Cyanopyridoimidazole/1,2-Aminothiol Click Reaction: A Novel Bioorthogonal Reaction for Synthesis of Radiotracers.

We herein described the design and synthesis of the cyanopyridoimidazoles (CPIs) as new bioorthogonal click reagents toward 1,2-aminothiol groups. Kinetic and density functional theory-based studies of the synthetic compounds revealed that incorporating an electron-withdrawing substituent into the CPI scaffold lowers its lowest unoccupied molecular orbital energy, consequently increasing reactivity. Optimized CPI 8a showed rapid reactivity and high stability in physiological conditions and has been demonstrated to be suitable for various radiotracer synthetic methods. Based on the new bioorthogonal reaction, a [67Ga]Ga-labeled prostate-specific membrane antigen-targeted probe was successfully prepared for in vivo imaging of prostate cancer in an animal model.

Evaluation of Chelator-to-Antibody Ratio on Development of 89Zr-iPET Tracer for Imaging of PD-L1 Expression on Tumor.

89Zr-iPET has been widely used for preclinical and clinical immunotherapy studies to predict patient stratification or evaluate therapeutic efficacy. In this study, we prepared and evaluated 89Zr-DFO-anti-PD-L1-mAb tracers with varying chelator-to-antibody ratios (CARs), including 89Zr-DFO-anti-PD-L1-mAb_3X (tracer_3X), 89Zr-DFO-anti-PD-L1-mAb_10X (tracer_10X), and 89Zr-DFO-anti-PD-L1-mAb_20X (tracer_20X). The DFO-anti-PD-L1-mAb conjugates with varying CARs were prepared using a random conjugation method and then subjected to quality control. The conjugates were radiolabeled with 89Zr and evaluated in a PD-L1-expressing CT26 tumor-bearing mouse model. Next, iPET imaging, biodistribution, pharmacokinetics, and ex vivo pathological and immunohistochemical examinations were conducted. LC-MS analysis revealed that DFO-anti-PD-L1-mAb conjugates were prepared with CARs ranging from 0.4 to 2.0. Radiochemical purity for all tracer groups was >99% after purification. The specific activity levels of tracer_3X, tracer_10X, and tracer_20X were 2.2 ± 0.6, 8.2 ± 0.6, and 10.5 ± 1.6 µCi/µg, respectively. 89Zr-iPET imaging showed evident tumor uptake in all tracer groups and reached the maximum uptake value at 24 h postinjection (p.i.). Biodistribution data at 168 h p.i. revealed that the tumor-to-liver, tumor-to-muscle, and tumor-to-blood uptake ratios for tracer_3X, tracer_10X, and tracer_20X were 0.46 ± 0.14, 0.58 ± 0.33, and 1.54 ± 0.51; 4.7 ± 1.3, 7.1 ± 3.9, and 14.7 ± 1.1; and 13.1 ± 5.8, 19.4 ± 13.8, and 41.3 ± 10.6, respectively. Significant differences were observed between tracer_3X and tracer_20X in the aforementioned uptake ratios at 168 h p.i. The mean residence time and elimination half-life for tracer_3X, tracer_10X, and tracer_20X were 25.4 ± 4.9, 24.2 ± 6.1, and 25.8 ± 3.3 h and 11.8 ± 0.5, 11.1 ± 0.7, and 11.7 ± 0.6 h, respectively. No statistical differences were found between-tracer in the aforementioned pharmacokinetic parameters. In conclusion, 89Zr-DFO-anti-PD-L1-mAb tracers with a CAR of 1.4-2.0 may be better at imaging PD-L1 expression in tumors than are traditional low-CAR 89Zr-iPET tracers.

Novel Imaging Probes: From Design to Applications.

Molecular imaging has emerged as a powerful tool for clinical diagnosis [...].

New Developments in Carbonic Anhydrase IX-Targeted Fluorescence and Nuclear Imaging Agents.

Carbonic anhydrase IX (CAIX) is a tumor-specific and hypoxia-induced biomarker for the molecular imaging of solid malignancies. The nuclear- and optical-imaging of CAIX-expressing tumors have received great attention due to their potential for clinical applications. Nuclear imaging is a powerful tool for the non-invasive diagnosis of primary and metastatic CAIX-positive tumors and for the assessment of responses to antineoplastic treatment. Intraoperative optical fluorescence imaging provides improved visualization for surgeons to increase the discrimination of tumor lesions, allowing for safer surgical treatment. Over the past decades, many CAIX-targeted molecular imaging probes, based on monoclonal antibodies, antibody fragments, peptides, and small molecules, have been reported. In this review, we outline the recent development of CAIX-targeted probes for single-photon emission computerized tomography (SPECT), positron emission tomography (PET), and near-infrared fluorescence imaging (NIRF), and we discuss issues yet to be addressed.

Towards Complete Tumor Resection: Novel Dual-Modality Probes for Improved Image-Guided Surgery of GRPR-Expressing Prostate Cancer.

Nuclear and optical dual-modality probes can be of great assistance in prostate cancer localization, providing the means for both preoperative nuclear imaging and intraoperative surgical guidance. We developed a series of probes based on the backbone of the established GRPR-targeting radiotracer NeoB. The inverse electron demand of the Diels-Alder reaction was used to integrate the sulfo-cyanine 5 dye. Indium-111 radiolabeling, stability studies and a competition binding assay were carried out. Pilot biodistribution and imaging studies were performed in PC-3 tumor-bearing mice, using the best two dual-labeled probes. The dual-modality probes were radiolabeled with a high yield (>92%), were proven to be hydrophilic and demonstrated high stability in mouse serum (>94% intact labeled ligand at 4 h). The binding affinity for the GRPR was in the nanomolar range (21.9-118.7 nM). SPECT/CT images at 2 h p.i. clearly visualized the tumor xenograft and biodistribution studies, after scanning confirmed the high tumor uptake (8.47 ± 0.46%ID/g and 6.90 ± 0.81%ID/g for probe [111In]In-12 and [111In]In-15, respectively). Receptor specificity was illustrated with blocking studies, and co-localization of the radioactive and fluorescent signal was verified by ex vivo fluorescent imaging. Although optimal tumor-to-blood and tumor-to-kidney ratios might not yet have been reached due to the prolonged blood circulation, our probes are promising candidates for the preoperative and intraoperative visualization of GRPR-positive prostate cancer.

Synthesis and Evaluation of Diverse N-Substituted Disaccharide Dipeptides for Human NOD2 Stimulation Activity.

A new strategy for the preparation of distinct N-substituted muropeptides is described. Different orthogonally N-protected disaccharide thioglycosides were designed and synthesized. Among them, compound 4, qualified as a key intermediate, was utilized for further chemical transformations to develop a series of diverse N-substituted-glucosaminyl N-substituted-muramyl dipeptides (GMDPs). These unique muropeptides were applied for the study of human NOD2 stimulation. Intriguingly, structural modification of the MurNAc residue to N-non-substituted muramic acid (MurNH2 ) in GMDP dramatically impaired NOD2 stimulatory activity, but GMDPs possessing the glucosamine residue with a free amino group retained NOD2 stimulation activity. This work is the first study to illustrate the impact of both N-substituents of GMDPs on immunostimulatory activities of human NOD2.

IEDDA: An Attractive Bioorthogonal Reaction for Biomedical Applications.

The pretargeting strategy has recently emerged in order to overcome the limitations of direct targeting, mainly in the field of radioimmunotherapy (RIT). This strategy is directly dependent on chemical reactions, namely bioorthogonal reactions, which have been developed for their ability to occur under physiological conditions. The Staudinger ligation, the copper catalyzed azide-alkyne cycloaddition (CuAAC) and the strain-promoted [3 + 2] azide-alkyne cycloaddition (SPAAC) were the first bioorthogonal reactions introduced in the literature. However, due to their incomplete biocompatibility and slow kinetics, the inverse-electron demand Diels-Alder (IEDDA) reaction was advanced in 2008 by Blackman et al. as an optimal bioorthogonal reaction. The IEDDA is the fastest bioorthogonal reaction known so far. Its biocompatibility and ideal kinetics are very appealing for pretargeting applications. The use of a trans-cyclooctene (TCO) and a tetrazine (Tz) in the reaction encouraged researchers to study them deeply. It was found that both reagents are sensitive to acidic or basic conditions. Furthermore, TCO is photosensitive and can be isomerized to its cis-conformation via a radical catalyzed reaction. Unfortunately, the cis-conformer is significantly less reactive toward tetrazine than the trans-conformation. Therefore, extensive research has been carried out to optimize both click reagents and to employ the IEDDA bioorthogonal reaction in biomedical applications.

Patient satisfaction and quality of life after orthodontic treatment for cleft lip and palate deformity.

OBJECTIVES: Patients with cleft lip-cleft palate (CLP) often require orthodontic treatment, with or without orthognathic surgery. Patient satisfaction is the most important outcome parameter in orthodontic treatment. This study aimed to (1) determine patient satisfaction and quality of life (QoL) after orthodontic treatment and (2) identify associated factors. MATERIAL AND METHODS: This prospective cross-sectional study recruited patients with CLP who had completed orthodontic treatment at a craniofacial center in Taiwan. Participants (N=213) had received treatment for unilateral CLP (n=99), bilateral CLP (n=50), cleft lip and alveolus (n=39), and isolated cleft palate (n=25). Self-report questionnaires evaluated satisfaction with appearance and QoL; multiple regression analysis examined associated factors. Participants' expectations of treatment results were also reported. RESULTS: Participants reported moderate satisfaction with facial appearance and QoL. Satisfaction with treatment was lower or much lower than expected for 13% of participants. Treatment for bilateral CLP was associated with the lowest satisfaction with overall appearance (r = -8.123, P < 0.05); participants who had received orthognathic surgery had the highest satisfaction (r = 5.534, P < 0.05). Treatment for unilateral and bilateral CLP was associated with low QoL for smile (both P < 0.05). CONCLUSIONS: Orthodontic treatment had a positive effect on facial appearance and quality of life in patients with CLP. Type of cleft and orthognathic surgery significantly influenced satisfaction with facial appearance. CLINICAL RELEVANCE: Efforts must be taken to modify treatment strategies for patients with bilateral CLP in order to improve satisfaction with appearance following treatment.

A novel clickable MSAP agent for dual fluorescence/nuclear labeling of biovectors.

We herein describe the development of a novel dual-modality optical/radio-imaging agent for general and site-specific labeling of biovectors through a 2-cyanobenzothiazole (CBT)/1,2-aminothiol click reaction. The CBT-based multifunctional single-attachment-point (MSAP) agent enables a single-step synthesis of various dual-modality probes characterized by rapid conjugation, high labeling yields, metabolically stable products and applicability to orthogonal two-step labeling of sensitive biomolecules. In addition, the two-step radiolabeling protocol and click reaction were optimized by using CBT scavengers to improve the reaction rate and molar activity of the imaging probes. Our methodology allows for a simple and efficient synthetic route to produce a variety of dual-modality imaging agents for preoperative surgical planning and intraoperative surgical guidance.

The SNP rs560426 Within ABCA4-ARHGAP29 Locus and the Risk of Nonsyndromic Oral Clefts.

OBJECTIVE: Nonsyndromic oral clefts are common birth defect with complex etiology. In the present study, we attempt to further validate the possible role for ABCA4 and ARHGAP29 in the susceptibility to nonsyndromic oral clefts. DESIGN: We performed allelic transmission disequilibrium test analysis, on 10 eligible single nucleotide polymorphisms (SNPs) and SNP haplotypes using the Family-Based Association Test. PARTICIPANTS: The study sample consisted of 334 case-parent trios of nonsyndromic oral clefts from Taiwanese population, separated into nonsyndromic cleft lip with or without cleft palate (NSCL/P) and nonsyndromic cleft palate only (NSCPO) groups. RESULTS: We found only the SNP rs560426 within the ABCA4 gene showed strong association with NSCPO (P = .03498; Permuted P = .05382). No association between other 9 selected SNPs in ABCA4-ARHGAP29 region and the risk of nonsyndromic oral clefts was found. For the haplotype analyses, we found only haplotype T-C (rs570926 and rs3789431) in ABCA4 block 2 showed significant association with nonsyndromic NSCL/P in these Taiwanese trios. CONCLUSIONS: We used a family-based analysis in 334 Taiwanese case-parent trios to validate the possible role for ABCA4 and ARHGAP29 in the susceptibility to nonsyndromic oral clefts. This study provides a new evidence for an association between the intron variant rs560426 within ABCA4 and nonsyndromic cleft palate which may contribute their regulatory role in craniofacial development.

Association Studies Between Regulatory Regions of IRF6/TP63 Genes and Nonsyndromic Oral Clefts.

OBJECTIVE: To evaluate genetic variants within the regulatory regions of interferon regulatory factor 6 (IRF6) and TP63 for the etiology of nonsyndromic oral clefts risk factors. DESIGN: We performed allelic transmission disequilibrium test analysis on 5 eligible single-nucleotide polymorphisms (SNPs) and SNP haplotypes using the Family-Based Association Test. PARTICIPANTS: The study sample consisted of 334 case-parent trios of nonsyndromic oral clefts from Taiwanese population, separated into nonsyndromic cleft lip/palate (NSCL/P) and nonsyndromic cleft palate only (NSCPO) groups. RESULTS: We found all 3 selected SNPs of the IRF6 gene show significant association with nonsyndromic oral clefts (rs2235371, P = 5.10E-07; rs642961, P = .00194; and rs77542756, P = 9.08E-07). Haplotype analyses identified 3 possible SNP combination haplotypes in the IRF6 gene and found that C-G-G showed significant undertransmission (P = .058), whereas 2 other haplotypes, T-G-A and C-A-G (P = 2.71E-06 and P = 5.00E-04, respectively), were significantly overtransmitted to the NSCL/P children but not to the NSCPO children. For the TP63 gene, we failed to detect evidence of nonsyndromic oral cleft association in the 2 SNPs within the TP63 large intron 1 region. CONCLUSIONS: We used a family-based analysis in 334 Taiwanese case-parent trios to evaluate selected SNPs of IRF6 genes and TP63 genes for a risk of orofacial clefting. This study provides additional evidence for an association between IRF6 and NSCL/P, including the genetic variants within the 5'-noncoding region of the gene. We also confirmed that NSCL/P and NSCPO individuals belong to different groups. For the TP63, our data did not favor the direct involvement of TAp63 isoforms during orofacial development.

A Flexible Synthesis of 68Ga-Labeled Carbonic Anhydrase IX (CAIX)-Targeted Molecules via CBT/1,2-Aminothiol Click Reaction.

We herein describe a flexible synthesis of a small library of 68Ga-labeled CAIX-targeted molecules via an orthogonal 2-cyanobenzothiazole (CBT)/1,2-aminothiol click reaction. Three novel CBT-functionalized chelators (1⁻3) were successfully synthesized and labeled with the positron emitter gallium-68. Cross-ligation between the pre-labeled bifunctional chelators (BFCs) and the 1,2-aminothiol-acetazolamide derivatives (8 and 9) yielded six new 68Ga-labeled CAIX ligands with high radiochemical yields. The click reaction conditions were optimized to improve the reaction rate for applications with short half-life radionuclides. Overall, our methodology allows for a simple and efficient radiosynthetic route to produce a variety of 68Ga-labeled imaging agents for tumor hypoxia.

Genetic diagnosis of neurofibromatosis type 1: targeted next- generation sequencing with Multiple Ligation-Dependent Probe Amplification analysis.

BACKGROUND: Neurofibromatosis type 1 (NF1) is a dominantly inherited tumor predisposition syndrome that targets the peripheral nervous system. It is caused by mutations of the NF1 gene which serve as a negative regulator of the cellular Ras/MAPK (mitogen-activated protein kinases) signaling pathway. Owing to the complexity in some parts of clinical diagnoses and the need for better understanding of its molecular relationships, a genetic characterization of this disorder will be helpful in the clinical setting. METHODS: In this study, we present a customized targeted gene panel of NF1/KRAS/BRAF/p53 and SPRED1 genes combined with Multiple Ligation-Dependent Probe Amplification analysis for the NF1 mutation screening in a cohort of patients clinically suspected as NF1. RESULTS: In this study, we identified 73 NF1 mutations and two BRAF novel variants from 100 NF1 patients who were suspected as having NF1. These genetic alterations are heterogeneous and distribute in a complicated way without clustering in either cysteine-serine-rich domain or within the GAP-related domain. We also detected fifteen multi-exon deletions within the NF1 gene by MLPA Analysis. CONCLUSIONS: Our results suggested that a genetic screening using a NGS panel with high coverage of Ras-signaling components combined with Multiple Ligation-Dependent Probe Amplification analysis will enable differential diagnosis of patients with overlapping clinical features.

Early-Stage Incorporation Strategy for Regioselective Labeling of Peptides using the 2-Cyanobenzothiazole/ 1,2-Aminothiol Bioorthogonal Click Reaction.

Invited for this month's cover picture is the group of Dr. Yann Seimbille at the Life Sciences Division at TRIUMF (Canada). The cover picture shows how a simple and innovative methodology based on the bioorthogonal click reaction between 2-cyanobenzothiazole and 1,2-aminothiol has been elaborated to facilitate the labeling of peptide biovectors. Read the full text of their Communication at 10.1002/open.201700191.

Early-Stage Incorporation Strategy for Regioselective Labeling of Peptides using the 2-Cyanobenzothiazole/1,2-Aminothiol Bioorthogonal Click Reaction.

Herein, we describe a synthetic strategy for the regioselective labeling of peptides by using a bioorthogonal click reaction between 2-cyanobenzothiazole (CBT) and a 1,2-aminothiol moiety. This methodology allows for the facile and site-specific modification of peptides with various imaging agents, including fluorophores and radioisotope-containing prosthetic groups. We investigated the feasibility of an early-stage incorporation of dipeptide 1 into targeting vectors, such as c[RGDyK(C)] and HER2 pep, during solid-phase peptide synthesis. Then, the utility of the click reaction to label bioactive peptides with a CBT-modified imaging agent (FITC-CBT, 9) was assessed. The ligation reaction was found to be highly selective and efficient under various conditions. The fluorescently labeled peptides 2 and 3 were obtained in respective yields of 88 and 82 % under optimized conditions.

Effect of the lipid II sugar moiety on bacterial transglycosylase: the 4-hydroxy epimer of lipid II is a TGase inhibitor.

Lipid II analogues bearing major modifications on the second sugar (GlcNAc) were synthesized and evaluated for their substrate activity toward TGases. Unexpectedly, N-deacetyled lipid II decreased its activity dramatically, and the C4-axial OH lipid II became an inhibitor (IC50 = 8 µM) with an approximately 14-fold increase in binding affinity toward TGase (25 vs. 27).

Structural Investigation of Park's Nucleotide on Bacterial Translocase MraY: Discovery of Unexpected MraY Inhibitors.

Systematic structural modifications of the muramic acid, peptide, and nucleotide moieties of Park's nucleotide were performed to investigate the substrate specificity of B. subtilis MraY (MraYBS). It was found that the simplest analogue of Park's nucleotide only bearing the first two amino acids, l-alanine-iso-d-glutamic acid, could function as a MraYBS substrate. Also, the acid group attached to the Cα of iso-d-glutamic acid was found to play an important role for substrate activity. Epimerization of the C4-hydroxyl group of muramic acid and modification at the 5-position of the uracil in Park's nucleotide were both found to dramatically impair their substrate activity. Unexpectedly, structural modifications on the uracil moiety changed the parent molecule from a substrate to an inhibitor, blocking the MraYBS translocation. One unoptimized inhibitor was found to have a Ki value of 4 ± 1 µM against MraYBS, more potent than tunicamycins.

Nonsynonymous variants in MYH9 and ABCA4 are the most frequent risk loci associated with nonsyndromic orofacial cleft in Taiwanese population.

BACKGROUND: Nonsyndromic orofacial cleft is a common birth defect with a complex etiology, including multiple genetic and environmental risk factors. Recent whole genome analyses suggested associations between nonsyndromic orofacial cleft and up to 18 genetic risk loci (ABCA4, BMP4, CRISPLD2, GSTT1, FGF8, FGFR2, FOXE1, IRF6, MAFB, MSX1, MTHFR, MYH9, PDGFC, PVRL1, SUMO1, TGFA, TGFB3, and VAX1), each of which confers a different relative risk in different populations. We evaluate the nonsynonymous variants in these 18 genetic risk loci in nonsyndromic orofacial clefts and normal controls to clarify the specific variants in Taiwanese population. METHODS: We evaluated these 18 genetic risk loci in 103 cases of nonsyndromic orofacial clefts and 100 normal controls using a next-generation sequencing (NGS) customized panel and manipulated a whole-exon targeted-sequencing study based on the NGS system of an Ion Torrent Personal Genome Machine (IT-PGM). IT-PGM data processing, including alignment with the human genome build 19 reference genome (hg19), base calling, trimming of barcoded adapter sequences, and filtering of poor signal reads, was performed using the IT platform-specific pipeline software Torrent Suite, version 4.2, with the plug-in "variant caller" program. Further advanced annotation was facilitated by uploading the exported VCF file from Variant Caller to the commercial software package Ion Reporter; the free online annotation software Vanno and Mutation Taster. Benign or tolerated amino acid changes were excluded after analysis using sorting intolerant from tolerant and polymorphism phenotyping. Sanger sequencing was used to validate the significant variants identified by NGS. Furthermore, each variant was confirmed in asymptomatic controls using the Sequenom MassARRAY (San Diego, CA, USA). RESULTS: We identified totally 22 types of nonsynonymous variants specific in nonsyndromic orofacial clefts, including 19 single nucleotide variants, 2 deletions, and 1 duplication in 10 studied genes(ABCA4, MYH9, MTHFR, CRISPLD2, FGF8, PVRL1, FOXE1, VAX1, FGFR2, and IRF6). Nonsynonymous variants in MYH9 and ABCA4, which were detected in 6 and 5 individuals, respectively, were identified to be the most frequent risk loci in nonsyndromic orofacial clefts in the Taiwanese population. CONCLUSIONS: Nonsynonymous variants in MYH9 and ABCA4 were identified to be the most frequent risk loci in nonsyndromic orofacial clefts in the Taiwanese population. These findings in our study have provided additional information regarding specific variants associated with nonsyndromic orofacial clefts in different population and demonstrate the power of our customized NGS panel, which is clinically useful for the simultaneous detection of multiple genes associated with nonsyndromic orofacial clefts.

Synthesis of Diverse N-Substituted Muramyl Dipeptide Derivatives and Their Use in a Study of Human NOD2 Stimulation Activity.

A flexible synthetic strategy toward the preparation of diverse N-substituted muramyl dipeptides (N-substituted MDPs) from different protected monosaccharides is described. The synthetic MDPs include N-acetyl MDP and N-glycolyl MDP, known NOD2 ligands, and this methodology allows for structural variation at six positions, including the muramic acid, peptide, and N-substituted moieties. The capacity of these molecules to activate human NOD2 in the innate immune response was also investigated. It was found that addition of the methyl group at the C1 position of N-glycolyl MDP significantly enhanced the NOD2 stimulating activity.

Synthesis of 1-C-Glycoside-Linked Lipid†II Analogues Toward Bacterial Transglycosylase Inhibition.

Preparation of Lipid II analogues containing an enzymatically uncleavable 1-C-glycoside linkage between the disaccharide moiety and the pyrophosphate- or pyrophosphonate-lipid moiety is described. The synthesis of a common 1-C-vinyl disaccharide intermediate has been developed that allows easy preparation of both an elongated sugar-phosphate bond and a sugar-phosphonate moiety, which are coupled with the polyprenyl phosphate to give the desired molecules. Inhibition studies show how a subtle structural modification results in dramatically different potency toward bacterial transglycosylase (TGase), and the results identify Lipid II-C-O-PP (IC50 =25 µM) as a potential TGase inhibitor.