Reshaping cell line development and CMC strategy for fast responses to pandemic outbreak.

The global pandemic outbreak COVID-19 (SARS-COV-2), has prompted many pharmaceutical companies to develop vaccines and therapeutic biologics for its prevention and treatment. Most of the therapeutic biologics are common human IgG antibodies, which were identified by next-generation sequencing (NGS) with the B cells from the convalescent patients. To fight against pandemic outbreaks like COVID-19, biologics development strategies need to be optimized to speed up the timeline. Since the advent of therapeutic biologics, strategies of transfection and cell line selection have been continuously improved for greater productivity and efficiency. NGS has also been implemented for accelerated cell bank testing. These recent advances enable us to rethink and reshape the chemistry, manufacturing, and controls (CMC) strategy in order to start supplying Good Manufacturing Practices (GMP) materials for clinical trials as soon as possible. We elucidated an accelerated CMC workflow for biologics, including using GMP-compliant pool materials for phase I clinical trials, selecting the final clone with product quality similar to that of phase I materials for late-stage development and commercial production.

Effect of Encoding on Prospective Memory.

Event-based prospective memory (ProM) refers to remembering to execute planned actions in response to a target ProM cues. Encoding modality influences ProM performance; visual encoding has been studied more than auditory encoding. Further, it has not yet been examined whether different encoding may influence ProM performance in different encoding modalities. This study examines the effects of encoding modality (visual vs. auditory), cue-encoding specificity (specific cue vs. non-specific cue), and encoding modes (standard vs. implementation intention) on event-based ProM tasks. In Experiment 1, cue specificity and encoding modality were manipulated as a within-groups encoding of visual cues is more commonly and between-groups variable. Results revealed the facilitative effect of cue specificity on ProM performance. Also, with respect to encoding modality, participants showed better performance when receiving auditory instructions compared with the visual encoding condition. In Experiment 2, as in Experiment 1, cue specificity and encoding modality were manipulated. Encoding mode was added as a new between-group variable. Result revealed that there was a significant interaction between encoding modality and encoding modes. Visual implementation intention encoding was a more effective method for improving ProM performance compared with visual standard encoding. Furthermore, there was a significant interaction between cue-encoding specificity and encoding modes. Implementation intention encoding enhances ProM performance in non-specific cue-encoding conditions. Overall, the present study found that (1) auditory encoding modality showed superior ProM performance compared with visual encoding, although implementation intention had facilitative on ProM performance regardless of the encoding modalities, and (2) there was better ProM performance under specific encoding compared with non-specific encoding, and implementation intention had a facilitative effect on ProM performance in the non-specific condition.

Nucleosome acidic patch-targeting binuclear ruthenium compounds induce aberrant chromatin condensation.

The 'acidic patch' is a highly electronegative cleft on the histone H2A-H2B dimer in the nucleosome. It is a fundamental motif for protein binding and chromatin dynamics, but the cellular impact of targeting this potentially therapeutic site with exogenous molecules remains unclear. Here, we characterize a family of binuclear ruthenium compounds that selectively target the nucleosome acidic patch, generating intra-nucleosomal H2A-H2B cross-links as well as inter-nucleosomal cross-links. In contrast to cisplatin or the progenitor RAPTA-C anticancer drugs, the binuclear agents neither arrest specific cell cycle phases nor elicit DNA damage response, but rather induce an irreversible, anomalous state of condensed chromatin that ultimately results in apoptosis. In vitro, the compounds induce misfolding of chromatin fibre and block the binding of the regulator of chromatin condensation 1 (RCC1) acidic patch-binding protein. This family of chromatin-modifying molecules has potential for applications in drug development and as tools for chromatin research.

Structural basis of small molecule ATPase inhibition of a human mitotic kinesin motor protein.

Kinesin microtubule motor proteins play essential roles in division, including attaching chromosomes to spindles and crosslinking microtubules for spindle assembly. Human kinesin-14 KIFC1 is unique in that cancer cells with amplified centrosomes are dependent on the motor for viable division because of its ability to cluster centrosomes and form bipolar spindles, but it is not required for division in almost all normal cells. Screens for small molecule inhibitors of KIFC1 have yielded several candidates for further development, but obtaining structural data to determine their sites of binding has been difficult. Here we compare a previously unreported KIFC1 crystal structure with new structures of two closely related kinesin-14 proteins, Ncd and KIFC3, to determine the potential binding site of a known KIFC1 ATPase inhibitor, AZ82. We analyze the previously identified kinesin inhibitor binding sites and identify features of AZ82 that favor binding to one of the sites, the α4/α6 site. This selectivity can be explained by unique structural features of the KIFC1 α4/α6 binding site. These features may help improve the drug-like properties of AZ82 and other specific KIFC1 inhibitors.

Allosteric cross-talk in chromatin can mediate drug-drug synergy.

Exploitation of drug-drug synergism and allostery could yield superior therapies by capitalizing on the immensely diverse, but highly specific, potential associated with the biological macromolecular landscape. Here we describe a drug-drug synergy mediated by allosteric cross-talk in chromatin, whereby the binding of one drug alters the activity of the second. We found two unrelated drugs, RAPTA-T and auranofin, that yield a synergistic activity in killing cancer cells, which coincides with a substantially greater number of chromatin adducts formed by one of the compounds when adducts from the other agent are also present. We show that this occurs through an allosteric mechanism within the nucleosome, whereby defined histone adducts of one drug promote reaction of the other drug at a distant, specific histone site. This opens up possibilities for epigenetic targeting and suggests that allosteric modulation in nucleosomes may have biological relevance and potential for therapeutic interventions.

An Organometallic Compound which Exhibits a DNA Topology-Dependent One-Stranded Intercalation Mode.

Understanding how small molecules interact with DNA is essential since it underlies a multitude of pathological conditions and therapeutic interventions. Many different intercalator compounds have been studied because of their activity as mutagens or drugs, but little is known regarding their interaction with nucleosomes, the protein-packaged form of DNA in cells. Here, using crystallographic methods and molecular dynamics simulations, we discovered that adducts formed by [(η(6) -THA)Ru(ethylenediamine)Cl][PF6 ] (THA=5,8,9,10-tetrahydroanthracene; RAED-THA-Cl[PF6 ]) in the nucleosome comprise a novel one-stranded intercalation and DNA distortion mode. Conversely, the THA group in fact remains solvent exposed and does not disrupt base stacking in RAED-THA adducts on B-form DNA. This newly observed DNA binding mode and topology dependence may actually be prevalent and should be considered when studying covalently binding intercalating compounds.