Secondary Binding Pocket Research Articles

A bitopic agonist bound to the dopamine 3 receptor reveals a selectivity site

Although aminergic GPCRs are the target for ~25% of approved drugs, developing subtype selective drugs is a major challenge due to the high sequence conservation at their orthosteric binding site. Bitopic ligands are covalently joined orthosteric and allosteric pharmacophores with the potential to boost receptor selectivity and improve current medications by reducing off-target side effects. However, the lack of structural information on their binding mode impedes rational design. Here we determine the cryo-EM structure of the hD3R:GαOβγ complex bound to the D3R selective bitopic agonist FOB02-04A. Structural, functional and computational analyses provide insights into its binding mode and point to a new TM2-ECL1-TM1 region, which requires the N-terminal ordering of TM1, as a major determinant of subtype selectivity in aminergic GPCRs. This region is underexploited in drug development, expands the established secondary binding pocket in aminergic GPCRs and could potentially be used to design novel and subtype selective drugs.

Dopamine Receptor Ligand Selectivity-An In Silico/In Vitro Insight.

Different dopamine receptor (DR) subtypes are involved in pathophysiological conditions such as Parkinson's Disease (PD), schizophrenia and depression. While many DR-targeting drugs have been approved by the U.S. Food and Drug Administration (FDA), only a very small number are truly selective for one of the DR subtypes. Additionally, most of them show promiscuous activity at related G-protein coupled receptors, thus suffering from diverse side-effect profiles. Multiple studies have shown that combined in silico/in vitro approaches are a valuable contribution to drug discovery processes. They can also be applied to divulge the mechanisms behind ligand selectivity. In this study, novel DR ligands were investigated in vitro to assess binding affinities at different DR subtypes. Thus, nine D2R/D3R-selective ligands (micro- to nanomolar binding affinities, D3R-selective profile) were successfully identified. The most promising ligand exerted nanomolar D3R activity (Ki = 2.3 nM) with 263.7-fold D2R/D3R selectivity. Subsequently, ligand selectivity was rationalized in silico based on ligand interaction with a secondary binding pocket, supporting the selectivity data determined in vitro. The developed workflow and identified ligands could aid in the further understanding of the structural motifs responsible for DR subtype selectivity, thus benefitting drug development in D2R/D3R-associated pathologies such as PD.

Siglec-6 mediates the uptake of extracellular vesicles through a noncanonical glycolipid binding pocket

Immunomodulatory Siglecs are controlled by their glycoprotein and glycolipid ligands. Siglec-glycolipid interactions are often studied outside the context of a lipid bilayer, missing the complex behaviors of glycolipids in a membrane. Through optimizing a liposomal formulation to dissect Siglec–glycolipid interactions, it is shown that Siglec-6 can recognize glycolipids independent of its canonical binding pocket, suggesting that Siglec-6 possesses a secondary binding pocket tailored for recognizing glycolipids in a bilayer. A panel of synthetic neoglycolipids is used to probe the specificity of this glycolipid binding pocket on Siglec-6, leading to the development of a neoglycolipid with higher avidity for Siglec-6 compared to natural glycolipids. This neoglycolipid facilitates the delivery of liposomes to Siglec-6 on human mast cells, memory B-cells and placental syncytiotrophoblasts. A physiological relevance for glycolipid recognition by Siglec-6 is revealed for the binding and internalization of extracellular vesicles. These results demonstrate a unique and physiologically relevant ability of Siglec-6 to recognize glycolipids in a membrane.

Structure of human placental steroid sulfatase at 2.0 angstrom resolution: Catalysis, quaternary association, and a secondary ligand site

Human placental estrone (E1)/dehydroepiandrosterone (DHEA) sulfatase (human placental steroid sulfatase; hSTS) is an integral membrane protein of the endoplasmic reticulum. This Ca2+-dependent enzyme catalyzes the hydrolysis of sulfate esters of E1 and DHEA to yield the respective unconjugated steroids, which then act as precursors for the biosynthesis of 17β-estradiol (E2) and dihydrotestosterone (DHT), respectively, the most potent forms of estrogens and androgens. hSTS is a key enzyme for the local production of E2 and DHT in the breast and the prostate. The enzyme is known to be responsible for maintaining high levels of estrogens in the breast tumor cells. The crystal structure of hSTS purified from human placenta has previously been reported at 2.6 Å resolution. Here we present the structure of hSTS determined at the superior 2.0 Å resolution bringing new clarity to the atomic architecture of the active site. The molecular basis of catalysis and steroid-protein interaction are revisited in light of the new data. We also reexamine the enzyme’s quaternary association and its implication on the membrane integration. A secondary ligand binding pocket at the intermolecular interface and adjacent to the active site access channel, buried into the gill of the mushroom-shaped molecule, has been identified. Its role as well as that of a phosphate ion bound to an exposed histidine side chain are examined from the structure-function perspective. Higher resolution data also aids in the tracing of an important loop missing in the previous structure.

Theoretical Investigation of Repurposed Drugs Potentially Capable of Binding to the Catalytic Site and the Secondary Binding Pocket of Subunit A of Ricin.

Recently, we reported a library of 82 compounds, selected from different databanks through virtual screening and docking studies, and pointed to 6 among them as potential repurposed dual binders to both the catalytic site and the secondary binding pockets of subunit A of ricin (RTA). Here, we report additional molecular modeling studies of an extended list of compounds from the original library. Rounds of flexible docking followed by molecular dynamics simulations and further rounds of MM-PBSA calculations using a more robust protocol, enabled a better investigation of the interactions of these compounds inside RTA, the elucidation of their dynamical behaviors, and updating the list of the most important residues for the ligand binding. Four compounds were pointed as potential repurposed ricin inhibitors that are worth being experimentally investigated.

The Impact of the Secondary Binding Pocket on the Pharmacology of Class A GPCRs.

G-protein coupled receptors (GPCRs) are considered important therapeutic targets due to their pathophysiological significance and pharmacological relevance. Class A receptors represent the largest group of GPCRs that gives the highest number of validated drug targets. Endogenous ligands bind to the orthosteric binding pocket (OBP) embedded in the intrahelical space of the receptor. During the last 10 years, however, it has been turned out that in many receptors there is secondary binding pocket (SBP) located in the extracellular vestibule that is much less conserved. In some cases, it serves as a stable allosteric site harbouring allosteric ligands that modulate the pharmacology of orthosteric binders. In other cases it is used by bitopic compounds occupying both the OBP and SBP. In these terms, SBP binding moieties might influence the pharmacology of the bitopic ligands. Together with others, our research group showed that SBP binders contribute significantly to the affinity, selectivity, functional activity, functional selectivity and binding kinetics of bitopic ligands. Based on these observations we developed a structure-based protocol for designing bitopic compounds with desired pharmacological profile.

Evidence for Two Modes of Binding of the Negative Allosteric Modulator SB269,652 to the Dopamine D2 Receptor.

SB269,652 has been described as the first negative allosteric modulator (NAM) of the dopamine D2 receptor (D2R), however, the binding mode and allosteric mechanism of action of this ligand remain incompletely understood. SB269,652 comprises an orthosteric, primary pharmacophore and a secondary (or allosteric) pharmacophore joined by a hydrophilic cyclohexyl linker and is known to form corresponding interactions with the orthosteric binding site (OBS) and the secondary binding pocket (SBP) in the D2R. Here, we observed a surprisingly low potency of SB269,652 to negatively modulate the D2R-mediated activation of G protein-coupled inward-rectifier potassium channels (GIRK) and decided to perform a more detailed investigation of the interaction between dopamine and SB269,652. The results indicated that the SB269,652 inhibitory potency is increased 6.6-fold upon ligand pre-incubation, compared to the simultaneous co-application with dopamine. Mutagenesis experiments implicated both S193 in the OBS and E95 in the SBP in the effect of pre-application. The present findings extend previous knowledge about how SB269,652 competes with dopamine at the D2R and may be useful for the development of novel D2R ligands, such as antipsychotic drug candidates.

A Remote Secondary Binding Pocket Promotes Heteromultivalent Targeting of DC-SIGN.

Dendritic cells (DC) are antigen-presenting cells coordinating the interplay of the innate and the adaptive immune response. The endocytic C-type lectin receptors DC-SIGN and Langerin display expression profiles restricted to distinct DC subtypes and have emerged as prime targets for next-generation immunotherapies and anti-infectives. Using heteromultivalent liposomes copresenting mannosides bearing aromatic aglycones with natural glycan ligands, we serendipitously discovered striking cooperativity effects for DC-SIGN+ but not for Langerin+ cell lines. Mechanistic investigations combining NMR spectroscopy with molecular docking and molecular dynamics simulations led to the identification of a secondary binding pocket for the glycomimetics. This pocket, located remotely of DC-SIGN’s carbohydrate bindings site, can be leveraged by heteromultivalent avidity enhancement. We further present preliminary evidence that the aglycone allosterically activates glycan recognition and thereby contributes to DC-SIGN-specific cell targeting. Our findings have important implications for both translational and basic glycoscience, showcasing heteromultivalent targeting of DCs to improve specificity and supporting potential allosteric regulation of DC-SIGN and CLRs in general.

Structural Insights into the Mechanisms of Action of Functionally Distinct Classes of Chikungunya Virus Nonstructural Protein 1 Inhibitors.

ABSTRACTChikungunya virus (CHIKV) nonstructural protein 1 (nsP1) harbors the methyltransferase (MTase) and guanylyltransferase (GTase) activities needed for viral RNA capping and represents a promising antiviral drug target. We compared the antiviral efficacies of nsP1 inhibitors belonging to the MADTP, CHVB, and FHNA series (6′-fluoro-homoneplanocin A [FHNA], its 3′-keto form, and 6′-β-fluoro-homoaristeromycin). Cell-based phenotypic cross-resistance assays revealed that the CHVB and MADTP series had similar modes of action that differed from that of the FHNA series. In biochemical assays with purified Semliki Forest virus and CHIKV nsP1, CHVB compounds strongly inhibited MTase and GTase activities, while MADTP-372 had a moderate inhibitory effect. FHNA did not directly inhibit the enzymatic activity of CHIKV nsP1. The first-of-their-kind molecular-docking studies with the cryo-electron microscopy (cryo-EM) structure of CHIKV nsP1, which is assembled into a dodecameric ring, revealed that the MADTP and CHVB series bind at the S-adenosylmethionine (SAM)-binding site in the capping domain, where they would function as competitive or noncompetitive inhibitors. The FHNA series was predicted to bind at the secondary binding pocket in the ring-aperture membrane-binding and oligomerization (RAMBO) domain, potentially interfering with the membrane binding and oligomerization of nsP1. Our cell-based and enzymatic assays, in combination with molecular docking and mapping of compound resistance mutations to the nsP1 structure, allowed us to group nsP1 inhibitors into functionally distinct classes. This study identified druggable pockets in the nsP1 dodecameric structure and provides a basis for the rational design, optimization, and combination of inhibitors of this unique and promising antiviral drug target.

Controlling the selectivity of aminergic GPCR ligands from the extracellular vestibule

In addition to the orthosteric binding pocket (OBP) of GPCRs, recent structural studies have revealed that there are several allosteric sites available for pharmacological intervention. The secondary binding pocket (SBP) of aminergic GPCRs is located in the extracellular vestibule of these receptors, and it has been suggested to be a potential selectivity pocket for bitopic ligands. Here, we applied a virtual screening protocol based on fragment docking to the SBP of the orthosteric ligand-receptor complex. This strategy was employed for a number of aminergic receptors. First, we designed dopamine D3 preferring bitopic compounds from a D2 selective orthosteric ligand. Next, we designed 5-HT2B selective bitopic compounds starting from the 5-HT1B preferring ergoline core of LSD. Comparing the serotonergic profiles of the new derivatives to that of LSD, we found that these derivatives became significantly biased towards the desired 5-HT2B receptor target. Finally, addressing the known limitations of H1 antihistamines, our protocol was successfully used to eliminate the well-known side effects related to the muscarinic M1 activity of amitriptyline while preserving H1 potency in some of the designed bitopic compounds. These applications highlight the usefulness of our new virtual screening protocol and offer a powerful strategy towards bitopic GPCR ligands with designed receptor profiles.

Allelic characterization and protein structure analysis reveals the involvement of splice site mutation for growth habit differences in Lablab purpureus (L.) Sweet

BackgroundInterrelationship between growth habit and flowering played a key role in the domestication history of pulses; however, the actual genes responsible for these traits have not been identified in Indian bean. Determinate growth habit is desirable due to its early flowering, photo-insensitivity, synchronous pod maturity, ease in manual harvesting and short crop duration. The present study aimed to identify, characterize and validate the gene responsible for growth habit by using a candidate gene approach coupled with sequencing, multiple sequence alignment, protein structure prediction and binding pocket analysis. ResultsTerminal flowering locus was amplified from GPKH 120 (indeterminate) and GNIB-21 (determinate) using the primers designed from PvTFL1y locus of common bean. Gene prediction revealed that the length of the third and fourth exons differed between the two alleles. Allelic sequence comparison indicated a transition from guanine to adenine at the end of the third exon in GNIB 21. This splice site single-nucleotide polymorphism (SNP) was validated in germplasm lines by sequencing. Protein structure analysis indicated involvement of two binding pockets for interaction of terminal flowering locus (TFL) protein with other proteins. ConclusionThe splice site SNP present at the end of the third exon of TFL locus is responsible for the transformation of shoot apical meristem into a reproductive fate in the determinate genotype GNIB 21. The splice site SNP leads to absence of 14 amino acids in mutant TFL protein of GNIB 21, rendering the protein non-functional. This deletion disturbed previously reported anion-binding pocket and secondary binding pocket due to displacement of small β-sheet away from an external loop. This finding may enable the modulation of growth habit in Indian bean and other pulse crops through genome editing.

Fragment evolution for GPCRs: the role of secondary binding sites in optimization.

We developed a docking-based fragment evolution approach that extends orthosteric fragments towards a less conserved secondary binding pocket of GPCRs. Evaluating 13 000 extensions for the β1- and β2-adrenergic receptors we synthesized and tested 112 bitopic molecules. Our results confirmed the positive contribution of the secondary binding pocket to both potency and selectivity optimizations.

Evidence for a Stereoselective Mechanism for Bitopic Activity by Extended-Length Antagonists of the D3 Dopamine Receptor.

The D3 dopamine receptor (D3R) has been suggested as a drug target for the treatment of a number of neuropsychiatric disorders, including substance use disorders (SUD). Many D3R-selective antagonists are bivalent in nature in that they engage two distinct sites on the receptor-a primary pharmacophore binds to the orthosteric site, where dopamine binds, whereas a secondary pharmacophore interacts with a unique secondary binding pocket (SBP). When engagement of the secondary pocket exerts allosteric activity, the compound is said to be bitopic. We recently reported the synthesis and characterization of two bitopic antagonists of the D3R, (±)-VK04-87 and (±)-VK05-95, which incorporated a racemic trans-cyclopropylmethyl linking chain. To gain a better understanding of the role of chirality in determining the pharmacology of such compounds, we resolved the enantiomers of (±)-VK04-87. We found that the (+)-isomer displays higher affinity for the D3R and exhibits greater selectivity versus the D2R than the (-)-isomer. Strikingly, using functional assays, we found that (+)-VK04-87 inhibits the D3R in a noncompetitive manner, while (-)-VK04-87 behaves as a purely competitive antagonist, indicating that the apparent allosteric activity of the racemate is due to the (+)-isomer. Molecular dynamic simulations of (+)-VK04-87 and (-)-VK04-87 binding to the D3R suggest that the (+)-isomer is able to interact with the SBP of the receptor whereas the (-)-isomer bends away from this pocket, thus potentially explaining their differing pharmacology. These results emphasize the importance of the linker, and its isomeric conformations, within extended-length molecules for their positioning and engagement within GPCR binding pockets.

Ligand with Two Modes of Interaction with the Dopamine D2 Receptor-An Induced-Fit Mechanism of Insurmountable Antagonism.

A solid understanding of the mechanisms governing ligand binding is crucial for rational design of therapeutics targeting the dopamine D2 receptor (D2R). Here, we use G protein-coupled inward rectifier potassium (GIRK) channel activation in Xenopus oocytes to measure the kinetics of D2R antagonism by a series of aripiprazole analogues, as well as the recovery of dopamine (DA) responsivity upon washout. The aripiprazole analogues comprise an orthosteric and a secondary pharmacophore and differ by the length of the saturated carbon linker joining these two pharmacophores. Two compounds containing 3- and 5-carbon linkers allowed for a similar extent of recovery from antagonism in the presence of 1 or 100 μM DA (>25 and >90% of control, respectively), whereas recovery was less prominent (∼20%) upon washout of the 4-carbon linker compound, SV-III-130, both with 1 and 100 μM DA. Prolonging the coincubation time with SV-III-130 further diminished recovery. Curve-shift experiments were consistent with competition between SV-III-130 and DA. Two mutations in the secondary binding pocket (V91A and E95A) of D2R decreased antagonistic potency and increased recovery from SV-III-130 antagonism, whereas a third mutation (L94A) only increased recovery. Our results suggest that the secondary binding pocket influences recovery from inhibition by the studied aripiprazole analogues. We propose a mechanism, supported by in silico modeling, whereby SV-III-130 initially binds reversibly to the D2R, after which the drug-receptor complex undergoes a slow transition to a second ligand-bound state, which is dependent on secondary binding pocket integrity and irreversible during the time frame of our experiments.

Analysis of the structure and substrate scope of chitooligosaccharide oxidase reveals high affinity for C2-modified glucosamines.

Chitooligosaccharide oxidase (ChitO) is a fungal carbohydrate oxidase containing a bicovalently bound FAD cofactor. The enzyme is known to catalyse the oxidation of chitooligosaccharides, oligomers of N-acetylated glucosamines derived from chitin degradation. In this study, the unique substrate acceptance was explored by testing a range of N-acetyl-d-glucosamine derivatives, revealing that ChitO preferentially accepts carbohydrates with a hydrophobic group attached to C2. The enzyme also accepts streptozotocin, a natural product used to treat tumours. Elucidation of the crystal structure provides an explanation for the high affinity towards C2-decorated glucosamines: the active site has a secondary binding pocket that accommodates groups attached at C2. Docking simulations are fully in line with the observed substrate preference. This work expands the knowledge on this versatile enzyme.

Controlling receptor function from the extracellular vestibule of G-protein coupled receptors.

Receptor function is traditionally controlled from the orthosteric binding site of G-protein coupled receptors. Here, we show that the functional activity and signalling of human dopamine D2 and D3 receptor ligands can be fine-tuned from the extracellular secondary binding pocket (SBP) located far from the signalling interface suggesting optimization of the SBP binding part of bitopic ligands might be a useful strategy to develop GPCR ligands with designed functional and signalling profile.

Crystal structure of dopamine receptor D4 bound to the subtype selective ligand, L745870.

Multiple subtypes of dopamine receptors within the GPCR superfamily regulate neurological processes through various downstream signaling pathways. A crucial question about the dopamine receptor family is what structural features determine the subtype-selectivity of potential drugs. Here, we report the 3.5-angstrom crystal structure of mouse dopamine receptor D4 (DRD4) complexed with a subtype-selective antagonist, L745870. Our structure reveals a secondary binding pocket extended from the orthosteric ligand-binding pocket to a DRD4-specific crevice located between transmembrane helices 2 and 3. Additional mutagenesis studies suggest that the antagonist L745870 prevents DRD4 activation by blocking the relative movement between transmembrane helices 2 and 3. These results expand our knowledge of the molecular basis for the physiological functions of DRD4 and assist new drug design.

Molecular Determinants of the Intrinsic Efficacy of the Antipsychotic Aripiprazole.

Partial agonists of the dopamine D2 receptor (D2R) have been developed to treat the symptoms of schizophrenia without causing the side effects elicited by antagonists. The receptor-ligand interactions that determine the intrinsic efficacy of such drugs, however, are poorly understood. Aripiprazole has an extended structure comprising a phenylpiperazine primary pharmacophore and a 1,2,3,4-tetrahydroquinolin-2-one secondary pharmacophore. We combined site-directed mutagenesis, analytical pharmacology, ligand fragments, and molecular dynamics simulations to identify the D2R-aripiprazole interactions that contribute to affinity and efficacy. We reveal that an interaction between the secondary pharmacophore of aripiprazole and a secondary binding pocket defined by residues at the extracellular portions of transmembrane segments 1, 2, and 7 determines the intrinsic efficacy of aripiprazole. Our findings reveal a hitherto unappreciated mechanism for fine-tuning the intrinsic efficacy of D2R agonists.

Identification of a secondary binding site in human macrophage galactose-type lectin by microarray studies: Implications for the molecular recognition of its ligands

The human macrophage galactose-type lectin (MGL) is a C-type lectin characterized by a unique specificity for terminal GalNAc residues present in the tumor-associated Tn antigen (αGalNAc-Ser/Thr) and its sialylated form, the sialyl-Tn antigen. However, human MGL has multiple splice variants, and whether these variants have distinct ligand-binding properties is unknown. Here, using glycan microarrays, we compared the binding properties of the short MGL 6C (MGLshort) and the long MGL 6B (MGLlong) splice variants, as well as of a histidine-to-threonine mutant (MGLshort H259T). Although the MGLshort and MGLlong variants displayed similar binding properties on the glycan array, the MGLshort H259T mutant failed to interact with the sialyl-Tn epitope. As the MGLshort H259T variant could still bind a single GalNAc monosaccharide on this array, we next investigated its binding characteristics to Tn-containing glycopeptides derived from the MGL ligands mucin 1 (MUC1), MUC2, and CD45. Strikingly, in the glycopeptide microarray, the MGLshort H259T variant lost high-affinity binding toward Tn-containing glycopeptides, especially at low probing concentrations. Moreover, MGLshort H259T was unable to recognize cancer-associated Tn epitopes on tumor cell lines. Molecular dynamics simulations indicated that in WT MGLshort, His259 mediates H bonds directly or engages the Tn-glycopeptide backbone through water molecules. These bonds were lost in MGLshort H259T, thus explaining its lower binding affinity. Together, our results suggest that MGL not only connects to the Tn carbohydrate epitope, but also engages the underlying peptide via a secondary binding pocket within the MGL carbohydrate recognition domain containing the His259 residue.

New Dopamine D3-Selective Receptor Ligands Containing a 6-Methoxy-1,2,3,4-tetrahydroisoquinolin-7-ol Motif.

A series of analogues featuring a 6-methoxy-1,2,3,4-tetrahydroisoquinolin-7-ol unit as the arylamine "head" group of a classical D3 antagonist core structure were synthesized and evaluated for affinity at dopamine D1, D2, and D3 receptors (D1R, D2R, D3R). The compounds generally displayed strong affinity for D3R with very good D3R selectivity. Docking studies at D2R and D3R crystal structures revealed that the molecules are oriented such that their arylamine units are positioned in the orthosteric binding pocket of D3R, with the arylamide "tail" units residing in the secondary binding pocket. Hydrogen bonding between Ser 182 and Tyr 365 at D3R stabilize extracellular loop 2 (ECL2), which in turn contributes to ligand binding by interacting with the "tail" units of the ligands in the secondary binding pocket. Similar interactions between ECL2 and the "tail" units were absent at D2R due to different positioning of the D2R loop region. The presence of multiple H-bonds with the phenol moiety of the headgroup of 7 and Ser192 accounts for its stronger D3R affinity as compared to the 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-containing analogue 8.