Yeng-Tseng Wang

Carbapenem-resistant Acinetobacter baumannii (CRAB) has emerged as a critical public health menace. Its resistance to last-resort antibiotics highlights the urgent need for innovative treatment approaches. Antimicrobial peptides (AMPs) are promising candidates to address this challenge. AMPs have distinct mechanisms and a low likelihood of inducing resistance. In this study, we designed a water-soluble cationic AMP, "T2-02." This was achieved using AMP database screening and in silico modeling with genetic algorithms (GAs). T2-02 has a net +7 charge at physiological pH and is composed of 21 amino acid residues. This charge facilitates strong electrostatic interactions with negatively charged microbial membranes. Moreover, the helical secondary structure of T2-02 enhances amphipathicity, enabling effective membrane insertion. When tested against Gram-negative CRAB isolates, T2-02 showed strong antibacterial activity. It also demonstrated outstanding biocompatibility, with low cytotoxicity and a minimal inhibitory concentration (MIC) of 8-16 μg/mL. Its therapeutic potential was further enhanced by the use of a liposomal nanodelivery method. This significantly improved T2-02's loading efficiency. The liposomal strategy amplified its antimicrobial efficacy, reducing MICs by 2- to 4-fold. It also further minimized cytotoxicity. These results position T2-02 as a promising candidate for combating CRAB infections.

The dual luciferase reporter (DLR) assay is a well-known tool for gene expression analysis. Its ability to provide batch-to-batch, side-by-side normalization makes it a valuable method through which to explore actual sample signals. DLRs identify a real signal based on the stimulant's efficacy and can reflect the slightest change in downstream signaling with its unique signal adjustment ability. However, DLR substrates (e.g., d-luciferin and coelenterazine) are expensive and not stable enough to deliver a laborless operating environment. In this study, we introduce a dual-structured chromogenic enzyme (DSCE) platform that uses horseradish peroxidase (HRP) as a proof of concept. The HRP was engineered to be either tethered to the cell membrane or secreted into the extracellular compartment. Optimizing this technology with substrates (ABTS and TMB), we found that sHRP with ABTS as an internal control and mHRP and TMB for sample signal detection provided the most optimized output. Furthermore, we compared the signal sensitivity and durability of DSCE with the DLR. The DSCE provided a broader dynamic range and signal durability. Finally, substrates of the DSCE had a monetary cost that was 30-fold lower than the DLR. In summary, the DSCE platform utilizes enzymes with substrates to provide rapid detection and a durable signal for over 8 h. The platform is cost-friendly and does not compromise the normalization ability.

The Zika virus (ZIKV), a member of the Flaviviridae family, poses a major threat to human health because of the lack of effective antiviral drugs. Although the NS2B–NS3 protease of ZIKV (NS2B–NS3pro) is regarded as a major target for antiviral inhibitors, viral mutations can lead to ineffective competitive inhibitors. Allosteric inhibitors bind to highly conserved nonprotease active sites, induce conformational changes in the protease active site, and prevent substrate binding. Currently, no molecular simulation techniques are available for accurately predicting and analysing conformational changes in the protease catalytic domain. In this study, we developed a combined approach that involves blind docking, Gaussian accelerated molecular dynamics, two-dimensional potential of mean force profiling, density functional theory (DFT) calculations, and interaction region indicator (IRI) analysis and employed it to examine the allosteric inhibitor-01 molecule and its interaction with ZIKV NS2B–NS3pro. Our results indicated that the binding of inhibitor-01 to NS2B–NS3pro resulted in two major conformational states, state I and state II, which in turn changed the volume of the protease active site from 1014 ų to 710 and 820 ų, respectively. These two states had an inactive catalytic domain (residues His116, Asp140, and Ser200). DFT and IRI analyses revealed that, in state I, Lys138 and Gln139 formed hydrogen bonds with inhibitor-01, whereas Lys138, Leu214, Asn217, Val220, and Ile221 engaged in van der Waals interactions with inhibitor-01. Advancements in computational techniques and power are expected to facilitate further progress in overcoming challenges associated with designing allosteric inhibitors for viral proteases.

Reporter gene assays are essential for high-throughput analysis, such as drug screening or determining downstream signaling activation/inhibition. However, use of this technology has been hampered by the high cost of the substrate (e.g., d-Luciferin (d-Luc)) in the most common firefly luciferase (FLuc) reporter gene assay. Although alternate luciferase is available worldwide, its substrate has remained expensive, and a more affordable option is still in demand. Here, we present a membrane-tethered horseradish peroxidase (mHRP), a new reporter system composed of a cell membrane expressing HRP that can preserve its enzymatic function on the cell surface, facilitates contact with HRP substrates (e.g., ABTS and TMB), and avoids the cell lysis process and the use of the high-priced luciferase substrate. An evaluation of the light signal sensitivity of mHRP compared to FLuc showed that both had comparable signal sensitivity. We also identified an extended substrate half-life of more than 5-fold that of d-Luc. Of note, this strategy provided a more stable detection signal, and the cell lysis process is not mandatory. Furthermore, with this strategy, we decreased the total amount of time taken for analysis and increased the time of detection limit of the reporter assay. Pricing analysis showed a one-third to one twenty-eighth price drop per single test of reporter assay. Given the convenience and stability of the mHRP reporter system, we believe that our strategy is suitable for use as an alternative to the luciferase reporter assay.

Coronavirus 3C-like protease (3CLpro) is found in SARS-CoV-2 virus, which causes COVID-19. 3CLpro controls virus replication and is a major target for target-based antiviral discovery. As reported by Pfizer, Nirmatrelvir (PF-07321332) is a competitive protein inhibitor and a clinical candidate for orally delivered medication. However, the binding mechanisms between Nirmatrelvir and 3CLpro complex structures remain unknown. This study incorporated ligand Gaussian accelerated molecular dynamics, the one-dimensional and two-dimensional potential of mean force, normal molecular dynamics, and Kramers’ rate theory to determine the binding and dissociation rate constants (k_off and k_on) associated with the binding of the 3CLpro protein to the Nirmatrelvir inhibitor. The proposed approach addresses the challenges in designing small-molecule antiviral drugs.

Background: Humanization of mouse monoclonal antibodies (mAbs) is crucial for reducing their immunogenicity in humans. However, humanized mAbs often lose their binding affinities. Therefore, an in silico humanization method that can prevent the loss of the binding affinity of mAbs is needed. Methods: We developed an in silico V(D)J recombination platform in which we used V(D)J human germline gene sequences to design five humanized candidates of anti-tumor necrosis factor (TNF)-α mAbs (C1–C5) by using different human germline templates. The candidates were subjected to molecular dynamics simulation. In addition, the structural similarities of their complementarity-determining regions (CDRs) to those of original mouse mAbs were estimated to derive the weighted interatomic root mean squared deviation (wRMSD_i) value. Subsequently, the correlation of the derived wRMSDi value with the half maximal effective concentration (EC50) and the binding affinity (K_D) of the humanized anti-TNF-α candidates was examined. To confirm whether our in silico estimation method can be used for other humanized mAbs, we tested our method using the anti-epidermal growth factor receptor (EGFR) a4.6.1, anti-glypican-3 (GPC3) YP9.1 and anti-α4β1 integrin HP1/2L mAbs. Results: The R² value for the correlation between the wRMSD_i and log(EC50) of the recombinant Remicade and those of the humanized anti-TNF-α candidates was 0.901, and the R² value for the correlation between wRMSD_i and log(K_D) was 0.9921. The results indicated that our in silico V(D)J recombination platform could predict the binding affinity of humanized candidates and successfully identify the high-affinity humanized anti-TNF-α antibody (Ab) C1 with a binding affinity similar to that of the parental chimeric mAb (5.13 × 10⁻¹⁰). For the anti-EGFR a4.6.1, anti-GPC3 YP9.1, and anti-α4β1 integrin HP1/2L mAbs, the wRMSD_i and log(EC50) exhibited strong correlations (R² = 0.9908, 0.9999, and 0.8907, respectively). Conclusions: Our in silico V(D)J recombination platform can facilitate the development of humanized mAbs with low immunogenicity and high binding affinities. This platform can directly transform numerous mAbs with therapeutic potential to humanized or even human therapeutic Abs for clinical use. Graphical

Correction for ‘Development of a structure-based computational simulation to optimize the blocking efficacy of pro-antibodies’ by Bo-Cheng Huang et al., Chem. Sci., 2021, DOI: 10.1039/D1SC01748A.