Nonperturbative Fluorogenic Labeling of Immunophilins Enables the Wash-free Detection of Immunosuppressants.

Immunosuppressants are clinically approved drugs to treat the potential rejection of transplanted organs and require frequent monitoring due to their narrow therapeutic window. Immunophilins are small proteins that bind immunosuppressants with high affinity, yet there are no examples of fluorogenic immunophilins and their potential application as optical biosensors for immunosuppressive drugs in clinical biosamples. In the present work, we designed novel diazonium BODIPY salts for the site-specific labeling of tyrosine residues in peptides via solid-phase synthesis as well as for late-stage functionalization of whole recombinant proteins. After the optimization of a straightforward one-step labeling procedure for immunophilins PPIA and FKBP12, we demonstrated the application of a fluorogenic analogue of FKBP12 for the selective detection of the immunosuppressant drug tacrolimus, including experiments in urine samples from patients with functioning renal transplants. This chemical methodology opens new avenues to rationally design wash-free immunophilin-based biosensors for rapid therapeutic drug monitoring.

The prohibitin-binding compound fluorizoline induces the pro-inflammatory cytokines interleukin-8 and interleukin-6 through the activation of JNK and p38 MAP kinases.

Fluorizoline is a prohibitin (PHB)-binding compound that induces apoptosis in several cancer cell lines as well as in primary cells from hematologic malignancies. In this study, we show that fluorizoline treatment triggers the activation of the stress-activated kinases c-Jun N-terminal kinase (JNK) and p38 prior to caspase activation in human cell lines. However, the blockage of p38 and JNK activity with chemical inhibitors or siRNA-mediated downregulation of MAPK14 (p38) does not prevent fluorizoline-induced apoptosis, suggesting that the activation of these kinases plays an alternative role in the cell response to fluorizoline treatment. Here, we describe that fluorizoline treatment leads to the secretion of pro-inflammatory cytokines interleukin-8 (IL-8) and interleukin-6 (IL-6). Importantly, we demonstrate that the activation of the stress-activated kinases JNK and p38 mediates the secretion of both IL-8 and IL-6. This study shows novel insights into the pro-inflammatory role exhibited by a compound that binds to PHB, thus supporting the potential of PHBs as anti-inflammatory proteins.

Charting the Chemical Reaction Space around a Multicomponent Combination: Controlled Access to a Diverse Set of Biologically Relevant Scaffolds.

Charting the chemical reaction space around the combination of carbonyls, amines, and isocyanoacetates allows the description of new multicomponent processes leading to a variety of unsaturated imidazolone scaffolds. The resulting compounds display the chromophore of the green fluorescent protein and the core of the natural product coelenterazine. Despite the competitive nature of the pathways involved, general protocols provide selective access to the desired chemotypes. Moreover, we describe unprecedented reactivity at the C-2 position of the imidazolone core to directly afford C, S, and N-derivatives featuring natural products (e.g. leucettamines), potent kinase inhibitors, and fluorescent probes with suitable optical and biological profiles.

The Prohibitin-Binding Compound Fluorizoline Activates the Integrated Stress Response through the eIF2α Kinase HRI.

Fluorizoline is a synthetic molecule that induces apoptosis, by selectively targeting prohibitins (PHBs), through induction of the BH3-only protein NOXA. This induction is transcriptionally regulated by the integrated stress response (ISR)-related transcription factors ATF3 and ATF4. Here, we evaluate the role of the four eIF2α kinases, to decipher which is responsible for the mechanism of ISR activation triggered by fluorizoline in HeLa and HAP1 cells. First, we demonstrated the involvement of the eIF2α kinases using ISR inhibitor (ISRIB) and by simultaneous downregulation of all four eIF2α kinases, as both approaches were able to increase cell resistance to fluorizoline-induced apoptosis. Furthermore, we confirmed that fluorizoline treatment results in endoplasmic reticulum (ER) stress, as evidenced by PERK activation. Despite PERK activation, this kinase was not directly involved in the ISR activation by fluorizoline. In this regard, we found that the eIF2α kinases are capable of compensating for each other's loss of function. Importantly, we demonstrated that the mitochondrial-stress-related eIF2α kinase HRI mediates ISR activation after fluorizoline treatment.

Efficient AntiMycolata Agents by Increasing the Lipophilicity of Known Antibiotics through Multicomponent Reactions.

New antibiotic agents were prepared using Povarov and Ugi multicomponent reactions upon the known drugs sulfadoxine and dapsone. The prepared derivatives, with increased lipophilicity, showed improved efficiency against Mycolata bacteria. Microbiological guidance for medicinal chemistry is a powerful tool to design new and effective antimicrobials. In this case, the readily synthesized compounds open new possibilities in the search for antimicrobials active on mycolic acid-containing bacteria.

Charting the Chemical Reaction Space around a Multicomponent Combination: Controlled Access to a Diverse Set of Biologically Relevant Scaffolds.

Charting the chemical reaction space around the combination of carbonyls, amines, and isocyanoacetates allows the description of new multicomponent processes leading to a variety of unsaturated imidazolone scaffolds. The resulting compounds display the chromophore of the green fluorescent protein and the core of the natural product coelenterazine. Despite the competitive nature of the pathways involved, general protocols provide selective access to the desired chemotypes. Moreover, we describe unprecedented reactivity at the C-2 position of the imidazolone core to directly afford C, S, and N-derivatives featuring natural products (e.g. leucettamines), potent kinase inhibitors, and fluorescent probes with suitable optical and biological profiles.

Heterocycle-Based Multicomponent Reactions in Drug Discovery: From Hit Finding to Rational Design.

In the context of the structural complexity necessary for a molecule to selectively display a therapeutical action and the requirements for suitable pharmacokinetics, a robust synthetic approach is essential. Typically, thousands of relatively similar compounds should be prepared along the drug discovery process. In this respect, heterocycle-based multicomponent reactions offer advantages over traditional stepwise sequences in terms of synthetic economy, as well as the fast access to chemsets to study the structure activity relationships, the fine tuning of properties, and the preparation of larger amounts for preclinical phases. In this account, we briefly summarize the scientific methodology backing the research line followed by the group. We comment on the main results, clustered according to the targets and, finally, in the conclusion section, we offer a general appraisal of the situation and some perspectives regarding future directions in academic and private research.

A Bivalent Activatable Fluorescent Probe for Screening and Intravital Imaging of Chemotherapy-Induced Cancer Cell Death.

The detection and quantification of apoptotic cells is a key process in cancer research, particularly during the screening of anticancer therapeutics and in mechanistic studies using preclinical models. Intravital optical imaging enables high-resolution visualisation of cellular events in live organisms; however, there are few fluorescent probes that can reliably provide functional readouts in situ without interference from tissue autofluorescence. We report the design and optimisation of the fluorogenic probe Apotracker Red for real-time detection of cancer cell death. The strong fluorogenic behaviour, high selectivity, and excellent stability of Apotracker Red make it a reliable optical reporter for the characterisation of the effects of anticancer drugs in cells in vitro and for direct imaging of chemotherapy-induced apoptosis in vivo in mouse models of breast cancer.

A Bivalent Activatable Fluorescent Probe for Screening and Intravital Imaging of Chemotherapy-Induced Cancer Cell Death.

The detection and quantification of apoptotic cells is a key process in cancer research, particularly during the screening of anticancer therapeutics and in mechanistic studies using preclinical models. Intravital optical imaging enables high-resolution visualisation of cellular events in live organisms; however, there are few fluorescent probes that can reliably provide functional readouts in situ without interference from tissue autofluorescence. We report the design and optimisation of the fluorogenic probe Apotracker Red for real-time detection of cancer cell death. The strong fluorogenic behaviour, high selectivity, and excellent stability of Apotracker Red make it a reliable optical reporter for the characterisation of the effects of anticancer drugs in cells in vitro and for direct imaging of chemotherapy-induced apoptosis in vivo in mouse models of breast cancer.

New Trimethoprim-Like Molecules: Bacteriological Evaluation and Insights into Their Action.

This work reports a detailed characterization of the antimicrobial profile of two trimethoprim-like molecules (compounds 1a and 1b) identified in previous studies. Both molecules displayed remarkable antimicrobial activity, particularly when combined with sulfamethoxazole. In disk diffusion assays on Petri dishes, compounds 1a and 1b showed synergistic effects with colistin. Specifically, in combinations with low concentrations of colistin, very large increases in the activities of compounds 1a and 1b were determined, as demonstrated by alterations in the kinetics of bacterial growth despite only slight changes in the fractional inhibitory concentration index. The effect of colistin may be to increase the rate of antibiotic entry while reducing efflux pump activity. Compounds 1a and 1b were susceptible to extrusion by efflux pumps, whereas the inhibitor phenylalanine arginyl ß-naphthylamide (PAßN) exerted effects similar to those of colistin. The interactions between the target enzyme (dihydrofolate reductase), the coenzyme nicotinamide adenine dinucleotide phosphate (NADPH), and the studied molecules were explored using enzymology tools and computational chemistry. A model based on docking results is reported.

Extended Multicomponent Reactions with Indole Aldehydes: Access to Unprecedented Polyheterocyclic Scaffolds, Ligands of the Aryl Hydrocarbon Receptor.

The participation of reactants undergoing a polarity inversion along a multicomponent reaction allows the continuation of the transformation with productive domino processes. Thus, indole aldehydes in Groebke-Blackburn-Bienaymé reactions lead to an initial adduct which spontaneously triggers a series of events leading to the discovery of novel reaction pathways together with direct access to a variety of linked, fused, and bridged polyheterocyclic scaffolds. Indole 3- and 4-carbaldehydes with suitable isocyanides and aminoazines afford fused adducts through oxidative Pictet-Spengler processes, whereas indole 2-carbaldehyde yields linked indolocarbazoles under mild conditions, and a bridged macrocycle at high temperature. These novel structures are potent activators of the human aryl hydrocarbon receptor signaling pathway.

Multicomponent Reactions Upon the Known Drug Trimethoprim as a Source of Novel Antimicrobial Agents.

Novel antibiotic compounds have been prepared through a selective multicomponent reaction upon the known drug Trimethoprim. The Groebke-Blackburn-Bienaymé reaction involving this α-aminoazine, with a range of aldehydes and isocyanides afforded the desired adducts in one-step. The analogs display meaningful structural features of the initial drug together with relevant modifications at several points, keeping antibiotic potency and showing satisfactory antimicrobial profile (good activity levels and reduced growth rates), especially against methicillin-resistant Staphylococcus aureus. The new products may open new possibilities to fight bacterial infections.

Selectivity in multiple multicomponent reactions: types and synthetic applications.

Multiple multicomponent reactions reach an unparalleled level of connectivity, leading to highly complex adducts. Usually, only one type of transformation involving the same set of reactants takes place. However, in some occasions this is not the case. Selectivity issues then arise, and different scenarios are analyzed. The structural pattern of the reactants, the reaction design and the experimental conditions are the critical factors dictating selectivity in these processes.

Fluorogenic Trp(redBODIPY) cyclopeptide targeting keratin 1 for imaging of aggressive carcinomas.

Keratin 1 (KRT1) is overexpressed in squamous carcinomas and associated with aggressive pathologies in breast cancer. Herein we report the design and preparation of the first Trp-based red fluorogenic amino acid, which is synthetically accessible in a few steps and displays excellent photophysical properties, and its application in a minimally-disruptive labelling strategy to prepare a new fluorogenic cyclopeptide for imaging of KRT1+ cells in whole intact tumour tissues.

Recent advances of the Povarov reaction in medicinal chemistry.

The Povarov multicomponent reaction consists on the condensation of an aniline, an aldehyde, and an activated olefin to generate a tetrahydroquinoline adduct with 3 diversity points. Hereby, we report the main features of this transformation and its uses in medicinal chemistry. Relevant examples of the impact of Povarov adducts in different therapeutic areas are provided.

Multiple Multicomponent Reactions: Unexplored Substrates, Selective Processes, and Versatile Chemotypes in Biomedicine.

Multiple multicomponent reactions rapidly assemble complex structures. Despite being very productive, the lack of selectivity and the reduced number of viable transformations restrict their general application in synthesis. Hereby, we describe a rationale for a selective version of these processes based in the preferential generation of intermediates which are less reactive than the initial substrates. In this way, applying the Groebke-Blackburn-Bienaymé reaction on a range of α-polyamino-polyazines, we prepared a family compact heterocyclic scaffolds with relevant applications in medicinal and biological chemistry (live cell imaging probes, selective binders for DNA quadruplexes, and antiviral agents against human adenoviruses). The approach has general character and yields complex molecular targets in a selective, tunable and direct manner.

Tetrasubstituted Imidazolium Salts as Potent Antiparasitic Agents against African and American Trypanosomiases.

Imidazolium salts are privileged compounds in organic chemistry, and have valuable biological properties. Recent studies show that symmetric imidazolium salts with bulky moieties can display antiparasitic activity against T. cruzi. After developing a facile methodology for the synthesis of tetrasubstituted imidazolium salts from propargylamines and isocyanides, we screened a small library of these adducts against the causative agents of African and American trypanosomiases. These compounds display nanomolar activity against T. brucei and low (or sub) micromolar activity against T. cruzi, with excellent selectivity indexes and favorable molecular properties, thereby emerging as promising hits for the treatment of Chagas disease and sleeping sickness.

Insertion of Isocyanides into N-Si Bonds: Multicomponent Reactions with Azines Leading to Potent Antiparasitic Compounds.

Trimethylsilyl chloride is an efficient activating agent for azines in isocyanide-based reactions, which then proceed through a key insertion of the isocyanide into a N-Si bond. The reaction is initiated by N activation of the azine, followed by nucleophilic attack of an isocyanide in a Reissert-type process. Finally, a second equivalent of the same or a different isocyanide inserts into the N-Si bond leading to the final adduct. The use of distinct nucleophiles leads to a variety of α-substituted dihydroazines after a selective cascade process. Based on computational studies, a mechanistic hypothesis for the course of these reactions was proposed. The resulting products exhibit significant activity against Trypanosoma brucei and T. cruzi, featuring favorable drug-like properties and safety profiles.

Studies on the interaction of isocyanides with imines: reaction scope and mechanistic variations.

The interaction of imines with isocyanides has been studied. The main product results from a sequential process involving the attack of two units of isocyanide, under Lewis acid catalysis, upon the carbon-nitrogen double bond of the imine to form the 4-membered ring system. The scope of the reaction regarding the imine and isocyanide ranges has been determined, and also some mechanistic variations and structural features have been described.