Expanding a peptide-covalent probe hybrid for PET imaging of S. aureus driven focal infections.

BACKGROUND: The urgent demand for innovative theranostic strategies to combat bacterial resistance to antibiotics is evident, with substantial implications for global health. Rapid diagnosis of life-threatening infections can expedite treatment, improving patient outcomes. Leveraging diagnostic modalities i.e., positron emission tomography (PET) and single photon emission computed tomography (SPECT) for detecting focal infections has yielded promising results. Augmenting the sensitivity of current PET and SPECT tracers could enable effective imaging of pathogenic bacteria, including drug-resistant strains.UBI (29-41), an antimicrobial peptide (AMP) fragment recognizes the S. aureus membrane through electrostatic binding. Radiolabeled UBI (29-41) is a promising SPECT and PET-based tracer for detecting focal infections. 2-APBA (2-acetyl-phenyl-boronic acid), a non-natural amino acid, specifically targets lysyl-phosphatidyl-glycerol (lysyl-PG) on the S. aureus membranes, particularly in AMP-resistant strains. We propose that combining UBI with 2-APBA could enhance the diagnostic potential of radiolabeled UBI. RESULTS: Present work aimed to compare the diagnostic potential of two radiolabeled peptides, namely UBI (29-41) and 2-APBA modified UBI (29-41), referred to as UBI and UBI-APBA. APBA modification imparted antibacterial activity to the initially non-bactericidal UBI against S. aureus by inducing a loss of membrane potential. The antibacterial activity demonstrated by UBI-APBA can be ascribed to the synergistic interaction of both UBI and UBI-APBA on the bacterial membrane. To enable PET imaging, we attached the chelator 1,4,7-triazacyclononane 1-glutaric acid 4,7-acetic acid (NODAGA) to the peptides for complexation with the positron emitter Gallium-68 (68Ga). Both NODAGA conjugates were radiolabeled with 68Ga with high radiochemical purity. The resultant 68Ga complexes were stable in phosphate-buffered saline and human serum. Uptake of these complexes was observed in S. aureus but not in mice splenocytes, indicating the selective nature of their interaction. Additionally, the APBA conjugate exhibited superior uptake in S. aureus while preserving the selectivity of the parent peptide. Furthermore, [68Ga]Ga-UBI-APBA demonstrated accumulation at the site of infection in rats, with an improved target-to-non-target ratio, as evidenced by ex-vivo biodistribution and PET imaging. CONCLUSIONS: Our findings suggest that linking UBI, as well as AMPs in general, with APBA shows promise as a strategy to augment the theranostic potential of these molecules.

Capturing Sialyl-glycan on Live Cancer Cells by Tailored Boronopeptide.

Boronic acid-containing molecules are substantially popularized in chemical biology and medicinal chemistry due to the broad spectrum of covalent conjugations as well as interaction modules offered by the versatile boron atom. Apparently, the WGA peptide (wheat germ agglutinin, 62-73), which shows a considerably low binding affinity to sialic acid, turned into a selective and >5 folds potent binder with the aid of a suitable boronic acid probe installed chemoselectively. In silico studies prompted us to install BA probes on the cysteine residue, supposedly located in close proximity to the bound sialic acid. In vitro studies revealed that the tailored boronopeptides show enhanced binding ability due to the synergistic recognition governed by selective non-covalent interactions and cis-diol boronic acid conjugation. The intense binding is observed even in 10 % serum, thus enabling profiling of sialyl-glycan on cancer cells, as compared with the widely used lectin, Sambucus nigra. The synergistic binding mode between the best boronopeptide (P3) binder and sialic acid was analyzed via 1 H and 11 B NMR.

Identification, synthesis, and characterization of an unprecedented N-(2-carboxyethyl) adduct impurity in an injectable ganirelix formulation.

Ganirelix, a peptide-based drug used to treat female infertility, has been in high market demand, which attracted generic formulation. A hitherto unknown impurity of ganirelix was observed in our formulation process, which reached ~0.3% in 6 months and led to a detailed investigation of its structure. In-depth analysis of ESI-MS/MS data of this impurity coupled with an artificial intelligence prediction tool led to a highly unusual putative structure, that is, N-(2-carboxyethyl)-ganirelix (NCE-GA), which was authenticated by chemical synthesis from ganirelix and NMR analysis and via corroborated HPLC and MS/MS data with the formulation-derived impurity.

Sulfonyl Diazaborine 'Click' Chemistry Enables Rapid and Efficient Bioorthogonal Labeling.

Finding an ideal bioorthogonal reaction that responds to a wide range of biological queries and applications is of great interest in biomedical applications. Rapid diazaborine (DAB) formation in water by the reactions of ortho-carbonyl phenylboronic acid with α-nucleophiles is an attractive conjugation module. Nevertheless, these conjugation reactions demand to satisfy stringent criteria for bioorthogonal applications. Here we show that widely used sulfonyl hydrazide (SHz) offers a stable DAB conjugate by combining with ortho-carbonyl phenylboronic acid at physiological pH, competent for an optimal biorthogonal reaction. Remarkably, the reaction conversion is quantitative and rapid (k2 >103  M-1 s-1 ) at low micromolar concentrations, and it preserves comparable efficacy in a complex biological milieu. DFT calculations support that SHz facilitates DAB formation via the most stable hydrazone intermediate and the lowest energy transition state compared to other biocompatible α-nucleophiles. This conjugation is extremely efficient on living cell surfaces, enabling compelling pretargeted imaging and peptide delivery. We anticipate this work will permit addressing a wide range of cell biology queries and drug discovery platforms exploiting commercially available sulfonyl hydrazide fluorophores and derivatives.

Cysteine-Selective Installation of Functionally Diverse Boronic Acid Probes on Peptides.

The current methods for direct late-stage and residue-selective installation of a versatile boronic acid (BA) repertoire on peptides are inadequate for a wide range of applications. Here, we show the suitability and efficiency of thiol-ene radical click chemistry to install functionally versatile BA derivatives on numerous bioactive, native peptides. Our work highlights that the methodology is operationally simple and adaptable for applications with BA-modified peptides, such as cyclization, conjugation, and functional group alteration.

Synthesis and characterization of two potential impurities (des-ethyl-Ganirelix) generated in the Ganirelix manufacturing process.

Controlling certain diseases using peptide drugs has remarkably increased in the past two decades. In this regard, a generic formulation is an upfront solution to fulfill market demands. Ganirelix, a leading peptide active pharmaceutical ingredient (API) primarily used as a gonadotropin-releasing hormone antagonist (GnRH), has established a potential market value worldwide. But its generic formulation mandates detailed impurity profiles from a synthetic source and contemplates the sameness of a reference-listed drug (RLD). Post-chemical synthesis and processing of Ganirelix, some commercial sources have revealed two new potential impurities among many known, which show the deletion of an ethyl group from the hArg(Et)2 residue at the sixth and eighth positions, named des-ethyl-Ganirelix. These impurities are unprecedented in traditional peptide chemistry, and such monoethylated-hArg building blocks are not easily accessible commercially to synthesize these two impurities. Here, we have outlined the synthesis, purification, and enantiomeric purity characterization of the amino acids and their incorporation in the Ganirelix peptide sequence to synthesize these potential peptide impurities. This methodology will enable the convenient synthesis of side-chain substituted Arg and hArg derivatives in peptide drug discovery platforms.

Rapid and Highly Productive Assembly of a Disulfide Bond in Solid-Phase Peptide Macrocyclization.

Here we report a highly efficient disulfide-driven peptide macrocyclization in 15 min on a solid support using persulfate as a crucial additive in iodine-mediated oxidative cyclization. The method eliminates the side products of classical iodine-mediated peptide cyclization. It is operationally simple and convenient for cyclizing small to lengthier peptides embodying popular cysteine building blocks in a single step.

Integrating a covalent probe with ubiquicidin fragment enables effective bacterial infection imaging.

Developing potent and novel bacterial imaging agents remains formidable due to the rapid development of bacterial resistance. Ubiquicidin and its derivatives are the most studied antimicrobial peptides that bind to anionic membranes of a broad range of bacterial pathogens. Studies reveal that UBI (29-41) labeled with 99mTc and 68Ga could distinguish sterile inflammation from infection. A significant challenge that remains for cationic peptides is their poor salt tolerance. The present study deliberates the increment of UBI (29-41) peptide interaction with the bacterial membrane by incorporating 2-acetylphenylboronic acid (2-APBA) as a covalent probe and developing infection imaging probes with improved retention at the target. Given that both 99mTc-UBI (29-41) and 99mTc-UBI (29-41)-2-APBA peptide complexes are stable in serum over 16 h, 99mTc-UBI (29-41)-2-APBA shows enhanced uptake in S. aureus cells as compared to 99mTc-UBI (29-41). SPECT imaging in a mouse model of infection exhibited a higher target to non-target ratio after 2 h in the case of 99mTc-UBI (29-41)-2-APBA. The present study reveals a synergistic mechanism of target binding through covalent conjugation and non-covalent interaction, which could be a potential strategy for improving bacterial infection imaging. As a proof of concept, 99mTc-UBI (29-41)-2-APBA elicits our hypothesis by in vivo imaging of bacterial infection.

Compelling Cyclic Peptide Scaffolds for Antitubercular Action: An Account (2011-21) of the Natural Source.

Natural cyclic peptide scaffolds are indispensable in medicinal chemistry, chemical biology, and drug discovery platforms due to their chemical diversity, structural integrity, proteolytic stability and biocompatibility. Historically, their isolation and profound understanding of target engagement have been identified as lead pharmacophore discovery. Natural cyclic peptides are the largest class of pharmacologically active scaffold, in which most show activity against drug-resistant Mycobacterium tuberculosis (Mtb). Nevertheless, eight recently discovered cyclic peptide scaffolds exhibit promising antitubercular activity among numerous naturally occurring antitubercular peptides, and they are amenable scaffolds to drug development. We examined their biological origin, scaffolds, isolations, chemical synthesis, and reasons for biological actions against Mtb. Understanding these peptide scaffold details will further allow synthetic and medicinal chemists to develop novel peptide therapeutics against tuberculosis-infected deadly diseases. This review emphasizes these cyclic peptides' in vitro and in vivo activity profiles, including their structural and chemical features.

High throughput virtual screening (HTVS) of peptide library: Technological advancement in ligand discovery.

High-throughput virtual screening (HTVS) is a leading biopharmaceutical technology that employs computational algorithms to uncover biologically active compounds from large-scale collections of chemical compound libraries. In addition, this method often leverages the precedence of screening focused libraries for assessing their binding affinities and improving physicochemical properties. Usually, developing a drug sometimes takes ages, and lessons are learnt from FDA-approved drugs. This screening strategy saves resources and time compared to laboratory testing in certain stages of drug discovery. Yet in-silico investigations remain challenging in some cases of drug discovery. For the last few decades, peptide-based drug discoveries have received remarkable momentum for several advantages over small molecules. Therefore, developing a high-fidelity HTVS platform for chemically versatile peptide libraries is highly desired. This review summarises the modern and frequently appreciated HTVS strategies for peptide libraries from 2011 to 2021. In addition, we focus on the software used for preparing peptide libraries, their screening techniques and shortcomings. An index of various HTVS methods reported here should assist researchers in identifying tools that could be beneficial for their peptide library screening projects.

The modern role of boron as a 'magic element' in biomedical science: chemistry perspective.

Boron was misconstrued as a toxic element for animals, which retarded the growth of boron-containing drug discovery in the last century. Nevertheless, modern applications of boronic acid derivatives are attractive in biomedical applications after the declaration that boron is a 'probable essential element' for humans by the WHO. Additionally, the approval of five boronic acid-containing drugs by the FDA has vastly impacted the use of boron in medicinal chemistry, chemical biology, drug delivery, biomaterial exploration, pharmacological improvements, and nutrition. This review article focuses on the chemistries attributed to boronic acids at physiological pH, enticing chemists to multidisciplinary applications. Prospective uses of boronic acid in pharma and chemical biology, along with prospects and challenges, are also part of the deliberation. Understanding these fundamental chemistries and interactions of boronic acid in biological systems will enable solving future challenges in drug discovery and executing space-age applications.

A periodic development of BPA and BSH based derivatives in boron neutron capture therapy (BNCT).

Boron neutron capture therapy (BNCT) is a particular type of radiotherapy that requires a selective and high concentration of boron accumulation in neoplastic cells. To distinguish the distribution of boron compounds between tumour and normal cells, multiple research groups have been involved and successively innovated a wide variety of boron-based compounds. Despite the development of numerous boron compounds, only boronophenylalanine (BPA) and sodium mercaptoundecahydro-closo-dodecaborate (BSH) have emerged as effective in clinical trials. Here, we highlight the detailed progress in the molecular design of BPA and BSH derivatives from the historical perspective to the latest advances in light of the widely accepted performance required for effective BNCT. In this report, we have provided an overview of a variety of derivatives of BPA and BSH, including amino acids, peptides, polymers, monoclonal antibodies and chelated complexes, and it is observed that such derivatives of BPA and BSH are judicious choices for BNCT. Finally, we have summarised the critical issues for BPA and BSH that must be addressed if BNCT is to become a more widely accepted clinical modality.

Site-Selective, Chemical Modification of Protein at Aromatic Side Chain and Their Emergent Applications.

Site-selective chemical modification of protein side chain has probed enormous opportunities in the fundamental understanding of cellular biology and therapeutic applications. Primarily, in the field of biopharmaceuticals, the formulation of bioconjugates has been found to have more potential than an individual constituent. In this regard, Lysine and Cysteine are the most widely used endogenous amino acid for these purposes. Recently, the aromatic side chain residues (Trp, Tyr, and His) that are low abundant in protein have gained more attention in therapeutic applications due to their advantages of chemical reactivity and specificity. This review discusses the site-selective bioconjugation methods for aromatic side chains (Trp, Tyr and His) and highlights the developed strategies in the last three years, along with their applications. Also, the review highlights the prevalent methods published earlier. We have examined that metal-catalyzed and photocatalytic reactions are gaining more attention for bioconjugation, though their practical operation is under development. The review has been summarized with the future perspective of protein and peptide conjugations contemplating therapeutic applications and challenges.

Boronic acid based dynamic click chemistry: recent advances and emergent applications.

Recently, reversible click reactions have found numerous applications in chemical biology, supramolecular chemistry, and biomedical applications. Boronic acid (BA)-mediated cis-diol conjugation is one of the best-studied reactions among them. An excellent understanding of the chemical properties and biocompatibility of BA-based compounds has inspired the exploration of novel chemistries using boron to fuel emergent sciences. This topical review focuses on the recent progress of iminoboronate and salicylhydroxamic-boronate constituted reversible click chemistries in the past decade. We highlight the mechanism of reversible kinetics and its applications in chemical biology, medicinal chemistry, biomedical devices, and material chemistry. This article also emphasizes the fundamental reactivity of these two conjugate chemistries with assorted nucleophiles at variable pHs, which is of utmost importance to any stimuli-responsive biological and material chemistry explorations.

An explicitly designed paratope of amyloid-ß prevents neuronal apoptosis in vitro and hippocampal damage in rat brain.

Synthetic antibodies hold great promise in combating diseases, diagnosis, and a wide range of biomedical applications. However, designing a therapeutically amenable, synthetic antibody that can arrest the aggregation of amyloid-ß (Aß) remains challenging. Here, we report a flexible, hairpin-like synthetic paratope (SP1, ∼2 kDa), which prevents the aggregation of Aß monomers and reverses the preformed amyloid fibril to a non-toxic species. Structural and biophysical studies further allowed dissecting the mode and affinity of molecular recognition events between SP1 and Aß. Subsequently, SP1 reduces Aß-induced neurotoxicity, neuronal apoptosis, and ROS-mediated oxidative damage in human neuroblastoma cells (SH-SY5Y). The non-toxic nature of SP1 and its ability to ameliorate hippocampal neurodegeneration in a rat model of AD demonstrate its therapeutic potential. This paratope engineering module could readily implement discoveries of cost-effective molecular probes to nurture the basic principles of protein misfolding, thus combating related diseases.

Author Correction: In-solution enrichment identifies peptide inhibitors of protein-protein interactions.

In the version of this article originally published, the peptide sequences of compounds 90, 92 and 93 in Fig. 5b and Supplementary Table 7 contained several errors. In Fig. 5b, position 6 of compound 90 should be Tyr instead of Phe. In both Fig. 5b and Supplementary Table 7, position 9 of compounds 92 and 93 should be Gln instead of Glu. Additionally, the surname of co-author Anupam Bandyopadhyay was incorrectly spelled as Bandyopdhyay. The errors have been corrected in the HTML and PDF versions of the paper and in the Supplementary Information PDF.

In-solution enrichment identifies peptide inhibitors of protein-protein interactions.

The use of competitive inhibitors to disrupt protein-protein interactions (PPIs) holds great promise for the treatment of disease. However, the discovery of high-affinity inhibitors can be a challenge. Here we report a platform for improving the affinity of peptide-based PPI inhibitors using non-canonical amino acids. The platform utilizes size exclusion-based enrichment from pools of synthetic peptides (1.5-4 kDa) and liquid chromatography-tandem mass spectrometry-based peptide sequencing to identify high-affinity binders to protein targets, without the need for 'reporter' or 'encoding' tags. Using this approach-which is inherently selective for high-affinity binders-we realized gains in affinity of up to ~100- or ~30-fold for binders to the oncogenic ubiquitin ligase MDM2 or HIV capsid protein C-terminal domain, which inhibit MDM2-p53 interaction or HIV capsid protein C-terminal domain dimerization, respectively. Subsequent macrocyclization of select MDM2 inhibitors rendered them cell permeable and cytotoxic toward cancer cells, demonstrating the utility of the identified compounds as functional PPI inhibitors.

Educational and Wealth Inequalities in Smokeless Tobacco Use: An Analysis of Rural-Urban Areas of Bangladesh and India.

This study aims to investigate the educational and wealth inequalities in smokeless tobacco (SLT) use in rural and urban areas of Bangladesh and India, the 2 largest global SLT users. Using the Global Adult Tobacco Survey, both absolute and relative measures of inequality were estimated. The analysis reveals that the educational inequalities in SLT use were higher in urban areas of India and in rural areas of Bangladesh, whereas the wealth inequalities in SLT use were higher in urban areas of both the countries. Moreover, the logit model showed that the odds of SLT use declined with an increase in the level of education and wealth in rural and urban areas of India. However, no consistent pattern was observed in rural and urban areas of Bangladesh. The findings clearly delineate the subgroups which require immediate attention for SLT cessation interventions in these 2 countries.

Gender-based tobacco policies in Bangladesh and India: A prerequisite to counter the smokeless tobacco epidemic.

This study examined gender differences in patterns of smokeless tobacco (SLT) use among various socioeconomic and demographic segments in Bangladesh and India, which are home to 80 percent of global SLT users and share similar sociocultural milieus. The objective was to provide evidence of whether gender-focused interventions and policies might be helpful for SLT cessation programs. Data from the Global Adult Tobacco Survey were used for the analysis. In Bangladesh, data were collected between July and August 2009, and 9,629 individuals were successfully interviewed. In India, data were collected from June 2009 to January 2010, and 69,296 individuals were interviewed. Tests of proportions revealed that the prevalence of SLT use was highest among women in Bangladesh (27.9 percent) and among men (32.9 percent) in India. Logistic regression models revealed that the odds of SLT use was lower among Bangladeshi females in the highest age category. However, in India, a similar observation was made only for females with rural residences. In addition, the odds of SLT use was less among those with a higher level of education and wealth, irrespective of the sex or country. The present findings suggest a need for gender-specific policies and interventions for SLT control.

Radiolabeled Cationic Peptides for Targeted Imaging of Infection.

Molecular probes targeting bacteria provide opportunities to target bacterial infections in vivo for both imaging and therapy. In the current study, we report the development of positron emission tomography (PET) probes for imaging of live bacterial infection based on the small molecules HLys-DOTA, a polycationic peptide synthesized as the D-isomer (RYWVAWRNRG) conjugated to 1, 4, 7, 10-tetraazacyclododecane-N',N″,N‴,N-tetraacetic acid (DOTA) and AB1-HLys-DOTA, which includes an unnatural amino acid AB1 that preferentially binds to bacteria membrane lipids with amine groups via formation of iminoboronates. HLys-DOTA and AB1-HLys-DOTA peptides were radiolabeled with 64Cu and investigated as PET imaging agents to track bacterial infection in vitro and in intramuscularly infected (IM) mice models. Cell uptake studies at 37°C in Staphylococcus aureus (SA) show higher uptake of 64Cu-AB1-HLys-DOTA; 98.47 ± 3.54% vs 64Cu-HLys-DOTA; 39.12 ± 3.27% at 24 h. Standard uptake values (SUV) analysis of the PET images resulted in mean SUV of 0.70 ± 0.08, 0.49 ± 0.04, and 0.31 ± 0.01 for 64Cu-AB1-HLys-DOTA and 0.17 ± 0.06, 0.16 ± 0.02, and 0.13 ± 0.01 for 64Cu-HLys-DOTA at 1, 4, and 24 h post injection, respectively, in the infected muscles. Similarly, in the biodistribution studies, dose uptake in the infected muscles was 4 times higher in the targeted 64Cu-AB1-HLys-DOTA group than in the 64Cu-HLys-DOTA group and 2-3 times higher than in the PBS control group at 1, 4, and 24 h post injection. 64Cu-AB1-HLys-DOTA was able to distinguish between SA-infected muscle and Pseudomonas aeruginosa (PA) infected muscle with lower mean SUV of 0.28 ± 0.10 at 1 h post injection. This illustrates the utility of the AB1 covalently targeting group in synergy with the HLys peptide, which noncovalently binds to bacterial membranes. These results suggest that 64Cu-labeled AB1-HLys-DOTA peptide could be used as an imaging probe for detection of bacterial infection in vivo with specificity for Gram-positive bacteria.