In Silico Prediction and Validation of CB2 Allosteric Binding Sites to Aid the Design of Allosteric Modulators

Although the 3D structures of active and inactive cannabinoid receptors type 2 (CB2) are available, neither the X-ray crystal nor the cryo-EM structure of CB2-orthosteric ligand-modulator has been resolved, prohibiting the drug discovery and development of CB2 allosteric modulators (AMs). In the present work, we mainly focused on investigating the potential allosteric binding site(s) of CB2. We applied different algorithms or tools to predict the potential allosteric binding sites of CB2 with the existing agonists. Seven potential allosteric sites can be observed for either CB2-CP55940 or CB2-WIN 55,212-2 complex, among which sites B, C, G and K are supported by the reported 3D structures of Class A GPCRs coupled with AMs. Applying our novel algorithm toolset-MCCS, we docked three known AMs of CB2 including Ec2la (C-2), trans-β-caryophyllene (TBC) and cannabidiol (CBD) to each site for further comparisons and quantified the potential binding residues in each allosteric binding site. Sequentially, we selected the most promising binding pose of C-2 in five allosteric sites to conduct the molecular dynamics (MD) simulations. Based on the results of docking studies and MD simulations, we suggest that site H is the most promising allosteric binding site. We plan to conduct bio-assay validations in the future.

Novel enhancers of guanylyl cyclase-A activity via allosteric modulation

Natriuretic peptide receptor (NPR)-A (also known as NPR-A, NPR1 or guanylyl cyclase-A, GC-A) is an attractive but challenging target to activate with small molecules. GC-A is activated by endogenous atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), and this activation leads to the production of cyclic guanosine monophosphate (cGMP). This system plays an important role in the regulation of cardiovascular and renal homeostasis. However, utilization of this receptor as a drug target has so far been limited to peptides, even though small molecule modulators allow oral administration and longer half-life. We have identified small molecular allosteric enhancers of GC-A, which strengthened ANP or BNP activation in various in vitro and ex vivo systems. These compounds do not mediate their actions through previously described allosteric binding sites or via known mechanisms of action. In addition, their selectivity and activity are dependent on only one amino acid in GC-A. Our findings show that there is a novel allosteric binding site on GC-A, which can be targeted by small molecules that increase the signaling effects of ANP and BNP.

Nucleic acid binding by SAMHD1 contributes to the antiretroviral activity and is enhanced by the GpsN modification

SAMHD1 impedes infection of myeloid cells and resting T lymphocytes by retroviruses, and the enzymatic activity of the protein—dephosphorylation of deoxynucleotide triphosphates (dNTPs)—implicates enzymatic dNTP depletion in innate antiviral immunity. Here we show that the allosteric binding sites of the enzyme are plastic and can accommodate oligonucleotides in place of the allosteric activators, GTP and dNTP. SAMHD1 displays a preference for oligonucleotides containing phosphorothioate bonds in the Rp configuration located 3’ to G nucleotides (GpsN), the modification pattern that occurs in a mechanism of antiviral defense in prokaryotes. In the presence of GTP and dNTPs, binding of GpsN-containing oligonucleotides promotes formation of a distinct tetramer with mixed occupancy of the allosteric sites. Mutations that impair formation of the mixed-occupancy complex abolish the antiretroviral activity of SAMHD1, but not its ability to deplete dNTPs. The findings link nucleic acid binding to the antiretroviral activity of SAMHD1, shed light on the immunomodulatory effects of synthetic phosphorothioated oligonucleotides and raise questions about the role of nucleic acid phosphorothioation in human innate immunity.

Distant Residues Modulate Conformational Opening in SARS-CoV-2 Spike Protein

Infection by SARS-CoV-2 involves the attachment of the receptor binding domain (RBD) of its spike proteins to the ACE2 receptors on the peripheral membrane of host cells. Binding is initiated by a down to up conformational change in the spike protein, an opening which presents the RBD to the receptor. To date, computational and experimental studies for therapeutics have concentrated, for good reason, on the RBD. However, the RBD region is highly prone to mutations, and therefore will possibly arise drug resistance. In contrast, we here focus on the correlations between the RBD and residues distant to it in the spike protein. We thereby provide a deeper understanding of the role of distant residues in the molecular mechanism of infection. Predictions of key mutations in distant allosteric binding sites are provided, with implications for therapeutics. Identifying these emerging mutants can also go a long way towards pre-designing vaccines for future outbreaks. The model we use, based on time-independent component analysis (tICA) and protein graph connectivity network, is able to identify multiple residues that exhibit long-distance coupling with the RBD opening. Mutation on these residues can lead to new strains of coronavirus with different degrees of transmissibility and virulence. The most ubiquitous D614G mutation and the A570D mutation of the highly contageous UK SARS-CoV-2 variant are predicted ab-initio from our model. Conversely, broad spectrum therapeutics like drugs and monoclonal antibodies can be generated targeting these key distant but conserved regions of the spike protein.

Pharmacological properties and biochemical mechanisms of μ-opioid receptor ligands might be due to different binding poses: MD studies

Background: Central and peripheral analgesia without adverse effects relies on the identification of μ-opioid agonists that are able to activate ‘basal’ antinociceptive pathways. Recently developed μ-selective benzomorphan agonists that are not antagonized by naloxone do not activate G-proteins and β-arrestins. Which pathways do μ receptors activate? How can each of them be selectively activated? What role is played by allosteric binding sites? Methodology & results: Molecular modeling studies characterize the amino acid residues involved in the interaction with various classes of endogenous and exogenous ligands and with agonists and antagonists. Conclusions: Critical binding differences between various classes of agonists with different pharmacological profiles have been identified. MML series binding poses may be relevant in the search for an antinociception agent without side effects.