A cell-based screening method using an intracellular antibody for discovering small molecules targeting the translocation protein LMO2

Intracellular antibodies are tools that can be used directly for target validation by interfering with properties like protein-protein interactions. An alternative use of intracellular antibodies in drug discovery is developing small-molecule surrogates using antibody-derived (Abd) technology. We previously used this strategy with an in vitro competitive surface plasmon resonance method that relied on high-affinity antibody fragments to obtain RAS-binding compounds. We now describe a novel implementation of the Abd method with a cell-based intracellular antibody–guided screening method that we have applied to the chromosomal translocation protein LMO2. We have identified a chemical series of anti-LMO2 Abd compounds that bind at the same LMO2 location as the inhibitory anti-LMO2 intracellular antibody combining site. Intracellular antibodies could therefore be used in cell-based screens to identify chemical surrogates of their binding sites and potentially be applied to any challenging proteins, such as transcription factors that have been considered undruggable.

Pan RAS-binding compounds selected from a chemical library by inhibiting interaction between RAS and a reduced affinity intracellular antibody

Intracellular antibodies are valuable tools for target validation studies for clinical situations such as cancer. Recently we have shown that antibodies can be used for drug discovery in screening for chemical compounds surrogates by showing that compounds could be developed to the so-called undruggable RAS protein family. This method, called Antibody-derived compound (Abd) technology, employed intracellular antibodies binding to RAS in a competitive surface plasmon resonance chemical library screen. Success with this method requires a high affinity interaction between the antibody and the target. We now show that reduction in the affinity (dematuration) of the anti-active RAS antibody facilitates the screening of a chemical library using an in vitro AlphaScreen method. This identified active RAS specific-binding Abd compounds that inhibit the RAS-antibody interaction. One compound is shown to be a pan-RAS binder to KRAS, HRAS and NRAS-GTP proteins with a Kd of average 37 mM, offering the possibility of a new chemical series that interacts with RAS in the switch region where the intracellular antibody binds. This simple approach shows the druggability of RAS and is generally applicable to antibody-derived chemical library screening by affording flexibility through simple antibody affinity variation. This approach can be applied to find Abd compounds as surrogates of antibody-combining sites for novel drug development in a range of human diseases.

The Protective Effect of a Combination of Human Intracellular and Extracellular Antibodies against Highly Pathogenic Avian Influenza H5N1 Virus

Background:Highly pathogenic avian influenza H5N1 virus is a serious threat to humans. Due to its antiviral activity, antibody-based therapy is one of the possible effective countermeasures. Here,a combination of intracellular and extracellular human antibodies was investigated and showed a better protective effect. Methods: The scFv4F5-based intracellular antibody and full-length IgG1 extracellular antibody vectors were constructed or expressed, respectively. In vitro, the sensitivity, specificity and affinity of these antibodies were detected by western blotting, ELISA, flow cytometry, Biacore X100 SPR technique and microneutralization assay. In vivo, the protective effect of the combination of antibodies and the dynamics of viral replication were tested, and the related cytokines and proteins were detected by ELISA, western blotting and qPCR. Results: The intracellular antibody could inhibit H5N1 virus propagation in A549 cells in a dose-dependent manner. The protective effect of IgG1 was good in post-treatment therapy in a mouse model. When the intracellular antibody was pre-transfected in a combination regimen with IgG1, it had a better protective effect than IgG1 alone. The protective effect was primarily accomplished by inducing the secretion of cytokines, i.e., IFN-γ, IL-6, and IL-10, and the expression of apoptosis-related proteins, i.e., Bim and cleaved PARP. Conclusions: This antibody combination technique could be used as an appropriate and powerful alternative to antiviral therapy.

Structure-based development of new RAS-effector inhibitors from a combination of active and inactive RAS-binding compounds

The RAS gene family is frequently mutated in human cancers, and the quest for compounds that bind to mutant RAS remains a major goal, as it also does for inhibitors of protein–protein interactions. We have refined crystallization conditions for KRAS169Q61H-yielding crystals suitable for soaking with compounds and exploited this to assess new RAS-binding compounds selected by screening a protein–protein interaction-focused compound library using surface plasmon resonance. Two compounds, referred to as PPIN-1 and PPIN-2, with related structures from 30 initial RAS binders showed binding to a pocket where compounds had been previously developed, including RAS effector protein–protein interaction inhibitors selected using an intracellular antibody fragment (called Abd compounds). Unlike the Abd series of RAS binders, PPIN-1 and PPIN-2 compounds were not competed by the inhibitory anti-RAS intracellular antibody fragment and did not show any RAS-effector inhibition properties. By fusing the common, anchoring part from the two new compounds with the inhibitory substituents of the Abd series, we have created a set of compounds that inhibit RAS-effector interactions with increased potency. These fused compounds add to the growing catalog of RAS protein–protein inhibitors and show that building a chemical series by crossing over two chemical series is a strategy to create RAS-binding small molecules.