Humanization of druggable targets is an important tool for increasing drug translation to clinic. Current in vivo drug discovery platforms can be quite limited in their ability to identify drug candidates which can successfully transition into humans because primary amino acid sequences are not conserved between animal models (mammalian mouse, zebrafish, and C. elegans), and humans [see Figure 1]. The differences between species’ amino acids means that there will be differences in the optimal drug binding site — this is where Whole-gene Humanized Animal Models (WHAM) can greatly improve drug discovery efficiency (patent US11477970B2; PMID 36411139).
Figure 1. Binding Site Specificity: example of how designed molecules will have different binding site capacity between humans and animal models due to differences in primary amino acid sequence. When this amino acid difference is eliminated with WHAM humanization, better quality hits are identified from the animal model.
Figure 2. Whole-gene humanization technique. A native (WT) animal is CRISPR edited to make a gene KO animal and a whole-gene humanized animal has a gene swap replacement of ortholog locus.
Figure 3. Discovery of drugs using computational approaches use human protein sequences, while screening of in silico hits in unmodified animal models can generate false negatives due to structural differences in drug binding site.
The lack of sequence conservation in drug binding sites is a shortcoming in drug screening on animal models. By analyzing what was successful for CF therapeutics in humans, we can observe how screening on animal-derived sequences has two major issues. The lack of sequence conservation between humans and animal models predicts that many of the hits found on an animal model will fail to work in humans (False Positives). Conversely, there would also be a set of compounds that were missed in the animal model but would have been found positive in humans (False Negatives). However, by using the WHAM method to bypass the molecular differences between species, the likelihood of achieving translation of hits-in-the-animal to winners-in-the-clinic will be much higher.
Lopes-Pacheco M. CFTR Modulators: The Changing Face of Cystic Fibrosis in the Era of Precision Medicine. Front Pharmacol. 2019;10: 1662.
Capurro V, Tomati V, Sondo E, Renda M, Borrelli A, Pastorino C, et al. Partial Rescue of F508del-CFTR Stability and Trafficking Defects by Double Corrector Treatment. Int J Mol Sci. 2021;22. doi:10.3390/ijms22105262
Hopkins C, Onweni C, Zambito V, Fairweather D, McCormick K, Ebihara H, et al. Platforms for Personalized Polytherapeutics Discovery in COVID-19. J Mol Biol. 2021;433: 166945.
Laselva O, Ardelean MC, Bear CE. Phenotyping Rare CFTR Mutations Reveal Functional Expression Defects Restored by TRIKAFTA. J Pers Med. 2021;11. doi:10.3390/jpm11040301
Baatallah N, Elbahnsi A, Mornon J-P, Chevalier B, Pranke I, Servel N, et al. Pharmacological chaperones improve intra-domain stability and inter-domain assembly via distinct binding sites to rescue misfolded CFTR. Cell Mol Life Sci. 2021;78: 7813–7829
Fiedorczuk K, Chen J. Mechanism of CFTR correction by type I folding correctors. Cell. 2022;185: 158–168.e11.