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Small Bioactive Compounds for Down-Modulation of TSHR, CD4, Sortlin, and ACE2
AdvisorBell, Thomas W.
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Cyclotriazadisulfonamide (CADA) is the first small molecule found to down-modulate the human CD4 (hCD4) protein, hypothetically by binding with its signal peptide. In the early 1990s, CADA was found to inhibit human immunodeficiency virus (HIV) replication. However, recent studies on CADA analogs show that these compounds not only down-modulate CD4 but also down-modulate other transmembrane proteins like sortilin, hTSHR, ACE2, and amongst others. A CADA analog (TL020) also shows antiviral activity for hepatitis, dengue, chikungunya, and SARS-CoV-2. Alanine scan mutagenesis on the hCD4 signal peptide reveals that the hydrophobic H-region and polar C-region amino acids (AAs) are essential for the down-modulation of hCD4 by CADA. The AA residues of the signal peptide in the position of Arg-8, Gln-15, Pro-20, and Lys (26 and 27) in the nascent protein chain were essential for CADA activity. Furthermore, it was proposed that one sulfonamide sidearm of CADA analogs makes a strong dipole-dipole interaction with Gln-15, which helps anchor the signal peptide inside the channel across the endoplasmic reticulum (ER) membrane during co-translational translocation of the nascent protein.The thyroid-stimulating hormone receptor (TSHR) is a G-protein coupled receptor responsible for the activation of the thyroid gland and secretion of thyroid hormones. It is also a therapeutic target for the autoimmune hyperthyroidism condition known as Graves’ disease (GD). Some CADA analogs with only one-sidearm, such as VGD040, were found to be active towards TSHR down-modulation. Considering the structure of the hTSHR signal peptide, we proposed that a PEGylated/alkyl linker with a benzylguanidine on one end and benzonitrile on another end (TK006) might be highly potent for inhibiting hTSHR down-modulation. The signal peptide of hTSHR has a charged AA (aspartate), which can form a strong ion-pair interaction with guanidine. Furthermore, it has a glutamine that can also form a strong dipole-dipole interaction with a benzonitrile sidearm, like the interaction of CADA with the hCD4 signal peptide. Twenty-three compounds of this class (symmetrical and unsymmetrical) were synthesized for the hTSHR down-modulation study. Amongst them, TK037 was found to have highest hTSHR down-modulation activity (approx. 76%). In addition, PEGylated CADA analogs were synthesized to improve their pharmacokinetic properties. Analysis of these properties is currently underway. Also, some head modified, tail modified, and sidearm post-functionalized CADA analogs were synthesized to target different proteins like CD4, sortilin, hTSHR, and ACE2. Furthermore, ACE2 down-modulation and SARS-CoV-2 RdRp screening were performed for selected analogs. From the ACE2 screening results, VGD020 seems to be a lead compound for further study (having dual activity), whereas the RdRp SARS-CoV-2 inhibition assay shows TK003 as a promising candidate. Finally, CADA analogs with a photoactive sidearm (TK046 and RA018) were synthesized for photo-affinity labeling (PAL) experiments to investigate the interaction site of CADA analogs in the Sec61 translocon.