The rise of covalent proteolysis targeting chimeras

Targeted protein degradation offers several advantages over direct inhibition of protein activity and is gaining increasing interest in chemical biology and drug discovery. Proteolysis targeting chimeras (PROTACs) in particular are enjoying widespread application. However, PROTACs, which recruit an E3 ligase for degradation of a target protein, still suffer from certain challenges. These include a limited selection for E3 ligases on the one hand and the requirement for potent target binding on the other hand. Both issues restrict the target scope available for PROTACs. Degraders that covalently engage the target protein or the E3 ligase can potentially expand the pool of both targets and E3 ligases. Moreover, they may offer additional advantages by improving the kinetics of ternary complex formation or by endowing additional selectivity to the degrader. Here, we review the recent progress in the emerging field of covalent PROTACs.     

Sodium bicarbonate‐gelled chitosan beads as mechanically stable carriers for the covalent immobilization of enzymes

The poor mechanical stability of chitosan has long impeded its industrial utilization as an immobilization carrier. In this study, the mechanical properties of chitosan beads were greatly improved through utilizing the slow rate of the sodium bicarbonate‐induced chitosan gelation and combining it with the chemical cross‐linking action of glutaraldehyde (GA). The GA‐treated sodium bicarbonate‐gelled chitosan beads exhibited much better mechanical properties and up to 2.45‐fold higher observed activity of the immobilized enzyme (β‐D‐galactosidase (β‐gal)) when compared to the GA‐treated sodium tripolyphosphate (TPP)‐gelled chitosan beads. The differences between the sodium bicarbonate‐gelled and the TPP‐gelled chitosan beads were proven visually and also via scanning electron microscopy, elemental analysis, and differential scanning calorimetry. Moreover, the optimum pH, the optimum temperature, the apparent K_m, and the apparent V_max of the β‐gals immobilized onto the two aforementioned types of chitosan beads were determined and compared. A reusability study was also performed. This study proved the superiority of the sodium bicarbonate‐gelled chitosan beads as they retained 72.22 ± 4.57% of their initial observed activity during the 13^th reusability cycle whereas the TPP‐gelled beads lost their activity during the first four reusability cycles, owing to their fragmentation. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:347–361, 2018     

The use of cultured hepatocytes to investigate the mechanisms of drug hepatotoxicity

In the course of biotransformation reactions catalyzed both by cytochrome P450 and by conjugating enzymes, drug-derived reactive metabolites and active oxygen species can appear that may escape the detoxification process, initiating radical chain reactions (e.g., lipid peroxidation), covalently binding to macromolecules (proteins, DNA), or impairing the energetic balance of cells. This is usually followed by alterations of ion homeostasis that precede irreversible biochemical changes and cell death. There are, however, cellular mechanisms of defense that prevent, or repair, the damage caused by these reactive intermediates. Ultimately it is the balance between bioactivation, detoxification, and defense mechanisms that determines whether a compound will or will not elicit a toxic effect. Cultures of hepatocytes, including those of human origin, can be used to elucidate the mechanisms of drug toxicity. This is illustrated in the study of the mechanism of hepatotoxicity by diclofenac. Much less cytotoxicity is observed in nonmetabolizing hepatomas than in hepatocytes. The observed cell dysfunction parallels the biotransformation of the drug, and particularly the formation of the minor metabolite N,5-dihydroxydiclofenac by hepatocytes. This compound is able to inhibit mitochondrial ATP synthesis in hepatocytes.     

Preparation,characterization and laboratory-scale application of an immobilized glucoamylase

Glucoamylase (1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) has been covalently immobilized on a polyacrylamide-type support containing carboxylic groups activated by water-soluble carbodiimide. The activity was 5.5– 6.0 units g^?1solid. The optimum pH for catalytic activity was pH 3.8. The apparent optimum temperature was found at 60°C. With soluble starch as substrate the K_m value was 14 mg ml^?1. The pH for maximum stability was pH 4.0–4.5. In the presence of 8 m urea the immobilized glucoamylase retained most of its catalytic activity but it was more susceptible to guanidinium hydrochloride than the soluble enzyme. The practical applicability of immobilized glucoamylase was tested in batch process and continuous operation.     

Immobilization of cytochrome c on beaded poly(N-acrylylpyrrolidine)

In order to examine a procedure whereby the points of covalent attachment between the components of a protein-polymer conjugate could be determined, horse heart cytochrome c was attached to a beaded copolymer of N-acrylylpyrrolidine, N,N′-bis(acrylyl)-1,2-diaminoethane and N-acrylyl-1,6-aminohexane through a cleavable azo linkage. Studies of protein removed from this conjugate showed that attachment of the polymer to cytochrome occurred predominantly through single lysine residues on the protein surface; lysine-25 was tentatively identified as the residue most extensively utilized in this way. Protein was also linked to the polymer by two lysine residues and a significant amount of protein was irreversibly attached to the polymer under the reaction conditions used.     

Immobilization of native and dextran-free dextransucrases from Leuconostoc mesenteroides NRRL B-512F for the synthesis of glucooligosaccharides

Dextransucrase from Leuconostoc mesenteroides NRRL B-512F was immobilized using two different methods: covalent attachment to activated silica and entrapment in calcium alginate. For immobilization on silica, native enzyme and dextran-free enzyme were compared. However, the entrapment in calcium alginate beads gave the best results in terms of immobilization yield and stability. This biocatalyst was employed in the acceptor reaction with maltose showing similar glucooligosaccharide production than the native enzyme but increased operational stability.     

Inhibition of bFGF activity by complement C1s: covalent binding of C1s with bFGF

The first complement component C1s formed large aggregates with bFGF when bFGF and C1s were incubated at 37°C overnight. Under non-reducing conditions, a part of the aggregates did not penetrate into 5% polyacrylamide gel in the presence of SDS, and the rest penetrated into 5% gel but not into 12% gel. The aggregates were dissociated into monomers by reducing with 2-mercaptoethanol. Both active and inactive C1s formed aggregates with bFGF. In addition, a portion of bFGF was degraded by active C1s but not by inactive C1s. Aggregates were not formed when 2-mercaptoethanol (2 mM &base;) was added to the incubation mixture. After the incubation with C1s the growth-stimulating activity of bFGF was measured by using human umbilical vein endothelial cells (HUVEC) as indicator cells. The aggregate formation between C1s and bFGF significantly reduced the activity of bFGF. © 1998 John Wiley & Sons, Ltd.     

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

Base excision repair （BER） is an evolutionarily conserved process for maintaining genomic integrity by eliminating several dozen damaged （oxidized or aikylated） or inappropriate bases that are generated endogenously or induced by genotoxicants, predominantly, reactive oxygen species （ROS）. BER involves 4-5 steps starting with base excision by a DNA glycosylase, followed by a common pathway usually involving an AP-endonuclease （APE） to generate 3＇ OH terminus at the damage site, followed by repair synthesis with a DNA polymerase and nick sealing by a DNA iigase. This pathway is also responsible for repairing DNA single-strand breaks with blocked termini directly generated by ROS. Nearly all glycosylases, far fewer than their substrate lesions particularly for oxidized bases, have broad and overlapping substrate range, and could serve as back-up enzymes in vivo. In contrast, mammalian cells encode only one APE, APEI, unlike two APEs in lower organisms. In spite of overall similarity, BER with distinct subpathways in the mammals is more complex than in E. coli. The glycosylases form complexes with downstream proteins to carry out efficient repair via distinct subpathways one of which, responsible for repair of strand breaks with 3＇ phosphate termini generated by the NEIL family glycosylases or by ROS, requires the phosphatase activity of polynucleotide kinase instead of APE1. Different complexes may utilize distinct DNA polymerases and iigases. Mammalian glycosylases have nonconserved extensions at one of the termini, dispensable for enzymatic activity but needed for interaction with other BER and non-BER proteins for complex formation and organeile targeting. The mammalian enzymes are sometimes covalently modified which may affect activity and complex formation. The focus of this review is on the early steps in mammalian BER for oxidized damage.     

Mechanistic insights into the inhibition of prostate specific antigen by beta-lactam class compounds

Prostate Specific Antigen (PSA) is a biomarker used in the diagnosis of prostate cancer and to monitor therapeutic response. However, its precise role in prostate carcinogenesis and metastasis remains largely unknown. A number of studies arguing in the favor of an active role of PSA in prostate cancer development and progression have implicated this serine protease in the release and activation of growth factors such as insulin-like growth factor 1 (IGF1) through cleavage of insulin like growth factor binding protein 3 and Transforming Growth Factor beta (TGF-beta) through cleavage of Latent TGF-beta. In contrast, other studies suggest that PSA activity might hinder tumor development and progression. In light of these contradictory findings, efficient inhibitors of PSA are needed for exploring its biological role in tumor development and metastasis. Towards the goal of developing potent inhibitors of PSA, we have explored the molecular mechanism of a series of beta-lactam based compounds on binding to PSA using activity assays, matrix assisted laser desorption ionization with a time-of-flight mass spectrometry, and GOLD docking methodology. The mass spectrometry experiments and the activity assays confirmed the time-dependent and covalent nature of beta-lactam binding. To gain insights on the reaction intermediates at the molecular level, we docked beta-lactam inhibitors to a homology modeled PSA using the GOLD docking program in noncovalent and covalent binding modes. The docking studies elucidated the molecular details of the early noncovalent Michaelis complex, the acyl-enzyme covalent complex, and the nature of conformational reorganization required for the long term stability of the covalent complex. Additionally, the molecular basis for the effect of stereochemistry of the lactam ring on the inhibitory potency was elucidated through docking of beta-lactam enantiomers. As a validation of our docking methodology, two novel enantiomers were synthesized and evaluated for their inhibitory potency using fluorogenic substrate based activity assays. Additionally, cis enantiomers of eight beta-lactam compounds reported in a previous study were docked and their GOLD scores and binding modes were analyzed in order to assess the general applicability of our docking results. The close agreement of our docking results with the experimental data validates the mechanistic insights revealed through the docking studies and paves the way for the design and development of potent and specific inhibitors of PSA.     

Covalent bonded Gag multimers in human immunodeficiency virus type-1 particles

The oligomerization of HIV-1 Gag and Gag-Pol proteins, which are assembled at the plasma membrane, leads to viral budding. The budding generally places the viral components under non-reducing conditions. Here the effects of non-reducing conditions on Gag structures and viral RNA protection were examined. Using different reducing conditions and SDS-PAGE, it was shown that oligomerized Gag possesses intermolecular covalent bonds under non-reducing conditions. In addition, it was demonstrated that the mature viral core contains a large amount of covalent bonded Gag multimers, as does the immature core. Viral genomic RNA becomes sensitive to ribonuclease in reducing conditions. These results suggest that, under non-reducing conditions, covalent bonded Gag multimers are formed within the viral particles and play a role in protection of the viral genome.