Covalent linkage formation is a very important mechanism for many covalent drugs to work. However, partly due to the limitations of proper computational tools for covalent docking, most covalent drugs are not discovered systematically. In this article, we present a new covalent docking package, the CovalentDock, built on the top of the source code of Autodock. We developed an empirical model of free energy change estimation for covalent linkage formation, which is compatible with existing scoring functions used in docking, while handling the molecular geometry constrains of the covalent linkage with special atom types and directional grid maps. Integrated preparation scripts are also written for the automation of the whole covalent docking workflow. The result tested on existing crystal structures with covalent linkage shows that CovalentDock can reproduce the native covalent complexes with significant improved accuracy when compared with the default covalent docking method in Autodock. Experiments also suggest that CovalentDock is capable of covalent virtual screening with satisfactory enrichment performance. In addition, the investigation on the results also shows that the chirality and target selectivity along with the molecular geometry constrains are well preserved by CovalentDock, showing great capability of this method in the application for covalent drug discovery.

Background: Variable heavy chain (VH) family frameworks (FWRs) have been reported to affect antibody receptor and superantigen binding; however, such effects in IgE remain largely unknown. Given that VH family biases have been previously reported in IgE of certain allergies, there is a need to investigate this phenomenon for biotechnological and therapeutic purposes. Objective: We sought to investigate the effects of VH families on IgE interaction with FcεRIa, anti-IgE omalizumab, antigen, and superantigen protein A (spA) by using the pertuzumab and trastuzumab IgE models. Methods: Pertuzumab VH1-VH7 family variants of IgE with the same complementarity-determining regions were investigated with regard to their binding interactions to FcεRIa, Her2, omalizumab, and spA. Notable FcεRIa-IgE observations were cross-checked against appropriate trastuzumab IgE VH variants. Computational structural modeling and simulations were also performed for insight into the mechanism of interactions with various VH FWRs. Results: The pertuzumab VH5 IgE variant, but not the trastuzumab VH5 IgE, was found to interact with FcεRIa significantly longer than the respective VH family variants within each model antibody. No significant differences in interaction were found between IgE and omalizumab for the pertuzumab VH variants. Although trastuzumab VH3 interacted with spA, none of our pertuzumab VH variants, including VH3, associated with spA. Conclusion: We found unexpected varying allosteric communications caused by the VH family FWRs to the FcεRIa-, Her2-, and spA-binding regions of pertuzumab IgE, with implications for use of IgE/anti-IgE therapeutics to treat allergy and IgE therapeutics in allergo-oncology.

Antibody research has traditionally focused on heavy chains, often neglecting the important complementary role of light chains in antibody formation and secretion. In the light chain, the complementarity-determining region 3 (VL-CDR3) is specifically implicated in disease states. By modulating VL-CDR3 exposure on the scaffold through deletions in the framework region 3 (VL-FWR3), we further investigated the effects on secretion in recombinant production and antigen binding kinetics. Our random deletions of two residues in the VL-FWR3 of a Trastuzumab model showed that the single deletions could impact recombinant production without significant effect on Her2 binding. When both the selected residues were deleted, antibody secretion was additively decreased, and so was Her2 binding kinetics. Interestingly, we also found allosteric effects on the Protein L binding site at VL-FWR1 elicited by these deletions in VL- FWR3. Together, these findings demonstrate the importance of light chain FWR3 in antigen binding, recombinant production, and antibody purification using Protein L.