D-Peptide-Based Drug Discovery Aided by Chemical Protein Synthesis

Despite a sharp increase in the expenditures for drug research and development (R&D) in the past decade, the declining trend in the number of new drugs approved annually by the US Food and Drug Administration continues. This growing disparity between R&D investment and new drug approvals results in part from the deficiency in promising therapeutic targets and leads to a stagnation exacerbated by the lack of advanced drug discovery tools for harvesting the “high-hanging fruits” such as inhibitors of protein–protein interactions (PPIs). Small peptide inhibitors of PPIs can be of high affinity and specificity, promising an important class of therapeutic agents that target PPIs involved in a great variety of biological processes. However, susceptibility to proteolytic degradation in vivo still remains a major hurdle that limits their therapeutic potential. This limitation can be overcome by mirror-image phage display, a technique that allows, through phage-expressed peptide library screening against the D -enantiomer of a target protein, for the identification of proteolysis-resistant D -peptide inhibitors of PPIs. Recent advances in total protein synthesis via native chemical ligation have significantly expanded the scope of molecular targets for mirror-image phage display. This concise review focuses on the latest development in the combined use of mirror-image phage display and native chemical ligation for D -peptide based anticancer drug discovery.     

Chemical Protein Modification through Cysteine

The modification of proteins with non‐protein entities is important for a wealth of applications, and methods for chemically modifying proteins attract considerable attention. Generally, modification is desired at a single site to maintain homogeneity and to minimise loss of function. Though protein modification can be achieved by targeting some natural amino acid side chains, this often leads to ill‐defined and randomly modified proteins. Amongst the natural amino acids, cysteine combines advantageous properties contributing to its suitability for site‐selective modification, including a unique nucleophilicity, and a low natural abundance—both allowing chemo‐ and regioselectivity. Native cysteine residues can be targeted, or Cys can be introduced at a desired site in a protein by means of reliable genetic engineering techniques. This review on chemical protein modification through cysteine should appeal to those interested in modifying proteins for a range of applications.     

Lessons in Organic Fluorescent Probe Discovery

Fluorescent probes have gained profound use in biotechnology, drug discovery, medical diagnostics, molecular and cell biology. The development of methods for the translation of fluorophores into fluorescent probes continues to be a robust field for medicinal chemists and chemical biologists, alike. Access to new experimental designs has enabled molecular diversification and led to the identification of new approaches to probe discovery. This review provides a synopsis of the recent lessons in modern fluorescent probe discovery.     

Protein microarrays and biomarkers of infectious disease

Protein microarrays are powerful tools that are widely used in systems biology research. For infectious diseases, proteome microarrays assembled from proteins of pathogens will play an increasingly important role in discovery of diagnostic markers, vaccines, and therapeutics. Distinct formats of protein microarrays have been developed for different applications, including abundance-based and function-based methods. Depending on the application, design issues should be considered, such as the need for multiplexing and label or label free detection methods. New developments, challenges, and future demands in infectious disease research will impact the application of protein microarrays for discovery and validation of biomarkers.     

Current Status and Future Perspectives of Mass Spectrometry Imaging

Mass spectrometry imaging is employed for mapping proteins, lipids and metabolites in biological tissues in a morphological context. Although initially developed as a tool for biomarker discovery by imaging the distribution of protein/peptide in tissue sections, the high sensitivity and molecular specificity of this technique have enabled its application to biomolecules, other than proteins, even in cells, latent finger prints and whole organisms. Relatively simple, with no requirement for labelling, homogenization, extraction or reconstitution, the technique has found a variety of applications in molecular biology, pathology, pharmacology and toxicology. By discriminating the spatial distribution of biomolecules in serial sections of tissues, biomarkers of lesions and the biological responses to stressors or diseases can be better understood in the context of structure and function. In this review, we have discussed the advances in the different aspects of mass spectrometry imaging processes, application towards different disciplines and relevance to the field of toxicology.     

Recent Progress in Strategies for the Creation of Protein‐Based Fluorescent Biosensors

The creation of novel bioanalytical tools for the detection and monitoring of a range of important target substances and biological events in vivo and in vitro is a great challenge in chemical biology and biotechnology. Protein‐based fluorescent biosensors—integrated devices that convert a molecular‐recognition event to a fluorescent signal—have recently emerged as a powerful tool. As the recognition units various proteins that can specifically recognize and bind a variety of molecules of biological significance with high affinity are employed. For the transducer, fluorescent proteins, such as green fluorescent protein (GFP) or synthetic fluorophores, are mostly adopted. Recent progress in protein engineering and organic synthesis allows us to manipulate proteins genetically and/or chemically, and a library of such protein scaffolds has been significantly expanded by genome projects. In this review, we briefly describe the recent progress of protein‐based fluorescent biosensors on the basis of their platform and construction strategy, which are primarily divided into the genetically encoded fluorescent biosensors and chemically constructed biosensors.     

Expression of intein‐tagged fusion protein and its applications in downstream processing

The conventional methods of downstream purification of a recombinant protein are not only complicated and delicate but time consuming, and need to be improved. Since the intein, the protein splicing element, was discovered, this self‐cleaving element has been exploited and applied to the purification of recombinant proteins which could significantly simplify the purification procedure. Intein has the unique property that when it is combined with an affinity tag, it enables a target protein to be purified in a single chromatographic step. This review elucidates the properties of intein (the mechanism that unravels the intein‐based protein splicing), the advantages of an intein affinity expression system, the progress of intein‐based protein purification procedures, and recent advances in the applications of intein. Further development of the intein‐based purification system may lead to the applications of this system to industrial‐scale production of recombinant proteins. Copyright © 2009 Society of Chemical Industry     

Chiral vibrational structures of proteins at interfaces probed by sum frequency generation spectroscopy

We review the recent development of chiral sum frequency generation (SFG) spectroscopy and its applications to study chiral vibrational structures at interfaces. This review summarizes observations of chiral SFG signals from various molecular systems and describes the molecular origins of chiral SFG response. It focuses on the chiral vibrational structures of proteins and presents the chiral SFG spectra of proteins at interfaces in the C-H stretch, amide I, and N-H stretch regions. In particular, a combination of chiral amide I and N-H stretches of the peptide backbone provides highly characteristic vibrational signatures, unique to various secondary structures, which demonstrate the capacity of chiral SFG spectroscopy to distinguish protein secondary structures at interfaces. On the basis of these recent developments, we further discuss the advantages of chiral SFG spectroscopy and its potential application in various fields of science and technology. We conclude that chiral SFG spectroscopy can be a new approach to probe chiral vibrational structures of protein at interfaces, providing structural and dynamic information to study in situ and in real time protein structures and dynamics at interfaces.     

Harnessing the Chemistry of the Indole Heterocycle to Drive Discoveries in Biology and Medicine

Indole-containing compounds demonstrate an array of biological activities relevant to numerous human diseases. The biological activities of diverse indole-based agents are driven by molecular interactions between indole agent and critical therapeutic target. The chemical inventory of medicinally useful or promising indole compounds spans the entire structural spectrum, from simple synthetic indoles to highly complex indole alkaloids. In an analogous fashion, the chemistry behind the indole heterocycle is unique and provides rich opportunities for extensive synthetic chemistry, enabling the construction and development of novel indole compounds to explore chemical space. This review will present heterocyclic chemistry of the indole nucleus, indole compounds of clinical use, complex indole alkaloids and indole-inspired discovery efforts by multiple research groups interested in using novel indole-containing small molecules to drive discoveries in human biology and medicine.     

Exploiting Structural Modelling Tools to Explore Host-Translocated Effector Proteins

Oomycete and fungal interactions with plants can be neutral, symbiotic or pathogenic with different impact on plant health and fitness. Both fungi and oomycetes can generate so-called effector proteins in order to successfully colonize the host plant. These proteins modify stress pathways, developmental processes and the innate immune system to the microbes’ benefit, with a very different outcome for the plant. Investigating the biological and functional roles of effectors during plant–microbe interactions are accessible through bioinformatics and experimental approaches. The next generation protein modeling software RoseTTafold and AlphaFold2 have made significant progress in defining the 3D-structure of proteins by utilizing novel machine-learning algorithms using amino acid sequences as their only input. As these two methods rely on super computers, Google Colabfold alternatives have received significant attention, making the approaches more accessible to users. Here, we focus on current structural biology, sequence motif and domain knowledge of effector proteins from filamentous microbes and discuss the broader use of novel modelling strategies, namely AlphaFold2 and RoseTTafold, in the field of effector biology. Finally, we compare the original programs and their Colab versions to assess current strengths, ease of access, limitations and future applications.     

Structure-Based Drug Discovery with Deep Learning**

Artificial intelligence (AI) in the form of deep learning has promise for drug discovery and chemical biology, for example, to predict protein structure and molecular bioactivity, plan organic synthesis, and design molecules de novo. While most of the deep learning efforts in drug discovery have focused on ligand-based approaches, structure-based drug discovery has the potential to tackle unsolved challenges, such as affinity prediction for unexplored protein targets, binding-mechanism elucidation, and the rationalization of related chemical kinetic properties. Advances in deep-learning methodologies and the availability of accurate predictions for protein tertiary structure advocate for a renaissance in structure-based approaches for drug discovery guided by AI. This review summarizes the most prominent algorithmic concepts in structure-based deep learning for drug discovery, and forecasts opportunities, applications, and challenges ahead.     

Point-of-Care Pathogen Detection with CRISPR-based Programmable Nucleic Acid Binding Proteins

The contemporary discovery of extremely versatile engineered nucleic acid-binding proteins has transformed a brave new world in the genome-editing scientific area. Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated programmable nucleic acid-binding proteins have brought about a revolution in diagnostic platforms. The groundbreaking finding that bacteria and archaea that harbored prokaryotes have transmitted adaptive immunity through CRISPR and CRISPR-associated (Cas) proteins has driven revolutionary advances in molecular biology. Importantly, advances in gene editing focus how expanding visions in CRISPR-Cas biology are revolutionizing the area of molecular diagnostics for identifying DNA and RNA in emerging microbiological pathogens, for single nucleotide polymorphism (SNP) identifications, and for cell-free mutation. Recent advances, such as improvements in multiplexing and quantitative capabilities as well as instrument-free detection of nucleic acids, will potentially leverage the introduction of these novel technologies to detecting bacteria and viruses at the point of care (POC). In this review, we highlight the fundamental features of CRISPR/Cas-based molecular diagnostic technologies and summarize a vision of the next applications for identifying pathogens in POC settings.     

Charting a New Path Towards Degrading Every Protein

Strategies to directly alter protein abundance such as small-molecule-induced targeted protein degradation (TPD) are innovative pharmacological modalities with promising clinical potential. Herein, I describe my experience with the development of the degradation tag (dTAG) system, which is a chemical biology strategy to induce rapid and precise degradation of any target protein. Open-source collaborative discovery has been critical for advancing the versatility and accessibility of the dTAG system and will be necessary to understand the benefits and limits of TPD-based strategies in the clinic.     

蛋白质化学修饰的研究进展

蛋白质分子具有极其复杂的结构层次，用化学修饰的方法研究蛋白质分子的结构与功能的关系一直是生物化学和分子生物学领域的热点。人们研究出许多小分子化学修饰剂并进行了多种类型的化学修饰。综述了蛋白质化学修饰领域的研究现状与水平，同时强调蛋白质的化学修饰是生化药物研究开发的重要手段之一。     

金纳米棒的合成、光学特性、修饰及其在生物学中的应用

由于具有独特的光学和电子特性,金纳米棒受到人们越来越多的关注。金纳米棒的这些性质主要取决于它自身的形状、大小和长径比。尤其金纳米棒独特的、可调的表面等离子体共振特性,使其在生物标记、生物成像及生物医学等领域有非常广阔的应用前景。本论文详细介绍了金纳米棒的几种合成方法及其光学特性和金纳米棒的表面修饰手段,综述和比较了金纳米棒在生物分子探针技术、荧光探针和癌症诊断和光热治疗领域的研究进展,对其存在的问题做了具体分析并对金纳米棒在生物学中的应用方向进行了展望。     

Multiplexed Delivery of Synthetic (Un)Conjugatable Ubiquitin and SUMO2 Enables Simultaneous Monitoring of Their Localization and Function in Live Cells

Ubiquitin (Ub) and its related small Ub like modifier (SUMO) are among the most influential protein post-translational modifications in eukaryotes. Unfortunately, visualizing these modifications in live cells is a challenging task. Chemical protein synthesis offers great opportunities in studying and further understanding Ub and SUMO biology. Nevertheless, the low cell permeability of proteins limits these studies mainly for in vitro applications. Here, we introduce a multiplexed protein cell delivery approach, termed MBL (multiplexed bead loading), for simultaneous loading of up to four differentially labeled proteins with organic fluorophores. We applied MBL to visualize ubiquitination and SUMOylation events in live and untransfected cells without fluorescent protein tags or perturbation to their endogenous levels. Our study reveals unprecedented involvements of Ub and SUMO2 in lysosomes depending on conjugation states. We envision that this approach will improve our understanding of dynamic cellular processes such as formation and disassembly of membraneless organelles.     

Smart Design of Small-Molecule Libraries: When Organic Synthesis Meets Cheminformatics

Synthetic chemists are always looking for new methods to maximize the diversity and complexity of small-molecule libraries. Diversity-oriented synthesis can give access to new chemotypes with high chemical diversity, exploiting complexity-generating reactions and divergent approaches. However, there is a need for new tools to drive synthetic efforts towards unexplored and biologically relevant regions of chemical space. Because the number of publicly accessible biological data will increase in the years to come, cheminformatics can represent a real opportunity to develop better chemical libraries. This minireview focuses on novel cheminformatics approaches used to design molecular scaffolds, as well as to analyze their quality, giving a perspective of them in the field of chemical biology and drug discovery through some selected case studies.     

Circular Nucleic Acids: Discovery,Functions and Applications

Circular nucleic acids (CNAs) are nucleic acid molecules with a closed-loop structure. This feature comes with a number of advantages including complete resistance to exonuclease degradation, much better thermodynamic stability, and the capability of being replicated by a DNA polymerase in a rolling circle manner. Circular functional nucleic acids, CNAs containing at least a ribozyme/DNAzyme or a DNA/RNA aptamer, not only inherit the advantages of CNAs but also offer some unique application opportunities, such as the design of topology-controlled or enabled molecular devices. This article will begin by summarizing the discovery, biogenesis, and applications of naturally occurring CNAs, followed by discussing the methods for constructing artificial CNAs. The exploitation of circular functional nucleic acids for applications in nanodevice engineering, biosensing, and drug delivery will be reviewed next. Finally, the efforts to couple functional nucleic acids with rolling circle amplification for ultra-sensitive biosensing and for synthesizing multivalent molecular scaffolds for unique applications in biosensing and drug delivery will be recapitulated.