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Historically, covalent drugs were avoided in drug development process due to the possible toxicity linked to the covalent binding of such drugs and cellular proteins. However, recent years have witnessed the fast resurgence of the discovery of covalent drugs because of the realization of the advantages of covalent drugs in efficacy, duration of action, therapy-induced resistance, and targeting hard targets. Since December 2019, SARS-CoV-2 has caused nearly 40 million COVID-19 patients and over one million deaths in the whole world as of October 19, 2020. In contrast, effective drugs have not been found at the same time. Therefore, it is of great value to discover and design drugs for the prevention and cure of SARS-CoV-2 infection. Recently, we developed a simple but efficient method named as SCARdock for the discovery of covalent drugs. In this work, we present a detailed protocol of this method in discovering potential anti-SARS-CoV-2 drugs.

The post-translational modification of cellular proteins by ubiquitin (Ub), called ubiquitylation, is indispensable for the normal growth and development of eukaryotic organisms. In order to conduct studies that elucidate the precise mechanistic roles for Ub, access to site-specifically and homogenously ubiquitylated proteins and peptides is critical. However, the low abundance, heterogeneity, and dynamic nature of protein ubiquitylation are significant limitations toward such studies. Here we provide a facile expressed protein ligation method that does not require specialized apparatus and permits the rapid semisynthesis of ubiquitylated peptides by using the atom-efficient ligation auxiliary 2-aminooxyethanethiol.

The genome of equine arteritis virus (EAV) produces a 3' coterminal-nested set of six subgenomic (sg) viral RNAs during virus replication cycle, and each set possesses a common leader sequence of 206 nucleotides (nt) in length derived from the 5' end of the viral genome. Given the presence of the leader region within both genomic and sg mRNAs, it is likely to contain cis-acting signals that may interact with cellular or viral proteins for RNA synthesis. Gel mobility shift assays indicated that proteins in Vero cell cytoplasmic extracts formed complexes with the positive (+) and negative (-) strands of the EAV leader RNA. Several cell proteins with molecular masses ranging from 74 to 31 kDa and 58 to 32 kDa were detected in UV-induced cross-linking assays with the EAV leader RNA (+) and (-) strands, respectively. In both cases, intense bands were observed at the 58-52 kDa molecular weight markers. Results from competition gel mobility shift assays using overlapping cold RNA probes spanning the leader RNA (+) strand indicated that nt 140-206 are not necessary for binding to cell proteins.

Polyomavirus SV40 was discovered as an unrecognized contaminant of poliovaccines and has proven to be a premier model infectious agent of cancer. The virus usurps cellular processes to accomplish viral replication and can transform cells in culture and induce tumors in laboratory animals. Characterizations of viral functions and interactions with cellular targets have revealed fundamental insights into mammalian cell biology and mechanisms of carcinogenesis. This review focuses on basic properties of SV40, with an emphasis on large T-antigen, the viral oncoprotein, and on cellular proteins targeted by T-antigen. It describes small animal models and the insights provided into viral biology and cell transformation, and also summarizes the status of SV40 human infections and viral associations with human cancer.

Viruses are intracellular parasites that commandeer cellular processes, such as RNA processing or protein synthesis, to perform virus-specific functions. For this purpose, many viral proteins shuttle between the nuclear and cytoplasmic compartments, even when the viral genome is replicated in the cytoplasm. This shutding process is usually regulated by classical nuclear import and export signals (NLSs and NESs, respeaively), which are also found in many cellular proteins and are described elsewhere in this book. In this Chapter, we will focus on viruses that replicate in the nucleus, and in particular on the mechanisms by which they transport their genomes into and out of the nucleus.

Ubiquitin is a small, evolutionarily conserved eukaryotic protein that can be attached to a wide variety of intracellular proteins including itself. Covalent attachment of ubiquitin to other proteins serves various functions, but its major role is to target cellular proteins for destruction. Cellular components that activate, transfer, remove, or simply recognize ubiquitin number in the hundreds, perhaps even in the thousands. In light of this complexity the ubiquitin pathway is ideal for a systems biology approach.

Proteinases play key roles in many essential physiological processes, and are of great importance in maintaining various cellular functions. With the advent of molecular biology in the 20th Century, remarkable progress has been made in understanding the molecular structure and function, catalytic mechanism, and evolution of proteinases. Further, it has become evident that proteinases can be classified into families, with the members of each family having similar structures and catalytic mechanisms1. In parallel with this, we also have witnessed dramatic advances in understanding the term proteolysis, which is one of the most important enzymatic protein modifications. Careful control of the levels of structural proteins, enzymes, and regulatory proteins is essential to maintain the proper cellular functioning. Generally, structural proteins or proteins that are constitutively required are longlived, whereas regulatory proteins are otten rapidly degraded. Furthermore, their degradation must be precisely timed and regulated, because unregulated degradation would be quite hazardous to cells. Ultimately, the balance between the rate of synthesis and the breakdown of cellular proteins determines the fate of cells in vivo. Thus, the highly complex and regulated mechanism, termed proteolysis, has evolved to accomplish this purpose. As indicated in Table 1, the widely used term protease, which is synonymous to peptidase, can be applied equally to both exopeptidases and endopeptidases. In contrast, the term proteinase is applied only to endopeptidases, and four mechanistic classes of proteinases are recognized by the IUBMB as described below1. This chapter will mainly describe mammalian proteinases possessing endopeptidase activity. However, to describe all mammalian proteinases is beyond the scope of this chapter. It is the aim of this chapter to summarize mammalian proteinases briefly and to explain the structural and evolutionary characteristics of their genes on the basis of recent information.

Heat shock proteins are ubiquitous proteins, present in every free-living organism from the simplest bacterium to the most highly differentiated human cells. They are highly conserved proteins; some members of the family are constitutively expressed, while others are induced by heat-shock and other forms of cellular stress, to protect the cells from environmental damage [1, 2]. Heat shock proteins function as molecular chaperones, and they are essential for proper folding and intercellular transport of many vital cellular proteins [3,4].

For more than 25 years protein identification has largely depended on automated Edman chemistry (Hewick et al., 1981) or western blotting with an appropriate monoclonal antibody. Several limitations, however, have never been overcome. The Edman procedure is inherently slow (generally one or two peptide or protein samples per day) and does not allow direct identification of many post-translational modifications. In addition, current detection limits are in the low-picomole to upper-femtomole range (Totty et al., 1992). Protein identification by western blotting can be extremely rapid, but requires the ready availability of an extensive library of suitable antibody probes. Large-format 2D-electrophoresis systems now make it possible to resolve several thousand cellular proteins from whole-cell lysates in the low- to upper-femtomole concentration range (Patton et al., 1990), presenting significant analytical challenges. The recent introduction of matrix-assisted laser-desorption (MALD) time-of-flight mass spectrometers (Karas and Hillenkamp, 1988) has led to the rapid analysis (at high sensitivity) of peptide mixtures. New strategies have been developed using a combination of protease digestion, MALD mass spectrometry and searching of peptide-mass databases that promise rapid acceleration in the identification of proteins (Henzel et al., 1993; Pappin et al., 1993; Mann et al., 1993; James et al., 1993; Yates et al., 1993).

The adenovirus E1A gene products can transactivate viral and many cellular promoters while repressing others, drive quiescent cells to enter the cell cycle and synthesize DNA, and contribute to the oncogenic transformation of cells. These diverse effects of the ElA proteins appear to result from their ability to bind to a variety of cellular proteins, altering the activity of several different transcription factors.

The human immunodeficiency virus long terminal repeat (HIV LTR) is regulated by multiple cis-acting regulatory sequences.1 Cellular factors which bind to the enhancer, SP1, TATA, and TAR regions are involved in the transcriptional regulation of HIV.2 In addition to cellular proteins, the viral protein, tat, has been shown to greatly stimulate HIV gene expression from the HIV LTR.3, 4

Infection of mammalian cells by human adenoviruses induces dramatic alterations in the cells’ abilities to continue their normal business. Indeed, adenoviruses were first recognised by their ability to kill cells which supported the efficient replication of the virus (1, 2). Such cell death is the culmination of several processes which redirect the host cells’ biosynthetic apparatus. Thus, when adenoviruses infect rapidly-dividing, fully permissive cells, they induce rapid and complete inhibition of synthesis of both cellular DNA and cellular proteins. At first sight, such redirection of cellular functions appears of obvious advantage to the virus: the relevant cellular machinery becomes devoted to the production of the components which will form progeny virions, the raison d’etre of the infectious cycle. The complete cessation of cellular DNA or protein synthesis and the vast quantities of viral macromolecules produced attest to the efficiency of the molecular mechanisms by which the virus carries out such usurption. It is, therefore, quite surprising that as little as 10% of the newly made viral material is assembled into virions (see ref. 3 for a review).

The dynamic nature of cellular proteins was demonstrated over 40 years ago (Schoenheimer et al., 1939). But it was not until 1967–1972 that the measurement of protein turnover in vivo received systematic attention, notably by Wa-terlow’s group (Waterlow and Stephen, 1967; Picou and Taylor-Roberts, 1969; Garlick, 1969; Garlick and Marshall, 1972). It is now generally accepted that cellular proteins are always subject to continual degradation even when protein is neither gained nor lost from the body.

In 1947, Henshaw, Riley, and Stapleton first proposed that aging can be caused by alterations in DNA. Unfortunately, they were a decade ahead of their time, for it was not until the early 60’s that attempts were made to resolve the extent to which cellular proteins are vulnerable to aging, and the genetic bases for the changes. One of the driving forces behind this surge of activity was Orgel (1963), who proposed that errors in transcription and translation can bring about senescence through a progressive accumulation of errors in the protein-synthesizing machinery. Although most investigators focused their efforts at the transcriptional and translational levels, a few also considered the effects of aging at the post-translational level, especially after Robinson et al. (1970) observed that deamidation of asparagine and glutamine can cause conformational changes in proteins. Suffice to say, the efforts of the 60’s and 70’s clearly documented that aging at the cellular protein level is complex and heterogenous, and therefore could be polymorphic at the genetic level.

The use of acute labeling techniques (pulse labeling) has been successful in tracing and locating rapid metabolic pathways in vivo. This procedure was used in the classical experiments of Schoenheimer [1] on labile cellular proteins which gave rise to the concept of the dynamic state of body constituents. The concept described protein turnover as a continual process of synthesis and degradation as evidenced by the active uptake and release of isotopic compounds (see Fig. 1). Turnover represented the occurrence of synthesis which cannot be detected analytically because it was exactly balanced by breakdown [2]. Acute and chronic isotopic studies have demonstrated that the fibrous proteins of muscle and connective tissues have the least active turnover and are considered to be relatively inert metabolically [3, 4]—inert in the sense that the proteins were nonrenewable. However, these studies could not distinguish between essentially non isotopic turnover (nonsynthetic and nondestructive) of macromolecules and absolute inertness.