SCH23390 is a widely used D1 dopamine receptor (D1R) antagonist that also elicits some D1R-independent effects. We previously found that the benzazepine, SKF83959, an analog of SCH23390, produces positive allosteric modulation of the Sigma-1 receptor (Sig1R). SCH23390 does not bind to the orthodoxic site of Sig1R but enhances the binding of 3H (+)-pentazocine to Sig1R. In this study, we investigated whether SCH23390 functions as an allosteric modulator of Sig1R. We detected increased Sig1R dissociation from binding immunoglobulin protein (BiP) and translocation of Sig1R to the plasma membrane in response to SCH23390 in transfected HEK293T and SH-SY5Y cells, respectively. Activation of Sig1R by SCH23390 was further confirmed by inhibition of GSK3β activity in a time- and dose-dependent manner; this effect was blocked by pretreatment with the Sig1R antagonist, BD1047, and by knockdown of Sig1R. SCH23390 also inhibited GSK3β in wild-type mice but not in Sig1R knockout mice. Finally, we showed that SCH23390 allosterically modulated the effect of the Sig1R agonist SKF10047 on inhibition of GSK3β. This positive allosteric effect of SCH23390 was further confirmed via promotion of neuronal protection afforded by SKF10047 in primary cortical neurons challenged with MPP+. These results provide the first evidence that SCH23390 elicits functional allosteric modulation of Sig1R. Our findings not only reveal novel pharmacological effects of SCH23390 but also indicate a potential mechanism for SCH23390-mediated D1R-independent effects. Therefore, attention should be paid to these Sig1R-mediated effects when explaining pharmacological responses to SCH23390.

Positive allosteric modulators (PAMs) of the cannabinoid CB1 receptor (CB1) offer potential therapeutic advantages in the treatment of neuropathic pain and addiction by avoiding the adverse effects associated with orthosteric CB1 activation. Here, molecular modeling and mutagenesis were used to identify residues central to PAM activity at CB1. Six putative allosteric binding sites were identified in silico, including novel sites previously associated with cholesterol binding, and key residues within each site were mutated to alanine. The recently determined ZCZ011 binding site was found to be essential for allosteric agonism, as GAT228, GAT229 and ZCZ011 all increased wild-type G protein dissociation in the absence of an orthosteric ligand; activity that was abolished in mutants F191A3.27 and I169A2.56. PAM activity was demonstrated for ZCZ011 in the presence of the orthosteric ligand CP55940, which was only abolished in I169A2.56. In contrast, the PAM activity of GAT229 was reduced for mutants R220A3.56, L404A8.50, F191A3.27 and I169A2.56. This indicates that allosteric modulation may represent the net effect of binding at multiple sites, and that allosteric agonism is likely to be mediated via the ZCZ011 site. This study underlines the need for detailed understanding of ligand receptor interactions in the search for pure CB1 allosteric modulators.

Human serum albumin (HSA) is a multi-domain macromolecule with diverse ligand binding capability because of its ability to allow allosteric modulation despite being a monomeric protein. Physiologically, HSA act as the primary carrier for various exogenous and endogenous compounds and fatty acids, and alter the pharmacokinetic properties of several drugs. It has antioxidant properties and is utilized therapeutically to improve the drug delivery of pharmacological agents for the treatment of several disorders. The flexibility of albumin in holding various types of drugs coupled with a variety of modifications makes this protein a versatile drug carrier with incalculable potential in therapeutics. This review provides a brief outline of the different structural properties of HSA, and its various binding sites, moreover, an overview of the genetic, biomedical, and allosteric modulation of drugs and drug delivery aspects of HSA is also included, which may be helpful in guiding advanced clinical applications and further research on the therapeutic potential of this extraordinary protein.

Guanylyl cyclase-A (GC-A), activated by endogenous atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), plays an important role in the regulation of cardiovascular and renal homeostasis and is an attractive drug target. Even though small molecule modulators allow oral administration and longer half-life, drug targeting of GC-A has so far been limited to peptides. Thus, in this study we aimed to develop small molecular activators of GC-A.
Hits were identified through high-throughput screening and optimized by in silico design. Cyclic GMP was measured in QBIHEK293A cells expressing GC-A, GC-B or chimerae of the two receptors using AlphaScreen technology. Binding assays were performed in membrane preparations or whole cells using 125 I-ANP. Vasorelaxation was measured in aortic rings isolated from Wistar rats.
We have identified small molecular allosteric enhancers of GC-A, which enhanced ANP or BNP effects in cellular systems and ANP-induced vasorelaxation in rat aortic rings. The mechanism of action appears novel and not mediated through previously described allosteric binding sites. In addition, the selectivity and activity depend on a single amino acid residue that differs between the two similar receptors GC-A and GC-B.
We describe a novel allosteric binding site on GC-A, which can be targeted by small molecules to enhance ANP and BNP effects. These compounds will be valuable tools in further development and proof-of-concept of GC-A enhancement for the potential use in cardiovascular therapy.

Cell signaling by receptor protein tyrosine kinases (RTKs) is tightly controlled by the counterbalancing actions of receptor protein tyrosine phosphatases (RPTPs). Due to their role in attenuating the signal-initiating potency of RTKs, RPTPs have long been viewed as therapeutic targets. However, the development of activators of RPTPs has remained limited. We previously reported that the homodimerization of a representative member of the RPTP family (protein tyrosine phosphatase receptor J or PTPRJ) is regulated by specific transmembrane (TM) residues. Disrupting this interaction by single point mutations promotes PTPRJ access to its RTK substrates (e.g., EGFR and FLT3), reduces RTK\'s phosphorylation and downstream signaling, and ultimately antagonizes RTK-driven cell phenotypes. Here, we designed and tested a series of first-in-class pH-responsive TM peptide agonists of PTPRJ that are soluble in aqueous solution but insert as a helical TM domain in lipid membranes when the pH is lowered to match that of the acidic microenvironment of tumors. The most promising peptide reduced EGFR\'s phosphorylation and inhibited cancer cell EGFR-driven migration and proliferation, similar to the PTPRJ\'s TM point mutations. Developing tumor-selective and TM-targeting peptide binders of critical RPTPs could afford a potentially transformative approach to studying RPTP\'s selectivity mechanism without requiring less specific inhibitors and represent a novel class of therapeutics against RTK-driven cancers.

G protein-coupled receptors (GPCRs) represent the target for approximately a third of FDA-approved small molecule drugs. The adenosine A1 receptor (A1R), one of four adenosine GPCR subtypes, has important (patho)physiological roles in humans. A1R has well-established roles in the regulation of the cardiovascular and nervous systems, where it has been identified as a potential therapeutic target for a number of conditions, including cardiac ischemia-reperfusion injury, cognition, epilepsy, and neuropathic pain. A1R small molecule drugs, typically orthosteric ligands, have undergone clinical trials. To date, none have progressed into the clinic, predominantly due to dose-limiting unwanted effects. The development of A1R allosteric modulators that target a topographically distinct binding site represent a promising approach to overcome current limitations. Pharmacological parameters of allosteric ligands, including affinity, efficacy and cooperativity, can be optimized to regulate A1R activity with high subtype, spatial and temporal selectivity. This review aims to offer insights into the A1R as a potential therapeutic target and highlight recent advances in the structural understanding of A1R allosteric modulation.

Although drugs targeting the orthosteric binding site of cannabinoid receptors (CBRs) have several therapeutic effects on human physiological and pathological conditions, they can also cause serious adverse effects. Only a few orthosteric ligands have successfully passed clinical trials. Recently, allosteric modulation has become a novel option for drug discovery, with fewer adverse effects and the potential to avoid drug overdose. In this review, we highlight novel findings related to the drug discovery of allosteric modulators (AMs) targeting CBRs. We summarize newly synthesized AMs and the reported/predicted allosteric binding sites. We also discuss the structural determinants of the AMs binding as well as the molecular mechanism of CBR allostery.

The SARS-CoV-2 spike protein contains a functionally important fatty acid (FA) binding site, which is also found in some other coronaviruses, e.g. SARS-CoV and MERS-CoV. The occupancy of the FA site by linoleic acid (LA) reduces infectivity by \'locking\' the spike in a less infectious conformation. Here, we use dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations to compare the allosteric responses of spike variants to LA removal. D-NEMD simulations show that the FA site is coupled to other functional regions of the protein, e.g. the receptor-binding motif (RBM), N-terminal domain (NTD), furin cleavage site, and regions surrounding the fusion peptide. D-NEMD simulations also identify the allosteric networks connecting the FA site to these functional regions. The comparison between the wild-type spike and four variants (Alpha, Delta, Delta plus, and Omicron BA.1) shows that the variants differ significantly in their responses to LA removal. The allosteric connections to the FA site on Alpha are generally similar to those on the wild-type protein, with the exception of the RBM and the S71-R78 region, which show a weaker link to the FA site. In contrast, Omicron is the most different variant, exhibiting significant differences in the RBM, NTD, V622-L629, and furin cleavage site. These differences in the allosteric modulation may be of functional relevance, potentially affecting transmissibility and virulence. Experimental comparison of the effects of LA on SARS-CoV-2 variants, including emerging variants, is warranted.

Originally, it was thought that a single serum amyloid A (SAA) protein was involved in amyloid A amyloidosis, but in fact, SAA represents a four-membered family wherein SAA1 and SAA2 are acute phase proteins (A-SAA). SAA is highly conserved throughout evolution within a wide range of animal species suggestive of an important biological function. In fact, A-SAA has been linked to a number of divergent biological activities wherein a number of these functions are mediated via the G protein-coupled receptor (GPCR), formyl peptide receptor (FPR) 2. For instance, through the activation of FPR2, A-SAA has been described to regulate leukocyte activation, atherosclerosis, pathogen recognition, bone formation and cell survival. Moreover, A-SAA is subject to post-translational modification, primarily through proteolytic processing, generating a range of A-SAA-derived peptides. Although very little is known regarding the biological effect of A-SAA-derived peptides, they have been shown to promote neutrophil and monocyte migration through FPR2 activation via synergy with other GPCR ligands namely, the chemokines CXCL8 and CCL3, respectively. Within this review, we provide a detailed analysis of the FPR2-mediated functions of A-SAA. Moreover, we discuss the potential role of A-SAA-derived peptides as allosteric modulators of FPR2.

Neurotransmission mediated by diverse subtypes of N-methyl-D-aspartate receptors (NMDARs) is fundamental for basic brain functions and development as well as neuropsychiatric diseases and disorders. NMDARs are glycine- and glutamate-gated ion channels that exist as heterotetramers composed of obligatory GluN1 and GluN2(A-D) and/or GluN3(A-B). The GluN2C and GluN2D subunits form ion channels with distinct properties and spatio-temporal expression patterns. Here, we provide the structures of the agonist-bound human GluN1-2C NMDAR in the presence and absence of the GluN2C-selective positive allosteric potentiator (PAM), PYD-106, the agonist-bound GluN1-2A-2C tri-heteromeric NMDAR, and agonist-bound GluN1-2D NMDARs by single-particle electron cryomicroscopy. Our analysis shows unique inter-subunit and domain arrangements of the GluN2C NMDARs, which contribute to functional regulation and formation of the PAM binding pocket and is distinct from GluN2D NMDARs. Our findings here provide the fundamental blueprint to study GluN2C- and GluN2D-containing NMDARs, which are uniquely involved in neuropsychiatric disorders.