RESUMO
Noncanonical nucleic acid structures, particularly G-quadruplexes, have garnered significant attention as potential therapeutic targets in cancer treatment. Here, the recognition of G-quadruplex DNA by peptides derived from the Rap1 protein is explored, with the aim of developing novel peptide-based G-quadruplex ligands with enhanced selectivity and anticancer activity. Biophysical techniques were employed to assess the interaction of a peptide derived from the G-quadruplex-binding domain of the protein with various biologically relevant G-quadruplex structures. Through alanine scanning mutagenesis, key amino acids crucial for G-quadruplex recognition were identified, leading to the discovery of two peptides with improved G-quadruplex-binding properties. However, despite their in vitro efficacy, these peptides showed limited cell penetration and anticancer activity. To overcome this challenge, cell-penetrating peptide (CPP)-conjugated derivatives were designed, some of which exhibited significant cytotoxic effects on cancer cells. Interestingly, selected CPP-conjugated peptides exerted potent anticancer activity across various tumour types via a G-quadruplex-dependent mechanism. These findings underscore the potential of peptide-based G-quadruplex ligands in cancer therapy and pave the way for the development of novel therapeutic strategies targeting these DNA structures.
Assuntos
Antineoplásicos , Peptídeos Penetradores de Células , Quadruplex G , Quadruplex G/efeitos dos fármacos , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/química , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacologia , Linhagem Celular Tumoral , Peptídeos/química , Peptídeos/farmacologia , Ligantes , DNA/química , DNA/metabolismo , Complexo Shelterina/metabolismo , Complexo Shelterina/química , Ligação ProteicaRESUMO
A novel class of compounds designed to hit two anti-tumour targets, G-quadruplex structures and human carbonic anhydrases (hCAs) IX and XII is proposed. The induction/stabilisation of G-quadruplex structures by small molecules has emerged as an anticancer strategy, disrupting telomere maintenance and reducing oncogene expression. hCAs IX and XII are well-established anti-tumour targets, upregulated in many hypoxic tumours and contributing to metastasis. The ligands reported feature a berberine G-quadruplex stabiliser scaffold connected to a moiety inhibiting hCAs IX and XII. In vitro experiments showed that our compounds selectively stabilise G-quadruplex structures and inhibit hCAs IX and XII. The crystal structure of a telomeric G-quadruplex in complex with one of these ligands was obtained, shedding light on the ligand/target interaction mode. The most promising ligands showed significant cytotoxicity against CA IX-positive HeLa cancer cells in hypoxia, and the ability to stabilise G-quadruplexes within tumour cells.
Assuntos
Antineoplásicos , Anidrase Carbônica IX , Inibidores da Anidrase Carbônica , Anidrases Carbônicas , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Quadruplex G , Humanos , Quadruplex G/efeitos dos fármacos , Inibidores da Anidrase Carbônica/farmacologia , Inibidores da Anidrase Carbônica/química , Inibidores da Anidrase Carbônica/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Relação Estrutura-Atividade , Estrutura Molecular , Anidrase Carbônica IX/antagonistas & inibidores , Anidrase Carbônica IX/metabolismo , Anidrases Carbônicas/metabolismo , Proliferação de Células/efeitos dos fármacos , Ligantes , Células HeLa , Antígenos de Neoplasias/metabolismo , Modelos MolecularesRESUMO
Immunotherapy has emerged as a game-changing approach for cancer treatment. Although monoclonal antibodies (mAbs) targeting the programmed cell death protein 1/programmed cell death protein 1 ligand 1 (PD-1/PD-L1) axis have entered the market revolutionizing the treatment landscape of many cancer types, small molecules, although presenting several advantages including the possibility of oral administration and/or reduced costs, struggled to enter in clinical trials, suffering of water insolubility and/or inadequate potency compared with mAbs. Thus, the search for novel scaffolds for both the design of effective small molecules and possible synergistic strategies is an ongoing field of interest. In an attempt to find novel chemotypes, a virtual screening approach was employed, resulting in the identification of new chemical entities with a certain binding capability, the most versatile of which was the benzimidazole-containing compound 10. Through rational design, a small library of its derivatives was synthesized and evaluated. The homogeneous time-resolved fluorescence (HTRF) assay revealed that compound 17 shows the most potent inhibitory activity (IC50 ) in the submicromolar range and notably, differently from the major part of PD-L1 inhibitors, exhibits satisfactory water solubility properties. These findings highlight the potential of benzimidazole-based compounds as novel promising candidates for PD-L1 inhibition.
Assuntos
Compostos de Bifenilo , Inibidores de Checkpoint Imunológico , Receptor de Morte Celular Programada 1 , Antígeno B7-H1 , Ligantes , Relação Estrutura-Atividade , Benzimidazóis/farmacologia , ÁguaRESUMO
The aim of this Special Issue is to highlight significant and new aspects concerning the chemistry and biology of noncanonical nucleic acid structures, with emphasis on their structure, stability, and conformational equilibria, as well as on the biological relevance of their interactions with proteins and ligands [...].
Assuntos
Conformação de Ácido Nucleico , Ácidos Nucleicos , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , Humanos , Ligantes , RNA/química , RNA/metabolismoRESUMO
OBJECTIVES: Interleukin (IL) 17s cytokines are key drivers of inflammation that are functionally dysregulated in several human immune-mediated inflammatory diseases (IMIDs), such as rheumatoid arthritis (RA), psoriasis and inflammatory bowel disease (IBD). Targeting these cytokines has some therapeutic benefits, but issues associated with low therapeutic efficacy and immunogenicity for subgroups of patients or IMIDs reduce their clinical use. Therefore, there is an urgent need to improve the coverage and efficacy of antibodies targeting IL-17A and/or IL-17F and IL-17A/F heterodimer. METHODS AND RESULTS: Here, we initially identified a bioactive 20 amino acid IL-17A/F-derived peptide (nIL-17) that mimics the pro-inflammatory actions of the full-length proteins. Subsequently, we generated a novel anti-IL-17 neutralising monoclonal antibody (Ab-IPL-IL-17) capable of effectively reversing the pro-inflammatory, pro-migratory actions of both nIL-17 and IL-17A/F. Importantly, we demonstrated that Ab-IPL-IL-17 has less off-target effects than the current gold-standard biologic, secukinumab. Finally, we compared the therapeutic efficacy of Ab-IPL-IL-17 with reference anti-IL-17 antibodies in preclinical murine models and samples from patients with RA and IBD. We found that Ab-IPL-IL-17 could effectively reduce clinical signs of arthritis and neutralise elevated IL-17 levels in IBD patient serum. CONCLUSIONS: Collectively, our preclinical and in vitro clinical evidence indicates high efficacy and therapeutic potency of Ab-IPL-IL-17, supporting the rationale for large-scale clinical evaluation of Ab-IPL-IL-17 in patients with IMIDs.
Assuntos
Artrite Reumatoide , Produtos Biológicos , Doenças Inflamatórias Intestinais , Humanos , Camundongos , Animais , Interleucina-17 , Agentes de Imunomodulação , Citocinas , Doenças Inflamatórias Intestinais/tratamento farmacológico , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêuticoRESUMO
The recent disclosure of the ability of aromatic isocyanides to harvest visible light and act as single electron acceptors when reacting with tertiary aromatic amines has triggered a renewed interest in their application to the development of green photoredox catalytic methodologies. Accordingly, the present work explores their ability to promote the generation of both alkyl and acyl radicals starting from radical precursors such as Hantzsch esters, potassium alkyltrifluoroborates, and α-oxoacids. Mechanistic studies involving UV-visible absorption and fluorescence experiments, electrochemical measurements of the ground-state redox potentials along with computational calculations of both the ground- and the excited-state redox potentials of a set of nine different aromatic isocyanides provide key insights to promote a rationale design of a new generation of isocyanide-based organic photoredox catalysts. Importantly, the green potential of the investigated chemistry is demonstrated by a direct and easy access to deuterium labeled compounds.
RESUMO
Nowadays, RNA is an attractive target for the design of new small molecules with different pharmacological activities. Among several RNA molecules, long noncoding RNAs (lncRNAs) are extensively reported to be involved in cancer pathogenesis. In particular, the overexpression of lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays an important role in the development of multiple myeloma (MM). Starting from the crystallographic structure of the triple-helical stability element at the 3'-end of MALAT1, we performed a structure-based virtual screening of a large commercial database, previously filtered according to the drug-like properties. After a thermodynamic analysis, we selected five compounds for the in vitro assays. Compound M5, characterized by a diazaindene scaffold, emerged as the most promising molecule enabling the destabilization of the MALAT1 triplex structure and antiproliferative activity on in vitro models of MM. M5 is proposed as a lead compound to be further optimized for improving its affinity toward MALAT1.
Assuntos
RNA Longo não Codificante , RNA Longo não Codificante/genética , RNA Longo não Codificante/química , Relação Estrutura-AtividadeRESUMO
The development of electrochemical strips, as extremely powerful diagnostic tools, has received much attention in the field of sensor analysis and, in particular, the detection of nucleic acids in complex matrixes is a hot topic in the electroanalytical area, especially when directed toward the development of emerging technologies, for the purpose of facilitating personal healthcare. One of the major diseases for which early diagnosis is crucial is represented by Alzheimer's disease (AD). AD is a progressive neurodegenerative disease, and it is the most common cause of dementia worldwide. In this context microRNAs (miRNAs), which are small noncoding RNAs, have recently been highlighted for their promising role as biomarkers for early diagnosis. In particular, miRNA-29 represents a class of miRNAs known to regulate pathogenesis of AD. In this work we developed an electrochemical printed strip for the detection of miRNA-29a at low levels. The architecture was characterized by the presence of gold nanoparticles (AuNPs) and an anti-miRNA-29a probe labeled with a redox mediator. The novel analytical tool has been characterized with microscale thermophoresis and electrochemical methods, and it has been optimized by selection of the most appropriate probe density to detect low target concentration. The present tool was capable to detect miRNA-29a both in standard solution and in serum, respectively, down to 0.15 and 0.2 nM. The platform highlighted good repeatability (calculated as the relative standard deviation) of ca. 10% and satisfactory selectivity in the presence of interfering species. This work has the objective to open a way for the study and possible early diagnosis of a physically and socially devastating disease such as Alzheimer's. The results demonstrate the suitability of this approach in terms of ease of use, time of production, sensitivity, and applicability.
Assuntos
Doença de Alzheimer , Técnicas Biossensoriais , Nanopartículas Metálicas , MicroRNAs , Doenças Neurodegenerativas , Humanos , Ouro/química , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/genética , Nanopartículas Metálicas/química , Biomarcadores , MicroRNAs/análise , Técnicas Biossensoriais/métodosRESUMO
The promoter regions of important oncogenes such as BCL2 and KRAS contain GC-rich sequences that can form distinctive noncanonical DNA structures involved in the regulation of transcription: G-quadruplexes on the G-rich strand and i-motifs on the C-rich strand. Interestingly, BCL2 and KRAS promoter i-motifs are highly dynamic in nature and exist in a pH-dependent equilibrium with hairpin and even with hybrid i-motif/hairpin species. Herein, the effects of pH and presence of cell-mimicking molecular crowding conditions on conformational equilibria of the BCL2 and KRAS i-motif-forming sequences were investigated by ultraviolet resonance Raman (UVRR) and circular dichroism (CD) spectroscopies. Multivariate analysis of CD data was essential to model the presence and identity of the species involved. Analysis of UVRR spectra measured as a function of pH, performed also by the two-dimensional correlation spectroscopy (2D-COS) technique, showed the role of several functional groups in the DNA conformational transitions, and provided structural and dynamic information. Thus, the UVRR investigation of intramolecular interactions and of local and environmental dynamics in promoting the different species induced by the solution conditions provided valuable insights into i-motif conformational transitions. The combined use of the two spectroscopic tools is emphasized by the relevant possibility of working in the same DNA concentration range and by the heterospectral UVRR/CD 2D-COS analysis. The results of this study shed light on the factors that can influence at the molecular level the equilibrium between the different conformational species putatively involved in the oncogene expression.
Assuntos
Quadruplex G , Dicroísmo Circular , DNA/química , Conformação de Ácido Nucleico , Análise Espectral RamanRESUMO
Two analogues of the MS3 aptamer, which was previously shown to have an exquisite capability to selectively bind and modulate the activity of mutant huntingtin (mHTT), have been here designed and evaluated in their physicochemical and biological properties. Featured by a distinctive propensity to form complex G-quadruplex structures, including large multimeric aggregates, the original 36-mer MS3 has been truncated to give a 33-mer (here named MS3-33) and a 17-mer (here named MS3-17). A combined use of different techniques (UV, CD, DSC, gel electrophoresis) allowed a detailed physicochemical characterization of these novel G-quadruplex-forming aptamers, tested in vitro on SH-SY5Y cells and in vivo on a Drosophila Huntington's disease model, in which these shorter MS3-derived oligonucleotides proved to have improved bioactivity in comparison with the parent aptamer.
Assuntos
Aptâmeros de Nucleotídeos , Quadruplex G , Doença de Huntington , Neuroblastoma , Humanos , Aptâmeros de Nucleotídeos/farmacologia , Aptâmeros de Nucleotídeos/química , Proteína Huntingtina/genéticaRESUMO
A set of guanine-rich aptamers able to preferentially recognize full-length huntingtin with an expanded polyglutamine tract has been recently identified, showing high efficacy in modulating the functions of the mutated protein in a variety of cell experiments. We here report a detailed biophysical characterization of the best aptamer in the series, named MS3, proved to adopt a stable, parallel G-quadruplex structure and show high nuclease resistance in serum. Confocal microscopy experiments on HeLa and SH-SY5Y cells, as models of non-neuronal and neuronal cells, respectively, showed a rapid, dose-dependent uptake of fluorescein-labelled MS3, demonstrating its effective internalization, even in the absence of transfecting agents, with no general cytotoxicity. Then, using a well-established Drosophila melanogaster model for Huntington's disease, which expresses the mutated form of human huntingtin, a significant improvement in the motor neuronal function in flies fed with MS3 was observed, proving the in vivo efficacy of this aptamer.
Assuntos
Doença de Huntington , Animais , Modelos Animais de Doenças , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Doença de Huntington/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismoRESUMO
The possibility to harness aromatic isocyanides as visible-light photocatalysts in the α-amino C(sp3)-H functionalization is herein presented. Actually, the three-component cross-dehydrogenative coupling of aromatic tertiary amines with isocyanides and water leads to amide products under very mild conditions in high yields and with a good substrate scope. While the reaction with aromatic isocyanides proceeds upon direct photoexcitation, aliphatic isocyanides are able to form a photoactive electron-donor-acceptor complex with aromatic amines. Moreover, the use of a catalytic loading of an aromatic isocyanide promotes the oxidative coupling of N-phenyl-1,2,3,4-tetrahydroisoquinoline with an array of different (pro)nucleophiles in good to excellent yields, thus providing the proof-of-concept for the development of a new highly tunable class of organic visible-light photocatalysts.
RESUMO
Under slightly acidic conditions, cytosine-rich DNA sequences can form non-canonical secondary structures called i-motifs, which occur as four stretches of cytosine repeats form hemi-protonated C·C+ base pairs. The growing interest in the i-motif structures as important components in functional DNA-based nanotechnology or as potential targets of anticancer drugs, increases the need for a deep understanding of the energetics of their structural transitions. Here, a combination of spectroscopic and calorimetric techniques is used to unravel the thermodynamics of folding of an i-motif DNA under favorable conditions. The results give new insights into the energetic aspects of i-motifs and show that thermodynamic and thermal stability are related but not identical properties of such DNA structures.
Assuntos
DNA/química , Motivos de Nucleotídeos , Pareamento de Bases , Citosina/química , Concentração de Íons de Hidrogênio , Análise de Componente Principal , Relação Estrutura-Atividade , TermodinâmicaRESUMO
Transcription factors (TFs) have a remarkable role in the homeostasis of the organisms and there is a growing interest in how they recognize and interact with specific DNA sequences. TFs recognize DNA using a variety of structural motifs. Among those, the ribbon-helix-helix (RHH) proteins, exemplified by the MetJ and ARC repressors, form dimers that insert antiparallel ß-sheets into the major groove of DNA. A great chemical challenge consists of using the principles of DNA recognition by TFs to design minimized peptides that maintain the DNA affinity and specificity characteristics of the natural counterparts. In this context, a peptide mimic of an antiparallel ß-sheet is very attractive since it can be obtained by a single peptide chain folding in a ß-hairpin structure and can be as short as 14 amino acids or less. Herein, we designed eight linear and two cyclic dodeca-peptides endowed with ß-hairpins. Their DNA binding properties have been investigated using fluorescence spectroscopy together with the conformational analysis through circular dichroism and solution NMR. We found that one of our peptides, peptide 6, is able to bind DNA, albeit without sequence selectivity. Notably, it shows a topological selectivity for the major groove of the DNA which is the interaction site of ARC and many other DNA-binding proteins. Moreover, we found that a type I' ß-hairpin folding pattern is a favorite peptide structure for interaction with the B-DNA major groove. Peptide 6 is a valuable lead compound for the development of novel analogs with sequence selectivity.
Assuntos
DNA de Forma B/química , Peptídeos/química , Fatores de Transcrição/química , Estrutura Molecular , Peptídeos/síntese químicaRESUMO
HMGB1 is a ubiquitous non-histone protein, which biological effects depend on its expression and subcellular location. Inside the nucleus, HMGB1 is engaged in many DNA events such as DNA repair, transcription and telomere maintenance. HMGB1 has been reported to bind preferentially to bent DNA as well as to noncanonical DNA structures like 4-way junctions and, more recently, to G-quadruplexes. These are four-stranded conformations of nucleic acids involved in important cellular processes, including telomere maintenance. In this frame, G-quadruplex recognition by specific proteins represents a key event to modulate physiological or pathological pathways. Herein, to get insights into the telomeric G-quadruplex DNA recognition by HMGB1, we performed detailed biophysical studies complemented with biological analyses. The obtained results provided information about the molecular determinants for the interaction and showed that the structural variability of human telomeric G-quadruplex DNA may have significant implications in HMGB1 recognition. The biological data identified HMGB1 as a telomere-associated protein in both telomerase-positive and -negative tumor cells and showed that HMGB1 gene silencing in such cells induces telomere DNA damage foci. Altogether, these findings provide a deeper understanding of telomeric G-quadruplex recognition by HMGB1 and suggest that this protein could actually represent a new target for cancer therapy.
Assuntos
Quadruplex G , Proteína HMGB1/genética , Conformação de Ácido Nucleico , Telômero/genética , DNA/química , DNA/genética , Escherichia coli/genética , Proteína HMGB1/química , Humanos , Telomerase/química , Telomerase/genética , Telômero/químicaRESUMO
DNA G-quadruplex (G4) structures, either within gene promoter sequences or at telomeres, have been extensively investigated as potential small-molecule therapeutic targets. However, although G4s forming at the telomeric DNA have been extensively investigated as anticancer targets, few studies focus on the telomeric repeat-containing RNA (TERRA), transcribed from telomeres, as potential pharmacological targets. Here, a virtual screening approach to identify a library of drug-like putative TERRA G4 binders, in tandem with circular dichroism melting assay to study their TERRA G4-stabilizing properties, led to the identification of a new hit compound. The affinity of this compound for TERRA RNA and some DNA G4s was analyzed through several biophysical techniques and its biological activity investigated in terms of antiproliferative effect, DNA damage response (DDR) activation, and TERRA RNA expression in high vs. low TERRA-expressing human cancer cells. The selected hit showed good affinity for TERRA G4 and no binding to double-stranded DNA. In addition, biological assays showed that this compound is endowed with a preferential cytotoxic effect on high TERRA-expressing cells, where it induces a DDR at telomeres, probably by displacing TERRA from telomeres. Our studies demonstrate that the identification of TERRA G4-targeting drugs with potential pharmacological effects is achievable, shedding light on new perspectives aimed at discovering new anticancer agents targeting these G4 structures.
Assuntos
RNA/genética , Telômero/genética , Antineoplásicos/farmacologia , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , DNA/genética , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Quadruplex G/efeitos dos fármacos , Humanos , Ligantes , Neoplasias/tratamento farmacológico , Neoplasias/genética , Relação Estrutura-Atividade , Telômero/efeitos dos fármacosRESUMO
Besides the well-known double-helical conformation, DNA is capable of folding into various noncanonical arrangements, such as G-quadruplexes (G4s) and i-motifs (iMs), whose occurrence in gene promoters, replication origins, and telomeres highlights the breadth of biological processes that they might regulate. Particularly, previous studies have reported that G4 and iM structures may play different roles in controlling gene transcription. Anyway, molecular tools able to simultaneously stabilize/destabilize those structures are still needed to shed light on what happens at the biological level. Herein, a multicomponent reaction and a click chemistry functionalization were combined to generate a set of 31 bis-triazolyl-pyridine derivatives which were initially screened by circular dichroism for their ability to interact with different G4 and/or iM DNAs and to affect the thermal stability of these structures. All the compounds were then clustered through multivariate data analysis, based on such capability. The most promising compounds were subjected to a further biophysical and biological characterization, leading to the identification of two molecules simultaneously able to stabilize G4s and destabilize iMs, both in vitro and in living cells.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Compostos Azo/química , DNA/metabolismo , Quadruplex G , Osteossarcoma/tratamento farmacológico , Piridinas/química , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/patologia , DNA/química , Humanos , Osteossarcoma/patologia , Células Tumorais CultivadasRESUMO
The i-motif DNA, also known as i-DNA, is a non-canonical DNA secondary structure formed by cytosine-rich sequences, consisting of two intercalated parallel-stranded duplexes held together by hemi-protonated cytosine-cytosine+ (C:C+ ) base pairs. The growing interest in the i-DNA structure as a target in anticancer therapy increases the need for tools for a rapid and meaningful interpretation of the spectroscopic data of i-DNA samples. Herein, we analyzed the circular dichroism (CD) and thermal difference UV-absorbance spectra (TDS) of 255 DNA sequences by means of multivariate data analysis, aiming at unveiling peculiar spectral regions that could be used as diagnostic features during the analysis of i-DNA-forming sequences.
Assuntos
DNA/química , Dicroísmo Circular , Conformação de Ácido Nucleico , Espectrofotometria UltravioletaRESUMO
CD22 (Siglec-2) is a B-cell surface inhibitory protein capable of selectively recognising sialylated glycans, thus dampening autoimmune responses against self-antigens. Here we have characterised the dynamic recognition of complex-type N-glycans by human CD22 by means of orthogonal approaches including NMR spectroscopy, computational methods and biophysical assays. We provide new molecular insights into the binding mode of sialoglycans in complex with h-CD22, highlighting the role of the sialic acid galactose moieties in the recognition process, elucidating the conformational behaviour of complex-type N-glycans bound to Siglec-2 and dissecting the formation of CD22 homo-oligomers on the B-cell surface. Our results could enable the development of additional therapeutics capable of modulating the activity of h-CD22 in autoimmune diseases and malignancies derived from B-cells.
Assuntos
Simulação de Dinâmica Molecular , Polissacarídeos/química , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/química , Linfócitos B/química , Configuração de Carboidratos , Galactose/química , HumanosRESUMO
A new visible light-induced photocatalytic protocol enabling the formation of secondary amides from electron-poor organic bromides and isocyanides was developed. In addition, the in situ interception of ketenimine intermediates with nitrogen nucleophiles such as amines, hydrazines, and TMSN3 afforded, in a one-pot two-step procedure, valuable scaffolds such as ketene aminals, pyrazolones, and tetrazoles. Mechanistic evidence confirmed a radical pathway where isocyanides acted as radical geminal acceptors generating key imidoyl radical species.