Accelerating the speed of innovative anti-tumor drugs to first-in-human trials incorporating key de-risk strategies.

Pharmaceutical companies have recently focused on accelerating the timeline for initiating first-in-human (FIH) trials to allow quick assessment of biologic drugs. For example, a stable cell pool can be used to produce materials for the toxicology (Tox) study, reducing time to the clinic by 4-5 months. During the coronavirus disease 2019 (COVID-19) pandemic, the anti-COVID drugs timeline from DNA transfection to the clinical stage was decreased to 6 months using a stable pool to generate a clinical drug substrate (DS) with limited stability, virus clearance, and Tox study package. However, a lean chemistry, manufacturing, and controls (CMC) package raises safety and comparability risks and may leave extra work in the late-stage development and commercialization phase. In addition, whether these accelerated COVID-19 drug development strategies can be applied to non-COVID projects and established as a standard practice in biologics development is uncertain. Here, we present a case study of a novel anti-tumor drug in which application of "fast-to-FIH" approaches in combination with BeiGene's de-risk strategy achieved successful delivery of a complete CMC package within 10 months. A comprehensive comparability study demonstrated that the DS generated from a stable pool and a single-cell-derived master cell bank were highly comparable with regards to process performance, product quality, and potency. This accomplishment can be a blueprint for non-COVID drug programs that approach the pace of drug development during the pandemic, with no adverse impact on the safety, quality, and late-stage development of biologics.

Identification and characterization of an unexpected isomerization motif in CDRH2 that affects antibody activity.

Aspartic acid (Asp) isomerization is a spontaneous non-enzymatic post-translation modification causing a change in the structure of the protein backbone, which is commonly observed in therapeutic antibodies during manufacturing and storage. The Asps in Asp-Gly (DG), Asp-Ser (DS), and Asp-Thr (DT) motifs in the structurally flexible regions, such as complementarity-determining regions (CDRs) in antibodies, are often found to have high rate of isomerization, and they are considered "hot spots" in antibodies. In contrast, the Asp-His (DH) motif is usually considered a silent spot with low isomerization propensity. However, in monoclonal antibody mAb-a, the isomerization rate of an Asp residue, Asp55, in the aspartic acid-histidine-lysine (DHK) motif present in CDRH2 was found to be unexpectedly high. By determining the conformation of DHK motif in the crystal structure of mAb-a, we found that the Cgamma of the Asp side chain carbonyl group and the back bone amide nitrogen of successor His were in proximal contact, which facilitates the formation of succinimide intermediate, and the +2 Lys played an important role in stabilizing such conformation. The contributing roles of the His and Lys residues in DHK motif were also verified using a series of synthetic peptides. This study identified a novel Asp isomerization hot spot, DHK, and the structural-based molecular mechanism was revealed. When 20% Asp55 isomerization in this DHK motif occurred in mAb-a, antigen binding activity reduced to 54%, but the pharmacokinetics in rat was not affected significantly. Although Asp isomerization of DHK motif in CDR does not appear to have a negative impact on PK, DHK motifs in the CDRs of antibody therapeutics should be removed, considering the high propensity of isomerization and impact on antibody activity and stability.

Profiling Glutathionylome in CD38-Mediated Epithelial-Mesenchymal Transition.

Protein S-glutathionylation is an important posttranslational modification that regulates various cellular processes. However, changes in glutathionylome in epithelial-mesenchymal transition (EMT), a crucial cellular process for embryonic development, wound healing, and carcinoma progression and metastasis, have not been fully characterized. Our previous study revealed that CD38 overexpression decreased cellular nicotinamide adenine dinucleotide (NAD+) levels and caused cells to undergo EMT. In the present study, we engineered a cell system in which the glutathione synthetase (GS) mutant was expressed that catalyzed the formation of a glutathione analogue from azido-alanine to profile changes of glutathionylome in CD38-overexpressing cells. We identified 1298 glutathionylated proteins and revealed that proteins with changed glutathionylation levels involved in EMT associated pathways including epithelial adherens junction, actin cytoskeleton, and integrin signaling. Moreover, the glutathionylation level of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) was increased in CD38-overexpressing cells. We further demonstrated that glutathionylation of Cys63 residue in 15-PGDH led to decreased enzymatic activity that could promote EMT by increasing prostaglandin E2 (PGE2). Taken together, these results indicate that the clickable glutathione is an effective probe for glutathionylome profiling, and glutathionylation of 15-PGDH on Cys63 inhibits its enzymatic activity to promote EMT.

Characterization of the Fc-III-4C-based recombinant protein expression system by using carbonic anhydrase as the model protein.

Development of new affinity tags is important for recombinant protein expression and purification. Based on our earlier work, we devised an affinity tag by addition of two cysteine residues onto the N- and C-termini of the Fc-III peptide and designated as the Fc-III-4C tag, in which four cysteine residues form two disulfide linkages. The binding affinity of Fc-III-4C tag to human IgG is measured as 2.28 nM (Kd) and is 100 times higher than that of the Fc-III tag to IgG. Fc-III-4C tagged carbonic anhydrase (CA) can be effectively purified with IgG-immobilized beads, and Fc-III-4C tag does not possess adverse effects on the structure and stability of CA. Furthermore, the Fc-III-4C tagged protein binds to multiple transition metal ions, which enhances activities of enzymes that use metal ions as co-factors. These results suggest that Fc-III-4C tag is a useful tool for expression and purification of recombinant proteins and enhances the activities of some fusion proteins that use Zn2+ or Cu2+ as cofactors.

Glutathionylation Decreases Methyltransferase Activity of PRMT5 and Inhibits Cell Proliferation.

Glutathionylation is an important posttranslational modification that protects proteins from further oxidative damage as well as influencing protein structure and activity. In the present study, we demonstrate that the cysteine-42 residue in protein arginine N-methyltransferase 5 (PRMT5) is glutathionylated in aged mice or in cells that have been exposed to oxidative stress. Deglutathionylation of this protein is catalyzed by glutaredoxin-1 (Grx1). Using mutagenesis and subsequent biochemical analyses, we show that glutathionylation decreased the binding affinity of PRMT5 with methylosome protein-50 (MEP50) and reduced the methyltransferase activity of PRMT5. Furthermore, overexpression of PRMT5-C42A mutant caused a significant increase in histone methylation in HEK293T and A549 cells and promoted cell growth, whereas overexpression of the PRMT5-C42D mutant, a mimic of glutathionylated PRMT5, inhibited cell proliferation. Taken together, our results demonstrate a new mechanism of regulation of PRMT5 methyltransferases activity and suggest that PRMT5 glutathionylation is partly responsible for reactive oxygen species-mediated cell growth inhibition.

Redox-Regulated Adaptation of Streptococcus oligofermentans to Hydrogen Peroxide Stress.

Preexposure to a low concentration of H2O2 significantly increases the survivability of catalase-negative streptococci in the presence of a higher concentration of H2O2 However, the mechanisms of this adaptation remain unknown. Here, using a redox proteomics assay, we identified 57 and 35 cysteine-oxidized proteins in Streptococcus oligofermentans bacteria that were anaerobically cultured and then pulsed with 40 µM H2O2 and that were statically grown in a 40-ml culture, respectively. The oxidized proteins included the peroxide-responsive repressor PerR, the manganese uptake repressor MntR, thioredoxin system proteins Trx and Tpx, and most glycolytic proteins. Cysteine oxidations of these proteins were verified through redox Western blotting, immunoprecipitation, and liquid chromatography-tandem mass spectrometry assays. In particular, Zn2+-coordinated Cys139 and Cys142 mutations eliminated the H2O2 oxidation of PerR, and inductively coupled plasma mass spectrometry detected significantly decreased amounts of Zn2+ in H2O2-treated PerR, demonstrating that cysteine oxidation results in Zn2+ loss. An electrophoretic mobility shift assay (EMSA) determined that the DNA binding of Mn2+-bound PerR protein (PerR:Zn,Mn) was abolished by H2O2 treatment but was restored by dithiothreitol reduction, verifying that H2O2 inactivates streptococcal PerR:Zn,Mn through cysteine oxidation, analogous to the findings for MntR. Quantitative PCR and EMSA demonstrated that tpx, mntA, mntR, and dpr belonged to the PerR regulons but that only dpr was directly regulated by PerR; mntA was also controlled by MntR. Deletion of mntR significantly reduced the low-H2O2-concentration-induced adaptation of S. oligofermentans to a higher H2O2 concentration, while the absence of PerR completely abolished the self-protection. Therefore, a low H2O2 concentration resulted in the cysteine-reversible oxidations of PerR and MntR to derepress their regulons, which function in cellular metal and redox homeostasis and which endow streptococci with the antioxidative capability. This work reveals a novel Cys redox-based H2O2 defense strategy employed by catalase-negative streptococci in Mn2+-rich cellular environments.IMPORTANCE The catalase-negative streptococci produce as well as tolerate high levels of H2O2 This work reports the molecular mechanisms of low-H2O2-concentration-induced adaptation to higher H2O2 stress in a Streptococcus species, in which the peroxide-responsive repressor PerR and its redox regulons play the major role. Distinct from the Bacillus subtilis PerR, which is inactivated by H2O2 through histidine oxidation by the Fe2+-triggered Fenton reaction, the streptococcal PerR is inactivated by H2O2 oxidation of the structural Zn2+ binding cysteine residues and thus derepresses the expression of genes defending against oxidative stress. The reversible cysteine oxidation could provide flexibility for PerR regulation in streptococci, and the mechanism might be widely used by lactic acid bacteria, including pathogenic streptococci, containing high levels of cellular manganese, in coping with oxidative stress. The adaptation mechanism could also be applied in oral hygiene by facilitating the fitness and adaptability of the oral commensal streptococci to suppress the pathogens.

Glutaredoxin-1 Silencing Induces Cell Senescence via p53/p21/p16 Signaling Axis.

Glutaredoxin-1 (Grx1) catalyzes deglutathionylation with glutathione as a cofactor. Accumulating evidence indicates important roles for Grx1 and S-glutathionylation in the aging process; however, further exploration of Grx1-regulated cellular processes is important to understand the functions of Grx1 in aging. In the present study, we constructed stable Grx1 knockdown or overexpression human cell lines. Grx1 silencing significantly decreased the cellular ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) (GSH/GSSG ratio), resulting in excessive reactive oxygen species (ROS) accumulation, whereas Grx1 overexpression decreased cellular ROS levels. Grx1 silencing also increased glutathionylation of DJ-1 and HSP60, contributing to decreased mitochondrial spare respiration capacity and ATP production. We applied quantitative proteomics to identify differentially expressed proteins between Grx1 knockdown and control cells and showed that Grx1 silencing inactivated DNA replication and damage repair pathways. p53 signaling was activated by Grx1 silencing, which inhibited the CDK4-mediated G1-S transition, resulting in G1 phase cell-cycle arrest and cell senescence, a known hallmark of aging. Taken together, our results indicate that Grx1 regulates DNA replication and damage repair processes and is a potential therapeutic target for aging-related diseases.