensitive to external stimuli, such as the presence of acid or base, where protonated and deprotonated species have specific optical signatures. Moreover, a new pyridine-functionalized HDMI dye displays acid-sensitive AIE properties. The photophysical properties of all compounds have also been studied using ab initio calculations to support experiments in deciphering the nature of the various radiative transitions observed and the related excited rotameric species.

Is the C-Terminal Domain an Effective and Selective Target for the Design of Hsp90 Inhibitors against Candida Yeast?

Improving the armamentarium to treat invasive candidiasis has become necessary to overcome drug resistance and the lack of alternative therapy. In the pathogenic fungus Candida albicans, the 90-kDa Heat-Shock Protein (Hsp90) has been described as a major regulator of virulence and resistance, offering a promising target. Some human Hsp90 inhibitors have shown activity against Candida spp. in vitro, but host toxicity has limited their use as antifungal drugs. The conservation of Hsp90 across all species leads to selectivity issues. To assess the potential of Hsp90 as a druggable antifungal target, the activity of nine structurally unrelated Hsp90 inhibitors with different binding domains was evaluated against a panel of Candida clinical isolates. The Hsp90 sequences from human and yeast species were aligned. Despite the degree of similarity between human and yeast N-terminal domain residues, the in vitro activities measured for the inhibitors interacting with this domain were not reproducible against all Candida species. Moreover, the inhibitors binding to the C-terminal domain (CTD) did not show any antifungal activity, with the exception of one of them. Given the greater sequence divergence in this domain, the identification of selective CTD inhibitors of fungal Hsp90 could be a promising strategy for the development of innovative antifungal drugs.

Mechanistic Basis for Red Light Switching of Azonium Ions.

Azonium ions formed by the protonation of tetra-ortho-methoxy-substituted aminoazobenzenes photoisomerize with red light under physiological conditions. This property makes them attractive as molecular tools for the photocontrol of physiological processes, for example, in photopharmacology. However, a mechanistic understanding of the photoisomerization process and subsequent thermal relaxation is necessary for the rational application of these compounds as well as for guiding the design of derivatives with improved properties. Using a combination of sub-ps/ns transient absorption measurements and quantum chemical calculations, we show that the absorption of a photon by the protonated E-H+ form of the photoswitch causes rapid (ps) isomerization to the protonated Z-H+ form, which can also absorb red light. Proton transfer to solvent then occurs on a microsecond time scale, leading to an equilibrium between Z and Z-H+ species, the position of which depends on the solution pH. Whereas thermal isomerization of the neutral Z form to the neutral E form is slow (∼0.001 s-1), thermal isomerization of Z-H+ to E-H+ is rapid (∼100 s-1), so the solution pH also governs the rate at which E/E-H+ concentrations are restored after a light pulse. This analysis provides the first complete mechanistic picture that explains the observed intricate photoswitching behavior of azonium ions at a range of pH values. It further suggests features of azonium ions that could be targeted for improvement to enhance the applicability of these compounds for the photocontrol of biomolecules.

Interplay between Dual-State and Aggregation-Induced Emission with ESIPT Scaffolds Containing Triphenylamine Substituents: Experimental and Theoretical Studies.

We detail the synthesis of a series of fluorophores containing triphenylamine derivatives along with their photophysical, electrochemical, and electronic structure properties. These compounds include molecular structures derived from imino-phenol (anil) and hydroxybenzoxazole scaffolds originating from similar salicylaldehyde derivatives and display excited-state intramolecular proton transfer. We show that depending on the nature of the π-conjugated scaffold, different photophysical processes are observed: aggregation-induced emission or dual-state emission, with a modulation of the fluorescence color and redox properties. The photophysical properties are further rationalized with the help of ab initio calculations.

Synthesis and biological evaluation of DIDS analogues as efficient inhibitors of RAD51 involved in homologous recombination.

RAD51 is a pivotal protein of the homologous recombination DNA repair pathway, and is overexpressed in some cancer cells, disrupting then the efficiency of cancer-treatments. The development of RAD51 inhibitors appears as a promising solution to restore these cancer cells sensitization to radio- or chemotherapy. From a small molecule identified as a modulator of RAD51, the 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), two series of analogues with small or bulky substituents on the aromatic parts of the stilbene moiety were prepared for a structure-activity relationship study. Three compounds, the cyano analogue (12), and benzamide (23) or phenylcarbamate (29) analogues of DIDS were characterized as novel potent RAD51 inhibitors with HR inhibition in the micromolar range.

Identification of sulfonamide compounds active on the insect nervous system: Molecular modeling, synthesis and biological evaluation.

Insect nicotinic acetylcholine receptors (nAChRs) are a recognized target for insecticide design. In this work, we have identified, from a structure-based approach using molecular modeling tools, ligands with potential selective activity for pests versus pollinators. A high-throughput virtual screening with the Openeye software was performed using a library from the ZINC database, thiacloprid being used as the target structure. The top sixteen molecules were then docked in α6 cockroach and honeybee homomeric nAChRs to check from a theoretical point of view relevant descriptors in favor of pest selectivity. Among the selected molecules, one original sulfonamide compound has afterward been synthesized, together with various analogs. Two compounds of this family have been shown to behave as activators of the cockroach cholinergic synaptic transmission.

Synthesis and Photophysical Characterizations of Pyrroloquinolone Photosensitizers for Singlet Oxygen Production†.

A series of pyrroloquinolone photosensitizers bearing different halogen substituents (Cl, Br, I) on the heterocyclic framework was studied. These structures were readily prepared through a multi-step synthetic sequence involving an oxidative protocol as an important step to access the quinolone framework. Spectroscopic characterizations and computational investigations were carried out to study the dyes before and after the oxidative step. Interestingly, the fluorescence emission was significantly reduced upon oxidation. In spite of a low photostability under UV light, the pyrroloquinolone photosensitizers proved effective to produce singlet oxygen. Higher singlet oxygen quantum yields were obtained with photosensitizers bearing halogen atoms with a higher atomic number.

Excited-State Intramolecular Proton Transfer Dyes with Dual-State Emission Properties: Concept, Examples and Applications.

Dual-state emissive (DSE) fluorophores are organic dyes displaying fluorescence emission both in dilute and concentrated solution and in the solid-state, as amorphous, single crystal, polycrystalline samples or thin films. This comes in contrast to the vast majority of organic fluorescent dyes which typically show intense fluorescence in solution but are quenched in concentrated media and in the solid-state owing to π-stacking interactions; a well-known phenomenon called aggregation-caused quenching (ACQ). On the contrary, molecular rotors with a significant number of free rotations have been engineered to show quenched emission in solution but strong fluorescence in the aggregated-state thanks to restriction of the intramolecular motions. This is the concept of aggregation-induced emission (AIE). DSE fluorophores have been far less explored despite the fact that they are at the crossroad of ACQ and AIE phenomena and allow targeting applications both in solution (bio-conjugation, sensing, imaging) and solid-state (organic electronics, data encryption, lasing, luminescent displays). Excited-State Intramolecular Proton Transfer (ESIPT) fluorescence is particularly suitable to engineer DSE dyes. Indeed, ESIPT fluorescence, which relies on a phototautomerism between normal and tautomeric species, is characterized by a strong emission in the solid-state along with a large Stokes' shift, an enhanced photostability and a strong sensitivity to the close environment, a feature prone to be used in bio-sensing. A drawback that needs to be overcome is their weak emission intensity in solution, owing to detrimental molecular motions in the excited-state. Several strategies have been proposed in that regard. In the past few years, a growing number of examples of DSE-ESIPT dyes have indeed emerged in the literature, enriching the database of such attractive dyes. This review aims at a brief but concise overview on the exploitation of ESIPT luminescence for the optimization of DSE dyes properties. In that perspective, a synergistic approach between organic synthesis, fluorescence spectroscopy and ab initio calculations has proven to be an efficient tool for the construction and optimization of DSE-ESIPT fluorophores.

Structural insights into the catalytic mechanism of granzyme B upon substrate and inhibitor binding.

Human granzyme B (hGzmB), which is present in various immune cells, has attracted much attention due to its role in various pathophysiological conditions. The hGzmB activity is triggered at a catalytic triad (His59, Asp103, Ser198), cleaving its specific substrates. To date, the drug design strategy against hGzmB mainly targets the catalytic triad, which causes the non-specificity problem of inhibitors due to the highly conserved active site in serine proteases. In the present work, microsecond classical molecular dynamics simulations are devoted to exploring the structural dynamics of the hGzmB catalytic cycle in the presence of Ac-IEPD-AMC, a known substrate (active hGzmB), and Ac-IEPD-CHO, a known inhibitor (inactive hGzmB). By comparing active and inactive forms of hGzmB in the six different stages of the hGzmB catalytic cycle, we revealed, for the very first time, an additional network of interactions involving Arg216, a residue located outside the conventional binding site. Upon activation, the His59∙∙∙Asp103 hydrogen bond is broken due to the formation of the Asp103∙∙∙Arg216 salt bridge, expanding the active site to facilitate the substrate-binding. On the contrary, the binding of inhibitor Ac-IEPD-CHO to hGzmB prevents the Arg216-mediated interactions within the catalytic triad, thus preventing hGzmB activity. In silico Arg216Ala mutation confirms the role of Arg216 in enzyme activity, as the substrate Ac-IEPD-AMC failed to bind to the mutated hGzmB. Importantly, as Arg216 is not conserved amongst the various granzymes, the current findings can be a major step to guide the design of hGzmB specific therapeutics.

Exploring structural dynamics and optical properties of UnaG fluorescent protein upon N57 mutations.

UnaG is a new class of fluorescence protein in which an endogenous ligand, namely bilirubin (BLR), plays the role of chromophore. Upon photoexcitation, holoUnaG emits green light. A single mutation at residue 57 induces a decrease in the fluorescence quantum yield. To our knowledge, no atomic simulation at the atomic level has been carried out to date to explain this fluorescence decay in N57A and N57Q mutants. Herein molecular dynamics simulations were carried out on wild-type (WT) UnaG and both mutants to investigate the structural impact of the mutation on its global structure, on BLR and the absorption spectra. Our study reveals significant global changes upon mutation at the protein entrance (L3, H2, and, H3) governing a BLR modification. BLR in WT UnaG is rather rigid while when embedded into N57A or N57Q, dihedral angles between endo and exo vinyl moieties and between A and B rings at the entrance of UnaG are strongly modified along with the number of inter-/intramolecular interactions. The water molecules play an important role in the modification of the shape of the binding cavity. For the first time, we show that the structural modifications upon ligand mutations are tightly related to the key structural changes in the protein such as Loop3 (L3), ß sheet 2 (B2), and ß sheet 3 (B3) dynamics. The present work suggests that the quenching of the fluorescence properties of UnaG mutants is mainly a non-radiative process closely related to the BLR flexibility induced by global structural changes.

Dual Solution-/Solid-State Emissive Excited-State Intramolecular Proton Transfer (ESIPT) Dyes: A Combined Experimental and Theoretical Approach.

Excited-state intramolecular proton transfer (ESIPT) dyes typically show strong solid-state emission, but faint fluorescence intensity is observed in the solution state owing to detrimental molecular motions. This article investigates the influence of direct (hetero)arylation on the optical properties of 2-(2'-hydroxyphenyl)benzoxazole ESIPT emitters. The synthesis of two series of ESIPT emitters bearing substituted neutral or charged aryl, thiophene, or pyridine rings is reported herein along with full photophysical studies in solution and solid states, demonstrating the dual solution-/solid-state emission behavior. Depending on the nature of substitution, several excited-state dynamics are observed: quantitative or partially frustrated ESIPT process or deprotonation of the excited species. Protonation studies revealed that pyridine substitution triggered a strong increase of quantum yield in the solution state for the protonated species owing to favorable quinoidal stabilization. These attractive features led to the development of a second series of dyes with alkyl or aryl pyridinium moieties showing strong tunable solution/solid fluorescence intensity. For each series, ab initio calculations helped rationalize and ascertain their behavior in the excited state and the nature of the emission observed by the experimental results.

Tailoring the optical and dynamic properties of iminothioindoxyl photoswitches through acidochromism.

Multi-responsive functional molecules are key for obtaining user-defined control of the properties and functions of chemical and biological systems. In this respect, pH-responsive photochromes, whose switching can be directed with light and acid-base equilibria, have emerged as highly attractive molecular units. The challenge in their design comes from the need to accommodate application-defined boundary conditions for both light- and protonation-responsivity. Here we combine time-resolved spectroscopic studies, on time scales ranging from femtoseconds to seconds, with density functional theory (DFT) calculations to elucidate and apply the acidochromism of a recently designed iminothioindoxyl (ITI) photoswitch. We show that protonation of the thermally stable Z isomer leads to a strong batochromically-shifted absorption band, allowing for fast isomerization to the metastable E isomer with light in the 500-600 nm region. Theoretical studies of the reaction mechanism reveal the crucial role of the acid-base equilibrium which controls the populations of the protonated and neutral forms of the E isomer. Since the former is thermally stable, while the latter re-isomerizes on a millisecond time scale, we are able to modulate the half-life of ITIs over three orders of magnitude by shifting this equilibrium. Finally, stable bidirectional switching of protonated ITI with green and red light is demonstrated with a half-life in the range of tens of seconds. Altogether, we designed a new type of multi-responsive molecular switch in which protonation red-shifts the activation wavelength by over 100 nm and enables efficient tuning of the half-life in the millisecond-second range.

Structure-based identification of inhibitors disrupting the CD2-CD58 interactions.

The immune system has very intricate mechanisms of fighting against the invading infections which are accomplished by a sequential event of molecular interactions in the body. One of the crucial phenomena in this process is the recognition of T-cells by the antigen-presenting cells (APCs), which is initiated by the rapid interaction between both cell surface receptors, i.e., CD2 located on T-cells and CD58 located on APCs. Under various pathological conditions, which involve undesired immune response, inhibiting the CD2-CD58 interactions becomes a therapeutically relevant opportunity. Herein we present an extensive work to identify novel inhibiting agents of the CD2-CD58 interactions. Classical molecular dynamics (MD) simulations of the CD2-CD58 complex highlighted a series of crucial CD58 residues responsible for the interactions with CD2. Based on such results, a pharmacophore map, complementary to the CD2-binding site of CD58, was created and employed for virtual screening of ~ 300,000 available compounds. On the ~ 6000 compounds filtered from pharmacophore mapping, ADME screening leads to ~ 350 molecules. Molecular docking was then performed on these molecules, and fifteen compounds emerged with significant binding energy (< - 50 kcal/mol) for CD58. Finally, short MD simulations were performed in triplicate on each complex (i) to provide a microscopic view of the ligand binding and (ii) to rule out possibly weak binders of CD58 from the identified hits. At last, we suggest eight compounds for in vitro testing that were identified as promising hits to bind CD58 with a high binding affinity.

How To Make Nitroaromatic Compounds Glow: Next-Generation Large X-Shaped, Centrosymmetric Diketopyrrolopyrroles.

Red-emissive π-expanded diketopyrrolopyrroles (DPPs) with fluorescence reaching λ=750 nm can be easily synthesized by a three-step strategy involving the preparation of diketopyrrolopyrrole followed by N-arylation and subsequent intramolecular palladium-catalyzed direct arylation. Comprehensive spectroscopic assays combined with first-principles calculations corroborated that both N-arylated and fused DPPs reach a locally excited (S1 ) state after excitation, followed by internal conversion to states with solvent and structural relaxation, before eventually undergoing intersystem crossing. Only the structurally relaxed state is fluorescent, with lifetimes in the range of several nanoseconds and tens of picoseconds in nonpolar and polar solvents, respectively. The lifetimes correlate with the fluorescence quantum yields, which range from 6 % to 88 % in nonpolar solvents and from 0.4 % and 3.2 % in polar solvents. A very inefficient (T1 ) population is responsible for fluorescence quantum yields as high as 88 % for the fully fused DPP in polar solvents.

IL-15Rα membrane anchorage in either cis or trans is required for stabilization of IL-15 and optimal signaling.

Interleukin (IL)-15 plays an important role in the communication between immune cells. It delivers its signal through different modes involving three receptor chains: IL-15Rα, IL-2Rß and IL-2Rγc. The combination of the different chains result in the formation of IL-15Rα/IL-2Rß/γc trimeric or IL-2Rß/γc dimeric receptors. In this study, we have investigated the role of the IL-15Rα chain in stabilizing the cytokine in the IL-2Rß/γc dimeric receptor. By analyzing the key amino acid residues of IL-15 facing IL-2Rß, we provide evidence of differential interfaces in the presence or in the absence of membrane-anchored IL-15Rα. Moreover, we found that the anchorage of IL-15Rα to the cell surface regardless its mode of presentation - i.e. cis or trans - is crucial for complete signaling. These observations show how the cells can finely modulate the intensity of cytokine signaling through the quality and the level of expression of the receptor chains.

Investigation of Phospholipase Cγ1 Interaction with SLP76 Using Molecular Modeling Methods for Identifying Novel Inhibitors.

The enzyme phospholipase C gamma 1 (PLCγ1) has been identified as a potential drug target of interest for various pathological conditions such as immune disorders, systemic lupus erythematosus, and cancers. Targeting its SH3 domain has been recognized as an efficient pharmacological approach for drug discovery against PLCγ1. Therefore, for the first time, a combination of various biophysical methods has been employed to shed light on the atomistic interactions between PLCγ1 and its known binding partners. Indeed, molecular modeling of PLCγ1 with SLP76 peptide and with previously reported inhibitors (ritonavir, anethole, daunorubicin, diflunisal, and rosiglitazone) facilitated the identification of the common critical residues (Gln805, Arg806, Asp808, Glu809, Asp825, Gly827, and Trp828) as well as the quantification of their interaction through binding energies calculations. These features are in agreement with previous experimental data. Such an in depth biophysical analysis of each complex provides an opportunity to identify new inhibitors through pharmacophore mapping, molecular docking and MD simulations. From such a systematic procedure, a total of seven compounds emerged as promising inhibitors, all characterized by a strong binding with PLCγ1 and a comparable or higher binding affinity to ritonavir (∆Gbind < -25 kcal/mol), one of the most potent inhibitor reported till now.

Interactions of the Rad51 inhibitor DIDS with human and bovine serum albumins: Optical spectroscopy and isothermal calorimetry approaches.

Rad51 is a key protein in DNA repair by homologous recombination and an important target for development of drugs in cancer therapy. 4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) has been used in clinic during the past 30 years as an inhibitor of anion transporters and channels. Recently DIDS has been demonstrated to affect Rad51-mediated homologous pairing and strand exchange, key processes in homologous recombination. Consequently, DIDS has been considered as a potential revertant of radio- and chemo-resistance of cancer cells, the major causes of therapy failure. Here, we have investigated the behavior of DIDS towards serum albumins. The effects of environmental factors, primarily, solvent polarity, on DIDS stability were evaluated, and the mechanisms of interaction of DIDS with human or bovine serum albumin were analyzed using isothermal calorimetry, circular dichroism and fluorescence spectroscopies. DIDS interaction with both serum albumins have been demonstrated, and the interaction characteristics have been determined. By comparing these characteristics for several DIDS derivatives, we have identified the DIDS moiety essential for the interaction. Furthermore, site competition data indicate that human albumin has two DIDS-binding sites: a high-affinity site in the IIIA subdomain and a low-affinity one in the IB subdomain. Molecular docking has revealed the key molecular moieties of DIDS responsible for its interactions in each site and shown that the IB site can bind two ligands. These findings show that binding of DIDS to serum albumin may change the balance between the free and bound DIDS forms, thereby affecting its bioavailability and efficacy against Rad51.

Mechanistic and Structural Insights on the IL-15 System through Molecular Dynamics Simulations.

Interleukin 15 (IL-15), a four-helix bundle cytokine, is involved in a plethora of different cellular functions and, particularly, plays a key role in the development and activation of immune responses. IL-15 forms receptor complexes by binding with IL-2Rß- and common γ(γc)-signaling subunits, which are shared with other members of the cytokines family (IL-2 for IL-2Rß- and all other γc- cytokines for γc). The specificity of IL-15 is brought by the non-signaling α-subunit, IL-15Rα. Here we present the results of molecular dynamics simulations carried out on four relevant forms of IL-15: its monomer, IL-15 interacting individually with IL-15Rα (IL-15/IL-15Rα), with IL-2Rß/γc subunits (IL-15/IL-2Rß/γc) or with its three receptors simultaneously (IL-15/IL-15Rα/IL-2Rß/γc). Through the analyses of the various trajectories, new insights on the structural features of the interfaces are highlighted, according to the considered form. The comparison of the results with the experimental data, available from X-ray crystallography, allows, in particular, the rationalization of the importance of IL-15 key residues (e.g. Asp8, Lys10, Glu64). Furthermore, the pivotal role of water molecules in the stabilization of the various protein-protein interfaces and their H-bonds networks are underlined for each of the considered complexes.

Binding of Sulfoxaflor to Aplysia californica-AChBP: Computational Insights from Multiscale Approaches.

Structural features and binding properties of sulfoxaflor (SFX) with Ac-AChBP, the surrogate of the insect nAChR ligand binding domain (LBD), are reported herein using various complementary molecular modeling approaches (QM, molecular docking, molecular dynamics, and QM/QM'). The different SFX stereoisomers show distinct behaviors in terms of binding and interactions with Ac-AChBP. Molecular docking and Molecular Dynamics (MD) simulations highlight the specific intermolecular contacts involved in the binding of the different SFX isomers and the relative contribution of the SFX functional groups. QM/QM' calculations provide further insights and a significant refinement of the geometric and energetic contributions of the various residues leading to a preference for the SS and RR stereoisomers. Notable differences in terms of binding interactions are pointed out for the four stereoisomers. The results point out the induced fit of the Ac-AChBP binding site according to the SFX stereoisomer. In this process, the water molecules-mediated contacts play a key role, their energetic contribution being among the most important for the various stereoisomers. In all cases, the interaction with Trp147 is the major binding component, through CH···π and π···π interactions. This study provides a rationale for the binding of SFX to insect nAChR, in particular with respect to the new class of sulfoximine-based insect nAChR competitive modulators, and points out the requirements of various levels of theory for an accurate description of ligand-receptor interactions.