The process of structure-based drug design

The field of structure-based drug design is a rapidly growing area in which many successes have occurred in recent years. The explosion of genomic, proteomic, and structural information has provided hundreds of new targets and opportunities for future drug lead discovery. This review summarizes the process of structure-based drug design and includes, primarily, the choice of a target, the evaluation of a structure of that target, the pivotal questions to consider in choosing a method for drug lead discovery, and evaluation of the drug leads. Key principles in the field of structure-based drug design will be illustrated through a case study that explores drug design for AmpC beta-lactamase.     

Parallel and multiplexed bead-based assays and encoding strategies

Advances in high throughput screening (HTS), together with the rapid progress in combinatorial chemistry, genomic and proteomic sciences have dramatically stimulated the development of a variety tools to enable the drug discovery process to become more efficient. Major future challenges in HTS include obtaining high density and good quality data based on assays that are rapid, reliable, inexpensive, sensitive, simple and miniaturised. This paper reviews the development and role of bead-based assays for HTS including DNA and single nucleotide polymorphism (SNP) assays, particularly from a multiplex perspective and evaluating the recent advances in bead-based arrays. The encoding strategies that are commonly used in bead-based assays are highlighted, while the importance of magnetic beads in genomic and proteomic purifications is discussed. In conclusion, bead-based assays offer a powerful promising approach for many aspects of drug discovery.     

化学生物学新前沿——化学蛋白质组学

随着包括人类在内的主要模式生物的基因组计划的完成,生命科学的研究重心转向蛋白质组的研究--在对应基因组的整体蛋白质水平上系统研究调控细胞生命活动的蛋白质.化学蛋白质组学是化学生物学在后基因组时代的最新发展:化学蛋白质组学利用化学小分子为工具和手段,以基于靶蛋白质功能的新战略探测体内蛋白质组,是新一代的功能蛋白质组学.本文综述了化学蛋白质组学的最新进展、有关技术及其在生物医学和药物研发等方面的应用,并对化学蛋白质组学的发展趋势和前景进行了讨论.     

Proteomics and Its Application in Biomarker Discovery and Drug Development

Proteomics is a research field aiming to characterize molecular and cellular dynamics in protein expression and function on a global level. The introduction of proteomics has been greatly broadening our view and accelerating our path in various medical researches. The most significant advantage of proteomics is its ability to examine a whole proteome or sub-proteome in a single experiment so that the protein alterations corresponding to a pathological or biochemical condition at a given time c…     

How chemoproteomics can enable drug discovery and development

Creating first-in-class medications to treat human disease is an extremely challenging endeavor. While genome sequencing and genetics are making direct connections between mutations and human disorders at an unprecedented rate, matching molecular targets with a suitable therapeutic indication must ultimately be achieved by pharmacology. Here, we discuss how the integration of chemical proteomic platforms (such as activity-based protein profiling) into the earliest stages of the drug discovery process has the potential to greatly expand the scope of proteins that can be pharmacologically evaluated in living systems, and, through doing so, promote the identification and prioritization of new therapeutic targets.     

Proteome‐Wide Profiling of Targets of Cysteine reactive Small Molecules by Using Ethynyl Benziodoxolone Reagents

In this study, we present a highly efficient method for proteomic profiling of cysteine residues in complex proteomes and in living cells. Our method is based on alkynylation of cysteines in complex proteomes using a “clickable” alkynyl benziodoxolone bearing an azide group. This reaction proceeds fast, under mild physiological conditions, and with a very high degree of chemoselectivity. The formed azide‐capped alkynyl–cysteine adducts are readily detectable by LC‐MS/MS, and can be further functionalized with TAMRA or biotin alkyne via CuAAC. We demonstrate the utility of alkynyl benziodoxolones for chemical proteomics applications by identifying the proteomic targets of curcumin, a diarylheptanoid natural product that was and still is part of multiple human clinical trials as anticancer agent. Our results demonstrate that curcumin covalently modifies several key players of cellular signaling and metabolism, most notably the enzyme casein kinase I gamma. We anticipate that this new method for cysteine profiling will find broad application in chemical proteomics and drug discovery.     

Activity-based probes: discovering new biology and new drug targets

The development and application of chemical technologies enabling direct analysis of enzyme activity in living systems has undergone explosive growth in recent years. Activity-based protein profiling (ABPP) is a key constituent of this broad field, and is among the most powerful and mature chemical proteomic technologies. This tutorial review introduces the essential features of ABPP and the design and application of activity-based probes (ABPs) from drug target elucidation and in vivo visualisation of enzyme activity to comprehensive profiling of the catalytic content of living systems, and the discovery of new biological pathways.     

Clinical perspectives of high-resolution mass spectrometry-based proteomics in neuroscience: exemplified in amyotrophic lateral sclerosis biomarker discovery research

Biomarker discovery is a central application in today's proteomic research. There is an urgent need for valid biomarkers to improve diagnostic tools and treatment in many disorders, such as the rapidly progressing neurodegenerative disorder amyotrophic lateral sclerosis (ALS) that has a fatal outcome in about 3 years and yet no curative treatment. Screening for clinically relevant biomarkers puts high demands on high-throughput, rapid and precise proteomic techniques. There is a large variety in the methods of choice involving mainly gel-based approaches as well as chromatographic techniques for multi-dimensional protein and peptide separations followed by mass spectrometry (MS) analysis. This special feature article will discuss some important aspects of MS-based clinical proteomics and biomarker discovery in the field of neurodegenerative diseases and ALS research respectively, with the aim to provide a prospective view on current and future research aspects in the field. Furthermore, examples for application of high-resolution MS-based proteomic strategies for ALS biomarker discovery will be demonstrated with two studies previously reported by our group. These studies include among others, utilization of capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS) for advanced protein pattern classification in cerebrospinal fluid (CSF) samples of ALS patients as well as highly sensitive protein identification in minimal amounts of postmortem spinal cord tissue and laser micro-dissected motor neurons using FT-ICR-MS in conjunction with nanoflow LC coupled to matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (LC-MALDI-TOF-TOF-MS).     

Effect of quality characteristics of single sample preparation steps in the precision and coverage of proteomic studies—A review

Proteomic profiling and biomarker search are analytical tools as many other. Nevertheless, in the proteomic discovery phase considerable sample fractionation is inevitable before readout. Since these procedures are of notable complexity, proteomic tools need in particular analytical quality validation standards as prevail for other analytical methods. With acceptance of the rule of error propagation the values of imprecision and yield of each preparation step determine overall reproducibility and therewith information harvest of a propagated method series. Thereto, we examined recent proteomic reports with reproducibility data and with parallelization, and automation approaches. Based on the data available from literature it is highly probable, that at least a part of current proteomic platforms actually suffer from high technical variance.     

Limitations of current proteomics technologies

Application of proteomics technologies in the investigation of biological systems creates new possibilities in the elucidation of biopathomechanisms and the discovery of novel drug targets and early disease markers. A proteomic analysis involves protein separation and protein identification as well as characterization of the post-translational modifications. Proteomics has been applied in the investigation of various disorders, like neurological diseases, and the application has resulted in the detection of a large number of differences in the levels and the modifications of proteins between healthy and diseased states. However, the current proteomics technologies are still under development and show certain limitations. In this article, we discuss the major drawbacks and pitfalls of proteomics we have observed in our laboratory and in particular during the application of proteomics technologies in the investigation of the brain.     

A new class of HIV-1 inhibitors and the target identification via proteomic profiling

Anti-HIV screening with the MT-4/MTT assay on a focused library of structurally diverse natural products has led to the discovery of a group of steroids with potent activities, which include four new ergostane-type steroids, named amotsterols A-D (1-4), together with two known analogs. Among them, the most potent amotsterol D (4) exhibited anti-HIV activity against wildtype and some clinically relevant multidrug resistant HIV-1 strains. Subsequent studies on its target identification through a proteomic approach found that compound 4 might target PKM2, a rate limiting enzyme of glycolysis, in host cells to restrict HIV replication. The docking model of compound 4 to PKM2 showed that the two hydroxyl groups of 4 form hydrogen bonds with the two parallel Y390 in each subunit of PKM2 separately, and the ring C of 4 is sandwiched between the two parallel aromatic rings of F26. The identified hit compound may have the potential to be further developed as a novel anti-HIV agent. These results demonstrated that an integrated approach, which combines new chemical structures and phenotypic screening with a proteomic approach, could not only identify novel HIV-1 inhibitors, but also elucidate the unknown targets of compound interactions in antiviral drug discovery.     

Stalis: A Computational Method for Template-Based Ab Initio Ligand Design

Proteins interact with small molecules through specific molecular recognition, which is central to essential biological functions in living systems. Therefore, understanding such interactions is crucial for basic sciences and drug discovery. Here, we present S tructure t emplate-based a b initio li gand design s olution (Stalis), a knowledge-based approach that uses structure templates from the Protein Data Bank libraries of whole ligands and their fragments and generates a set of molecules (virtual ligands) whose structures represent the pocket shape and chemical features of a given target binding site. Our benchmark performance evaluation shows that ligand structure-based virtual screening using virtual ligands from Stalis outperforms a receptor structure-based virtual screening using AutoDock Vina, demonstrating reliable overall screening performance applicable to computational high-throughput screening. However, virtual ligands from Stalis are worse in recognizing active compounds at the small fraction of a rank-ordered list of screened library compounds than crystal ligands, due to the low resolution of the virtual ligand structures. In conclusion, Stalis can facilitate drug discovery research by designing virtual ligands that can be used for fast ligand structure-based virtual screening. Moreover, Stalis provides actual three-dimensional ligand structures that likely bind to a target protein, enabling to gain structural insight into potential ligands. Stalis can be an efficient computational platform for high-throughput ligand design for fundamental biological study and drug discovery research at the proteomic level. © 2019 Wiley Periodicals, Inc.     

A comprehensive protein resource for the study of bladder cancer: http://biobase.dk/cgi-bin/celis

In our laboratories we are exploring the possibility of using proteome expression profiles of fresh bladder tumors (transitional cell carcinomas, TCCs; squamous cell carcinomas, SCCs) and random biopsies as fingerprints to subclassify histopathological types and as a starting point to search for protein markers that may form the basis for diagnosis, prognosis, and treatment. Ultimately, the goal of these studies is to identify signaling pathways and components that are affected at various stages of bladder cancer progression and that may provide novel leads in drug discovery. Here we present our ongoing efforts to establish comprehensive two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) databases of TCCs and SCCs which are being constructed based on the proteomic and immunohistochemical analysis of hundreds of fresh tumors, random biopsies and cystectomies received shortly after operation (http://biobase.dk/cgi-bin/celis).     

A Non‐Competitive Inhibitor of VCP/p97 and VPS4 Reveals Conserved Allosteric Circuits in Type I and II AAA ATPases

AAA ATPases have pivotal functions in diverse cellular processes essential for survival and proliferation. Revealing strategies for chemical inhibition of this class of enzymes is therefore of great interest for the development of novel chemotherapies or chemical tools. Here, we characterize the compound MSC1094308 as a reversible, allosteric inhibitor of the type II AAA ATPase human ubiquitin‐directed unfoldase (VCP)/p97 and the type I AAA ATPase VPS4B. Subsequent proteomic, genetic and biochemical studies indicate that MSC1094308 binds to a previously characterized drugable hotspot of p97, thereby inhibiting the D2 ATPase activity. Our results furthermore indicate that a similar allosteric site exists in VPS4B, suggesting conserved allosteric circuits and drugable sites in both type I and II AAA ATPases. Our results may thus guide future chemical tool and drug discovery efforts for the biomedically relevant AAA ATPases.     

Microfluidic chip-based liquid chromatography coupled to mass spectrometry for determination of small molecules in bioanalytical applications

The development and integration of microfabricated liquid chromatography (LC) microchips have increased dramatically in the last decade due to the needs of enhanced sensitivity and rapid analysis as well as the rising concern on reducing environmental impacts of chemicals used in various types of chemical and biochemical analyses. Recent development of microfluidic chip-based LC mass spectrometry (chip-based LC-MS) has played an important role in proteomic research for high throughput analysis. To date, the use of chip-based LC-MS for determination of small molecules, such as biomarkers, active pharmaceutical ingredients (APIs), and drugs of abuse and their metabolites, in clinical and pharmaceutical applications has not been thoroughly investigated. This mini-review summarizes the utilization of commercial chip-based LC-MS systems for determination of small molecules in bioanalytical applications, including drug metabolites and disease/tumor-associated biomarkers in clinical samples as well as adsorption, distribution, metabolism, and excretion studies of APIs in drug discovery and development. The different types of commercial chip-based interfaces for LC-MS analysis are discussed first and followed by applications of chip-based LC-MS on biological samples as well as the comparison with other LC-MS techniques.     

A magnetic bead‐based serum proteomic fingerprinting method for parallel analytical analysis and micropreparative purification

ProteinChip surface‐enhanced laser desorption/ionization technology and magnetic beads‐based ClinProt system are commonly used for semi‐quantitative profiling of plasma proteome in biomarker discovery. Unfortunately, the proteins/peptides detected by MS are non‐recoverable. To obtain the protein identity of a MS peak, additional time‐consuming and material‐consuming purification steps have to be done. In this study, we developed a magnetic beads‐based proteomic fingerprinting method that allowed semi‐quantitative proteomic profiling and micropreparative purification of the profiled proteins in parallel. The use of different chromatographic magnetic beads allowed us to obtain different proteomic profiles, which were comparable to those obtained by the ProteinChip surface‐enhanced laser desorption/ionization technology. Our assays were semi‐quantitative. The normalized peak intensity was proportional to concentration measured by immunoassay. Both intra‐assay and inter‐assay coefficients of variation of the normalized peak intensities were in the range of 4–30%. Our method only required 2 μL of serum or plasma for generating enough proteins for semi‐quantitative profiling by MALDI‐TOF‐MS as well as for gel electrophoresis and subsequent protein identification. The protein peaks and corresponding gel spots could be easily matched by comparing their intensities and masses. Because of its high efficiency and reproducibility, our method has great potentials in clinical research, especially in biomarker discovery.