An intracellular VHH targeting the Luteinizing Hormone receptor modulates G protein-dependent signaling and steroidogenesis.

Luteinizing hormone (LH) is essential for reproduction, controlling ovulation and steroidogenesis. Its receptor (LHR) recruits various transducers leading to the activation of a complex signaling network. We recently identified iPRC1, the first variable fragment from heavy-chain-only antibody (VHH) interacting with intracellular loop 3 (ICL3) of the follicle-stimulating hormone receptor (FSHR). Because of the high sequence similarity of the human FSHR and LHR (LHCGR), here we examined the ability of the iPRC1 intra-VHH to modulate LHCGR activity. In this study, we demonstrated that iPRC1 binds LHCGR, to a greater extent when the receptor was stimulated by the hormone. In addition, it decreased LH-induced cAMP production, cAMP-responsive element-dependent transcription, progesterone and testosterone production. These impairments are not due to Gs nor ß-arrestin recruitment to the LHCGR. Consequently, iPRC1 is the first intra-VHH to bind and modulate LHCGR biological activity, including steroidogenesis. It should help further understand signaling mechanisms elicited at this receptor and their outcomes on reproduction.

A single-domain intrabody targeting the follicle-stimulating hormone receptor impacts FSH-induced G protein-dependent signalling.

Intracellular variable fragments of heavy-chain antibody from camelids (intra-VHH) have been successfully used as chaperones to solve the 3D structure of active G protein-coupled receptors bound to their transducers. However, their effect on signalling has been poorly explored, although they may provide a better understanding of the relationships between receptor conformation and activity. Here, we isolated and characterized iPRC1, the first intra-VHH recognizing a member of the large glycoprotein hormone receptor family, the follicle-stimulating hormone receptor (FSHR). This intra-VHH recognizes the FSHR third intracellular loop and decreases cAMP production in response to FSH, without altering Gαs recruitment. Hence, iPRC1 behaves as an allosteric modulator and provides a new tool to complete structure/activity studies performed thus far on this receptor.

Combined Multiplexed Phage Display, High-Throughput Sequencing, and Functional Assays as a Platform for Identifying Modulatory VHHs Targeting the FSHR.

Developing modulatory antibodies against G protein-coupled receptors is challenging. In this study, we targeted the follicle-stimulating hormone receptor (FSHR), a significant regulator of reproduction, with variable domains of heavy chain-only antibodies (VHHs). We built two immune VHH libraries and submitted them to multiplexed phage display approaches. We used next-generation sequencing to identify 34 clusters of specifically enriched sequences that were functionally assessed in a primary screen based on a cAMP response element (CRE)-dependent reporter gene assay. In this assay, 23 VHHs displayed negative or positive modulation of FSH-induced responses, suggesting a high success rate of the multiplexed strategy. We then focused on the largest cluster identified (i.e., PRC1) that displayed positive modulation of FSH action. We demonstrated that PRC1 specifically binds to the human FSHR and human FSHR/FSH complex while potentiating FSH-induced cAMP production and Gs recruitment. We conclude that the improved selection strategy reported here is effective for rapidly identifying functionally active VHHs and could be adapted to target other challenging membrane receptors. This study also led to the identification of PRC1, the first potential positive modulator VHH reported for the human FSHR.

Purine containing carbonucleoside phosphonate analogues as novel chemotype for Plasmodium falciparum Inhibition.

The nucleotidase ISN1 is a potential therapeutic target of the purine salvage pathway of the malaria parasite Plasmodium falciparum. We identified PfISN1 ligands by in silico screening of a small library of nucleos(t)ide analogues and by thermal shift assays. Starting from a racemic cyclopentyl carbocyclic phosphonate scaffold, we explored the diversity on the nucleobase moiety and also proposed a convenient synthetic pathway to access the pure enantiomers of our initial hit (compound (±)-2). 2,6-Disubstituted purine containing derivatives such as compounds 1, (±)-7e and ß-L-(+)-2 showed the most potent inhibition of the parasite in vitro, with low micromolar IC50 values. These results are remarkable considering the anionic nature of nucleotide analogues, which are known to lack activity in cell culture experiments due to their scarce capacity to cross cell membranes. For the first time, we report the antimalarial activity of a carbocyclic methylphosphonate nucleoside with an L-like configuration.

Intracellular VHHs to monitor and modulate GPCR signaling.

Single-domain antibody fragments, also known as VHHs or nanobodies, have opened promising avenues in therapeutics and in exploration of intracellular processes. Because of their unique structural properties, they can reach cryptic regions in their cognate antigen. Intracellular VHHs/antibodies primarily directed against cytosolic proteins or transcription factors have been described. In contrast, few of them target membrane proteins and even less recognize G protein-coupled receptors. These receptors are major therapeutic targets, which reflects their involvement in a plethora of physiological responses. Hence, they elicit a tremendous interest in the scientific community and in the industry. Comprehension of their pharmacology has been obscured by their conformational complexity, that has precluded deciphering their structural properties until the early 2010's. To that respect, intracellular VHHs have been instrumental in stabilizing G protein-coupled receptors in active conformations in order to solve their structure, possibly bound to their primary transducers, G proteins or ß-arrestins. In contrast, the modulatory properties of VHHs recognizing the intracellular regions of G protein-coupled receptors on the induced signaling network have been poorly studied. In this review, we will present the advances that the intracellular VHHs have permitted in the field of GPCR signaling and trafficking. We will also discuss the methodological hurdles that linger the discovery of modulatory intracellular VHHs directed against GPCRs, as well as the opportunities they open in drug discovery.

Exploring molecular determinants of polysaccharide lyase family 6-1 enzyme activity.

The polysaccharide lyase family 6 (PL6) represents one of the 41 polysaccharide lyase families classified in the CAZy database with the vast majority of its members being alginate lyases grouped into three subfamilies, PL6_1-3. To decipher the mode of recognition and action of the enzymes belonging to subfamily PL6_1, we solved the crystal structures of Pedsa0632, Patl3640, Pedsa3628 and Pedsa3807, which all show different substrate specificities and mode of action (endo-/exolyase). Thorough exploration of the structures of Pedsa0632 and Patl3640 in complex with their substrates as well as docking experiments confirms that the conserved residues in subsites -1 to +3 of the catalytic site form a common platform that can accommodate various types of alginate in a very similar manner but with a series of original adaptations bringing them their specificities of action. From comparative studies with existing structures of PL6_1 alginate lyases, we observe that in the right-handed parallel ß-helix fold shared by all these enzymes, the substrate-binding site harbors the same overall conserved structures and organization. Despite this apparent similarity, it appears that members of the PL6_1 subfamily specifically accommodate and catalyze the degradation of different alginates suggesting that this common platform is actually a highly adaptable and specific tool.

Disulfide reduction in CD4 domain 1 or 2 is essential for interaction with HIV glycoprotein 120 (gp120), which impairs thioredoxin-driven CD4 dimerization.

Human CD4 is a membrane-bound glycoprotein expressed on the surface of certain leukocytes, where it plays a key role in the activation of immunostimulatory T cells and acts as the primary receptor for human immunodeficiency virus (HIV) glycoprotein (gp120). Although growing evidence suggests that redox exchange reactions involving CD4 disulfides, potentially catalyzed by cell surface-secreted oxidoreductases such as thioredoxin (Trx) and protein disulfide isomerase, play an essential role in regulating the activity of CD4, their mechanism(s) and biological utility remain incompletely understood. To gain more insights in this regard, we generated a panel of recombinant 2-domain CD4 proteins (2dCD4), including wild-type and Cys/Ala variants, and used these to show that while protein disulfide isomerase has little capacity for 2dCD4 reduction, Trx reduces 2dCD4 highly efficiently, catalyzing the formation of conformationally distinct monomeric 2dCD4 isomers, and a stable, disulfide-linked 2dCD4 dimer. Moreover, we show that HIV gp120 is incapable of binding a fully oxidized, monomeric 2dCD4 in which both domain 1 and 2 disulfides are intact, but binds robustly to reduced counterparts that are the ostensible products of Trx-mediated isomerization. Finally, we demonstrate that Trx-driven dimerization of CD4, a process believed to be critical for the establishment of functional MHCII-TCR-CD4 antigen presentation complexes, is impaired when CD4 is bound to gp120. These observations reinforce the importance of cell surface redox activity for HIV entry and posit the intriguing possibility that one of the many pathogenic effects of HIV may be related to gp120-mediated inhibition of oxidoreductive CD4 isomerization.