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Allosteric modulator
Allosteric modulator
Allosteric modulator
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In pharmacology and biochemistry, allosteric modulators are a group of substances that bind to a receptor to change that receptor's response to stimuli. Some of them, like benzodiazepines or alcohol, function as psychoactive drugs.[1] The site that an allosteric modulator binds to (i.e., an allosteric site) is not the same one to which an endogenous agonist of the receptor would bind (i.e., an orthosteric site). Modulators and agonists can both be called receptor ligands.[2]

Allosteric modulators can be 1 of 3 types either: positive, negative or neutral. Positive types increase the response of the receptor by increasing the probability that an agonist will bind to a receptor (i.e. affinity), increasing its ability to activate the receptor (i.e. efficacy), or both. Negative types decrease the agonist affinity and/or efficacy. Neutral types don't affect agonist activity but can stop other modulators from binding to an allosteric site. Some modulators also work as allosteric agonists and yield an agonistic effect by themselves.[2]

The term "allosteric" derives from the Greek language. Allos means "other", and stereos, "solid" or "shape". This can be translated to "other shape", which indicates the conformational changes within receptors caused by the modulators through which the modulators affect the receptor function.[3]

Introduction

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Allosteric modulators can alter the affinity and efficacy of other substances acting on a receptor. A modulator may also increase affinity and lower efficacy or vice versa.[4] Affinity is the ability of a substance to bind to a receptor. Efficacy is the ability of a substance to activate a receptor, given as a percentage of the ability of the substance to activate the receptor as compared to the receptor's endogenous agonist. If efficacy is zero, the substance is considered an antagonist.[1]

Orthosteric agonist (A) binds to orthosteric site (B) of a receptor (E). Allosteric modulator (C) binds to allosteric site (D). Modulator increases/lowers the affinity (1) and/or efficacy (2) of an agonist. Modulator may also act as an agonist and yield an agonistic effect (3). Modulated orthosteric agonist affects the receptor (4). Receptor response (F) follows.

The site to which endogenous agonists bind to is named the orthosteric site. Modulators don't bind to this site. They bind to any other suitable sites, which are named allosteric sites.[2] Upon binding, modulators generally change the three-dimensional structure (i.e. conformation) of the receptor. This will often cause the orthosteric site to also change, which can alter the effect of an agonist binding.[4] Allosteric modulators can also stabilize one of the normal configurations of a receptor.[5]

In practice, modulation can be complicated. A modulator may function as a partial agonist, meaning it doesn't need the agonist it modulates to yield agonistic effects.[6] Also, modulation may not affect the affinities or efficacies of different agonists equally. If a group of different agonists that should have the same action bind to the same receptor, the agonists might not be modulated the same by some modulators.[4]

Classes

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A modulator can have 3 effects within a receptor. One is its capability or incapability to activate a receptor (2 possibilities). The other two are agonist affinity and efficacy. They may be increased, lowered or left unaffected (3 and 3 possibilities). This yields 17 possible modulator combinations.[4] There are 18 (=2*3*3) if neutral modulator type is also included.

For all practical considerations, these combinations can be generalized only to 5 classes[4] and 1 neutral:

  • positive allosteric modulators (PAM) increase agonist affinity and/or efficacy.[4] Clinical examples are benzodiazepines like diazepam, alprazolam and chlordiazepoxide, which modulate GABAA-receptors, and cinacalcet, which modulates calcium-sensing receptors.[7]
    • PAM-agonists work like PAMs, but also as agonists with and without the agonists they modulate.[4]
    • PAM-antagonists work like PAMs, but also function as antagonists and lower the efficacy of the agonists they modulate.[4]
  • negative allosteric modulators (NAM) lower agonist affinity and/or efficacy.[4] Maraviroc is a medicine that modulates CCR5. Fenobam, raseglurant and dipraglurant are experimental GRM5 modulators.[7]
    • NAM-agonists work like NAMs, but also as agonists with and without the agonists they modulate.[4]
  • neutral allosteric modulators don't affect agonist activity, but bind to a receptor and prevent PAMs and other modulators from binding to the same receptor thus inhibiting their modulation.[4] Neutral modulators are also called silent allosteric modulators (SAM)[6] or neutral allosteric ligands (NAL). An example is 5-methyl-6-(phenylethynyl)-pyridine (5MPEP), a research chemical, which binds to GRM5.[8]

Mechanisms

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Due to the variety of locations on receptors that can serve as sites for allosteric modulation, as well as the lack of regulatory sites surrounding them, allosteric modulators can act in a wide variety of mechanisms.[citation needed]

Modulating binding

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Some allosteric modulators induce a conformational change in their target receptor which increases the binding affinity and/or efficacy of the receptor agonist.[2] Examples of such modulators include benzodiazepines and barbiturates, which are GABAA receptor positive allosteric modulators. Benzodiazepines like diazepam bind between α and γ subunits of the GABAA receptor ion channels and increase the channel opening frequency, but not the duration of each opening. Barbiturates like phenobarbital bind β domains and increase the duration of each opening, but not the frequency.[9]

Modulating unbinding

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CX614, a PAM for an AMPA receptor binding to an allosteric site and stabilizing the closed conformation

Some modulators act to stabilize conformational changes associated with the agonist-bound state. This increases the probability that the receptor will be in the active conformation, but does not prevent the receptor from switching back to the inactive state. With a higher probability of remaining in the active state, the receptor will bind agonist for longer. AMPA receptors modulated by aniracetam and CX614 will deactivate slower, and facilitate more overall cation transport. This is likely accomplished by aniracetam or CX614 binding to the back of the "clam shell" that contains the binding site for glutamate, stabilizing the closed conformation associated with activation of the AMPA receptor.[5][9]

Preventing desensitization

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Overall signal can be increased by preventing the desensitization of a receptor. Desensitization prevents a receptor from activating, despite the presence of an agonist. This is often caused by repeated or intense exposures to an agonist. Eliminating or reducing this phenomenon increases the receptor's overall activation. AMPA receptors are susceptible to desensitization via a disruption of a ligand-binding domain dimer interface. Cyclothiazide has been shown to stabilize this interface and slow desensitization, and is therefore considered a positive allosteric modulator.[5]

Stabilizing active/inactive conformation

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Modulators can directly regulate receptors rather than affecting the binding of the agonist. Similar to stabilizing the bound conformation of the receptor, a modulator that acts in this mechanism stabilizes a conformation associated with the active or inactive state. This increases the probability that the receptor will conform to the stabilized state, and modulate the receptor's activity accordingly. Calcium-sensing receptors can be modulated in this way by adjusting the pH. Lower pH increases the stability of the inactive state, and thereby decreases the sensitivity of the receptor. It is speculated that the changes in charges associated with adjustments to pH cause a conformational change in the receptor favoring inactivation.[10]

Interaction with agonists

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Modulators that increase only the affinity of partial and full agonists allow their efficacy maximum to be reached sooner at lower agonist concentrations – i.e. the slope and plateau of a dose-response curve shift to lower concentrations.[4]

Efficacy increasing modulators increase maximum efficacy of partial agonists. Full agonists already activate receptors fully so modulators don't affect their maximum efficacy, but somewhat shift their response curves to lower agonist concentrations.[4]

  • Receptor response % as a function of logarithmic agonist concentration [Ago]
  • PAMs shift initial agonist response curve (solid curve) to lower agonist concentrations by increasing affinity and/or increase maximum response by increasing efficacy. Dashed curves are 2 examples out of many possible curves after PAM addition. Arrows show the approximate direction of the shifts in curves.[4]
    PAMs shift initial agonist response curve (solid curve) to lower agonist concentrations by increasing affinity and/or increase maximum response by increasing efficacy. Dashed curves are 2 examples out of many possible curves after PAM addition. Arrows show the approximate direction of the shifts in curves.[4]
  • PAM-agonists work like PAMs, but are agonists themselves. Thus they induce a response even at minimal concentrations of the agonists they modulate.[4]
    PAM-agonists work like PAMs, but are agonists themselves. Thus they induce a response even at minimal concentrations of the agonists they modulate.[4]
  • PAM-antagonists increase agonist affinities and shift their curves to lower concentrations, but as they work as antagonists, they also lower maximum responses.[4]
    PAM-antagonists increase agonist affinities and shift their curves to lower concentrations, but as they work as antagonists, they also lower maximum responses.[4]
  • NAMs shift curves to higher concentrations by decreasing affinities and/or lower maximum responses by decreasing efficacies. If compared to PAMs, the effects of NAMs are inverse.[4]
    NAMs shift curves to higher concentrations by decreasing affinities and/or lower maximum responses by decreasing efficacies. If compared to PAMs, the effects of NAMs are inverse.[4]
  • NAM-agonists work like NAMs, but are agonists themselves. Thus they induce a response even at minimal concentrations of the agonists they modulate.[4]
    NAM-agonists work like NAMs, but are agonists themselves. Thus they induce a response even at minimal concentrations of the agonists they modulate.[4]

Medical importance

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Benefits

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Related receptors have orthosteric sites that are very similar in structure, as mutations within this site may especially lower receptor function. This can be harmful to organisms, so evolution doesn't often favor such changes. Allosteric sites are less important for receptor function, which is why they often have great variation between related receptors. This is why, in comparison to orthosteric drugs, allosteric drugs can be very specific, i.e. target their effects only on a very limited set of receptor types. However, such allosteric site variability occurs also between species so the effects of allosteric drugs vary greatly between species.[11]

Modulators can't turn receptors fully on or off as modulator action depends on endogenous ligands like neurotransmitters, which have limited and controlled production within body. This can lower overdose risk relative to similarly acting orthosteric drugs. It may also allow a strategy where doses large enough to saturate receptors can be taken safely to prolong the drug effects.[4] This also allows receptors to activate at prescribed times (i.e. in response to a stimulus) instead of being activated constantly by an agonist, irrespective of timing or purpose.[12]

Modulators affect the existing responses within tissues and can allow tissue specific drug targeting. This is unlike orthosteric drugs, which tend to produce a less targeted effect within body on all of the receptors they can bind to.[4]

Some modulators have also been shown to lack the desensitizing effect that some agonists have. Nicotinic acetylcholine receptors, for example, quickly desensitize in the presence of agonist drugs, but maintain normal function in the presence of PAMs.[13]

Applications

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Allosteric modulation has demonstrated as beneficial to many conditions that have been previously difficult to control with other pharmaceuticals. These include:

See also

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References

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Revisions and contributorsEdit on WikipediaRead on Wikipedia

Allosteric modulator

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from Grokipedia
An allosteric modulator is a that binds to a specific allosteric site on a target protein, such as a receptor, which is topographically distinct from the orthosteric (primary -binding) site, thereby altering the protein's conformation to modulate the binding affinity, , or signaling properties of the orthosteric without directly competing for the . These modulators typically exert their effects only in the presence of an endogenous , providing a saturable to their action that preserves physiological signaling patterns and reduces the risk of receptor hyperactivation. In , allosteric modulators represent an emerging paradigm in , particularly for challenging targets like G protein-coupled receptors (GPCRs), where they enable subtype selectivity by binding to less conserved allosteric sites and allow for fine-tuned regulation akin to a "dimmer switch" rather than binary on/off control offered by traditional orthosteric ligands. They are classified primarily into positive allosteric modulators (PAMs), which enhance the orthosteric ligand's potency or ; negative allosteric modulators (NAMs), which diminish it; and silent allosteric modulators (SAMs), which occupy the site without altering function, though additional subtypes like ago-PAMs (with intrinsic activity) and neutral allosteric ligands also exist. The therapeutic potential of allosteric modulators spans diverse diseases, including disorders (e.g., and Parkinson's via or mGluR targets), endocrine conditions (e.g., ), infectious diseases (e.g., ), and metabolic issues (e.g., ), with advantages such as reduced side effects and the ability to probe-dependent biased signaling that targets specific pathways. Approved examples include cinacalcet, a PAM of the calcium-sensing receptor for treating , and maraviroc, a NAM of the for entry inhibition, while numerous candidates like mGluR5 NAMs (e.g., dipraglurant) are in clinical development for conditions such as depression and .

Fundamentals

Definition and Basic Principles

An allosteric modulator is a that binds to a site on a receptor or distinct from the orthosteric site, thereby altering the receptor's response to its endogenous or without directly activating or inhibiting the receptor itself. This binding modulates the affinity or of the orthosteric , influencing downstream signaling or enzymatic activity through indirect means. The orthosteric site refers to the primary binding location for the endogenous , while the allosteric site is a secondary, topographically distinct region that enables this regulatory effect. The fundamental principle of allostery involves a conformational change in the upon modulator binding, which propagates through the molecule—often between distant sites—to affect the orthosteric site's properties. This phenomenon underpins , where binding at one site enhances (positive cooperativity) or diminishes (negative cooperativity) the binding or activity at another site, allowing fine-tuned regulation of protein function. A seminal conceptualization of allostery came from the Monod-Wyman-Changeux (MWC) model proposed in , which describes proteins as existing in equilibrium between tense (T) and relaxed (R) conformational states, with ligands shifting this equilibrium to promote cooperative interactions, as exemplified in hemoglobin's oxygen binding. A classic example of allosteric modulation is provided by benzodiazepines, which bind to an allosteric site on GABA_A receptors to enhance the receptor's response to the GABA, increasing inhibitory signaling in the without directly gating the . This illustrates how allosteric modulators can achieve subtype-specific effects while preserving the receptor's natural activation profile.

Comparison to Orthosteric Ligands

Orthosteric ligands bind directly to the primary of a receptor, also known as the orthosteric site, where they compete with endogenous agonists or antagonists for binding, thereby mimicking or blocking the natural 's interaction. In contrast, allosteric modulators bind to distinct secondary sites, known as allosteric sites, which are topographically separate from the orthosteric site, allowing non-competitive binding that does not directly displace the endogenous . This fundamental difference in binding location enables allosteric modulators to influence receptor function without interfering with the orthosteric site's occupancy by the agonist. Regarding effect profiles, orthosteric agonists activate receptors by fully emulating the endogenous 's action, while orthosteric antagonists completely inhibit it, often leading to on/off binary responses. Allosteric modulators, however, lack intrinsic and instead fine-tune the receptor's response to the orthosteric by altering its affinity or , resulting in graded modulation rather than direct or blockade. For instance, positive allosteric modulators enhance the orthosteric 's potency or maximal effect, whereas negative allosteric modulators reduce it, providing a more nuanced control over signaling. Pharmacologically, the allosteric approach offers advantages by avoiding direct competition at the orthosteric site, which can reduce off-target effects since allosteric sites are often more receptor-specific and less conserved across subtypes. A key implication is the "ceiling effect," where allosteric modulation reaches a maximum influence regardless of increasing modulator concentration, due to the saturable nature of allosteric binding, thereby limiting potential toxicity from overdose. This contrasts with orthosteric ligands, which can produce dose-proportional effects without such inherent safeguards. Structurally, orthosteric sites in G protein-coupled receptors (GPCRs) are typically located within the transmembrane helix bundle, forming a pocket that accommodates the endogenous , while allosteric sites are often found in extracellular loops or intracellular domains, facilitating conformational changes without overlapping the primary binding region. These distinct locations contribute to the non-competitive nature of allosteric interactions, allowing simultaneous binding and cooperative effects on receptor dynamics.

Types

Positive Allosteric Modulators

Positive allosteric modulators (PAMs) are ligands that bind to sites distinct from the orthosteric -binding site on a receptor, thereby enhancing the receptor's response to the endogenous . Typical PAMs do not directly activate the receptor on their own, though subtypes such as ago-PAMs possess intrinsic activity. These modulators can potentiate signaling by increasing the affinity of the for the receptor, its in eliciting a response, or both, leading to amplified physiological effects at endogenous concentrations. PAMs that primarily modulate affinity produce a leftward shift in the dose-response curve, indicating that lower concentrations are sufficient to achieve half-maximal response, while those modulating increase the maximal response amplitude without altering potency, enabling fuller receptor activation even at saturating levels. This allows for more physiological tuning of receptor activity compared to orthosteric . Prominent examples of PAMs include compounds targeting metabotropic glutamate receptors (mGluRs), such as SAR218645, a selective mGluR2 PAM investigated for its potential in treating by enhancing glutamate signaling to alleviate cognitive and negative symptoms. Similarly, zolpidem serves as a subtype-selective PAM of GABA_A receptors, preferentially modulating α1-containing subtypes to promote sedation and treat sleep disorders like by augmenting GABA-mediated inhibition. Endogenous PAMs also play key physiological roles, exemplified by , which acts as a positive allosteric effector on various ion channels, such as nicotinic receptors and GABA_A receptors, by stabilizing open-channel conformations and enhancing -induced currents to fine-tune neuronal excitability.

Negative and Silent Allosteric Modulators

Negative allosteric modulators (NAMs) are ligands that bind to an allosteric site on a receptor, topographically distinct from the orthosteric -binding site, and decrease the affinity, , or both of the orthosteric for the receptor. This binding stabilizes inactive receptor conformations or increases the energy barrier for , resulting in reduced receptor signaling without intrinsic activity of their own. In functional assays, NAMs typically produce a rightward shift in the dose-response curve, reflecting decreased affinity, and/or a reduction in the maximal response, indicating suppressed . A prominent example of NAMs is the class of calcilytics, which act as antagonists of the calcium-sensing receptor (CaSR), a (GPCR) that regulates (PTH) secretion and calcium . By decreasing CaSR sensitivity to extracellular calcium, calcilytics transiently increase PTH levels, promoting bone formation and potentially treating without the sustained hypercalcemia risks associated with PTH analogs. Compounds like NPSP795 have demonstrated this effect in preclinical models by inducing rapid PTH release and enhancing density. Silent allosteric modulators (SAMs), also known as neutral allosteric ligands, bind to the same allosteric site as other modulators but exert no detectable effect on orthosteric agonist affinity or efficacy in standard functional assays. Instead, SAMs competitively occupy the allosteric site to prevent binding or activity of positive or negative allosteric modulators, effectively blocking their influence on receptor signaling. However, SAMs may reveal modulatory effects under probe-dependent conditions, where their impact varies with the specific orthosteric agonist or signaling pathway probed, due to differences in receptor conformational states induced by various ligands. In receptors, such as CXCR1 and CXCR2, SAMs have been explored for their potential to inhibit pathological by blocking allosteric enhancement of signaling without disrupting baseline immune responses; for instance, certain non-peptide ligands exhibit silent properties in assays using full-length but may show modulation with fragments. This probe dependence underscores how SAMs can appear neutral in routine screens yet influence biased signaling or heterodimer interactions in disease contexts like cancer or autoimmune disorders. NAMs and SAMs differ fundamentally in their functional impact: NAMs actively suppress -induced responses by altering receptor dynamics, whereas SAMs remain functionally inert toward orthosteric ligands under normal conditions but serve as gatekeepers against other allosteric influences, with their silence potentially unmasked by probe-specific or biased assay conditions. For contrast, while positive allosteric modulators enhance potency or maximal effect, NAMs provide targeted inhibition useful in hyperactive receptor pathologies. An example of a NAM in a neurological context is , which acts as a negative allosteric modulator at the , reducing serotonin-induced signaling and showing promise in modulating psychosis-like behaviors without broad dopaminergic interference.

Mechanisms of Action

Modulation of Ligand Binding and Unbinding

Allosteric modulators exert their effects on orthosteric binding primarily by altering the kinetic parameters of association and dissociation. The association rate constant (k_on) reflects the speed at which the ligand binds to the receptor, while the dissociation rate constant (k_off) determines how quickly it unbinds, with the equilibrium dissociation constant (K_d) given by K_d = k_off / k_on. Positive allosteric modulators (PAMs) often decrease k_off for orthosteric agonists, thereby prolonging ligand on the receptor and increasing apparent affinity without substantially affecting k_on. This stabilization of the bound state has been observed in (mGlu2), where PAMs like LY487379 reduce the k_off of glutamate, correlating with enhanced functional potency. In contrast, negative allosteric modulators (NAMs) typically accelerate agonist unbinding by increasing k_off, which can diminish affinity and shorten , as demonstrated in single-molecule studies of G protein-coupled receptors (GPCRs) where NAMs expedited dissociation rates by factors of 2- to 4-fold. The reciprocal nature of allosteric interactions is captured by the factor α, which quantifies the extent to which binding of one influences the affinity of the other: α=KA⋅KBKAB⋅KBA\alpha = \frac{K_A \cdot K_B}{K_{AB} \cdot K_{BA}} where KAK_A is the equilibrium of the orthosteric alone, KBK_B is that of the allosteric modulator alone, KABK_{AB} is the dissociation constant of the orthosteric in the presence of saturating allosteric modulator, and KBAK_{BA} is the of the allosteric modulator in the presence of saturating orthosteric . Values of α > 1 indicate positive , resulting in decreased KABK_{AB} (higher orthosteric affinity) and decreased KBAK_{BA} (higher allosteric affinity), whereas α < 1 signifies negative with the opposite effects. This factor arises from coupled changes in binding free energies and can manifest asymmetrically in kinetics, where the modulator may slow orthosteric k_off while the orthosteric reciprocally slows allosteric k_off, but effects on k_on are often oppositely directed due to entropic contributions in the transition state. In simplified models for binding assays, the apparent affinity of the orthosteric ligand can be modulated by the allosteric modulator through cooperativity, leading to leftward shifts in IC50 values for PAMs and rightward shifts for NAMs in radioligand competition assays. These models account for non-equilibrium conditions, emphasizing that allosteric effects on kinetics can produce probe-dependent outcomes, where the observed shift in IC50 reflects both equilibrium cooperativity and differential impacts on k_on and k_off. Experimental validation of these kinetic modulations frequently employs fluorescence-based assays to monitor real-time binding dynamics in GPCRs. For instance, fluorescence resonance energy transfer (FRET) and total internal reflection fluorescence (TIRF) microscopy have revealed that PAMs can slow orthosteric agonist dissociation in cells expressing muscarinic receptors. Similarly, studies on adenosine A1 receptors have shown NAMs increasing k_off of orthosteric ligands, confirming kinetic contributions to reduced binding affinity. These approaches highlight the practical utility of kinetic profiling in dissecting allosteric mechanisms beyond equilibrium measurements. These binding kinetic alterations often arise from subtle conformational adjustments in the receptor that propagate between sites.

Influence on Receptor Conformation and Signaling

Allosteric modulators exert their effects by binding to sites distinct from the orthosteric ligand-binding pocket, thereby inducing or stabilizing specific conformational states of the receptor that alter its functional properties. Positive allosteric modulators (PAMs) preferentially stabilize the active conformation (R*) of the receptor, enhancing the probability of orthosteric agonist-induced activation, while negative allosteric modulators (NAMs) favor the inactive conformation (R), reducing the receptor's responsiveness to agonists. This conformational selection is captured in the extended ternary complex model, which describes how allosteric modulators shift the equilibrium between inactive (R) and active (R*) receptor states in the presence of orthosteric ligands and G-proteins, thereby modulating the formation of the agonist-receptor-G-protein ternary complex. These conformational changes directly influence downstream signaling pathways by altering the receptor's interactions with intracellular effectors. For instance, PAMs can enhance G-protein coupling efficiency, increasing the activation of second messengers such as cyclic AMP in G_s-coupled receptors, or promote ion flux in ligand-gated ion channels. Conversely, NAMs suppress these interactions, reducing G-protein dissociation and subsequent signaling. In addition, allosteric binding can prevent receptor desensitization by inhibiting key regulatory mechanisms, such as phosphorylation by G-protein-coupled receptor kinases (GRKs) or β-arrestin recruitment, which would otherwise uncouple the receptor from G-proteins or promote internalization. For example, in , positive allosteric modulators like cyclothiazide stabilize the open-channel state by disrupting the dimer interface rearrangements that underlie desensitization, thereby prolonging ion conductance without altering agonist binding kinetics. Allosteric modulators can also induce biased signaling, selectively favoring certain downstream pathways over others to produce pathway-specific effects. In G-protein-coupled receptors (GPCRs), this bias manifests as differential recruitment of G-proteins versus β-arrestins; for instance, certain PAMs may enhance G-protein-mediated signaling while suppressing β-arrestin-dependent pathways, leading to distinct cellular outcomes compared to orthosteric agonists. This allosteric control over signaling ensembles arises from the stabilization of intermediate conformational states that differentially engage effectors, providing a mechanism for fine-tuned regulation of receptor activity.

Binding Sites and Molecular Interactions

Identification and Characterization of Sites

The identification and characterization of allosteric binding sites on (GPCRs) and other targets relies on a combination of experimental and computational techniques to map potential pockets distinct from orthosteric sites. These methods enable the discovery of sites that can modulate receptor function without competing directly with endogenous ligands, offering opportunities for subtype-selective drug design. Key challenges include distinguishing allosteric sites from transient pockets and validating their functional relevance, which requires integrating structural, biochemical, and pharmacological data. Experimental techniques such as have been pivotal in visualizing allosteric binding sites at atomic resolution, often revealing cryptic pockets that open upon conformational changes. For instance, high-resolution structures of co-crystallized with allosteric modulators have identified transmembrane and extracellular vestibule sites, providing templates for rational design. Cryo-electron microscopy (cryo-EM) complements crystallography by capturing dynamic states in near-native conditions, particularly for complexes with G proteins or arrestins, where allosteric sites at intracellular interfaces become apparent. (NMR) spectroscopy further aids in probing site flexibility and ligand-induced conformational shifts in solution, especially for smaller receptor domains or peptide modulators. To confirm the functional importance of identified residues, site-directed mutagenesis is routinely employed, introducing point mutations to disrupt binding and assess impacts on modulator potency via radioligand or functional assays. Computational approaches accelerate site prediction by simulating receptor dynamics and screening libraries for potential binders. Molecular docking tools, such as Glide within the Schrödinger suite, predict allosteric pocket occupancy by sampling ligand poses in receptor models derived from homology or experimental structures. Molecular dynamics (MD) simulations reveal transient pockets that stabilize upon ligand binding, often identifying lipid-facing or intracellular sites not evident in static structures. Fragment-based screening, combining virtual docking with biophysical validation like surface plasmon resonance or mass spectrometry, has successfully detected low-affinity fragments in allosteric sites, serving as starting points for optimization. A historical milestone was the 2014 determination of the first crystal structure of an allosteric site in a class C GPCR, the mGlu1 receptor transmembrane domain bound to a negative allosteric modulator, which illuminated a conserved pocket for modulator design across metabotropic glutamate receptors. Once potential sites are mapped, characterization involves functional assays to validate allosteric effects and probe dependence, where modulator activity varies with the orthosteric ligand used. High-throughput platforms like FLIPR calcium imaging measure real-time changes in intracellular calcium flux in cells expressing the receptor, allowing quantification of modulation on agonist-induced responses. These assays confirm site specificity by testing shifts in EC50 or maximal efficacy across different probes, ensuring the site supports non-competitive interactions. Integrating these data refines models of site-residue interactions, guiding iterative optimization of modulators.

Probe Dependence and Specificity

Probe dependence refers to the phenomenon where the effect of an allosteric modulator on receptor function varies depending on the orthosteric ligand (probe) used to assess activity. This occurs because allosteric modulators can differentially influence the binding affinity or efficacy of distinct orthosteric ligands, even at the same receptor. For instance, a modulator might enhance the affinity of one agonist while having no effect or even reducing the affinity of another, leading to context-specific outcomes in functional assays. The underlying mechanisms for probe dependence include the existence of multiple allosteric sites on the receptor or the dynamic nature of receptor conformational ensembles, where the modulator stabilizes different states that interact variably with orthosteric probes. In G protein-coupled receptors (GPCRs), such as the μ-opioid receptor, structural studies have revealed that probe-dependent effects arise from how allosteric ligands alter the receptor's energy landscape, favoring conformations that either cooperate or compete with specific orthosteric ligands. This variability complicates the classification of modulators as positive (PAMs) or negative (NAMs), as their behavior is probe-specific rather than intrinsic. Quantitative assessment of probe dependence often employs cooperativity indices, such as the binding cooperativity factor α, where log(α) values quantify the magnitude and direction of modulation for a given orthosteric probe. Positive log(α) (>0) indicates affinity enhancement (PAM-like), while negative values (<0) denote reduction (NAM-like), and these can differ significantly across probes; for example, in muscarinic receptors, log(α) shifts from positive to neutral depending on the agonist used. Efficacy modulation is similarly captured by β factors in extended operational models, highlighting how probe choice affects both binding and signaling outcomes. In the context of negative allosteric modulators (NAMs) at opioid receptors, probe dependence manifests in differential impacts on therapeutic versus adverse effects, such as enhanced analgesia with one agonist probe while mitigating respiratory depression with another due to biased conformational stabilization. For example, certain NAMs at the μ-opioid receptor exhibit probe-dependent antagonism, reducing respiratory depression induced by high-efficacy agonists like fentanyl without fully blocking analgesia from endogenous opioids. This selectivity arises from the modulator's influence on distinct signaling pathways activated by different probes. Achieving specificity remains a key challenge for allosteric modulators, as they often exhibit cross-reactivity across closely related receptor subtypes, such as between GABA_A and glycine receptors, which share allosteric sites sensitive to modulators like volatile anesthetics. This off-target binding can lead to unintended effects, complicating therapeutic development. Strategies to enhance specificity include the design of bitopic ligands, which combine orthosteric and allosteric pharmacophores in a single molecule to achieve higher subtype selectivity by exploiting unique site geometries across receptors. For instance, bitopic ligands at muscarinic receptors demonstrate improved discrimination between M1 and M2 subtypes compared to pure allosteric agents.

Interactions with Orthosteric Ligands

Effects on Agonist Affinity

Allosteric modulators exert their effects on agonist affinity through non-competitive interactions that alter the binding equilibrium of orthosteric ligands via allosteric coupling between distinct sites on the receptor. This coupling induces conformational changes that either stabilize or destabilize the agonist-bound state, leading to shifts in the apparent dissociation constant (K_D) or half-maximal effective concentration (EC_{50}) without directly competing for the orthosteric site. Positive allosteric modulators (PAMs) enhance agonist affinity, typically manifesting as a leftward shift in concentration-response curves (decreased EC_{50}), while negative allosteric modulators (NAMs) reduce affinity, producing rightward shifts (increased EC_{50}). These shifts are concentration-dependent and reflect the degree of cooperativity between the modulator and agonist binding events. Quantitative assessment of affinity modulation employs models such as the allosteric ternary complex model, which describes the observed agonist affinity in the presence of a modulator. The apparent dissociation constant for the agonist (A) is given by: KAapp=KA1+[B]KB1+α[B]KBK_A^{app} = K_A \frac{1 + \frac{[B]}{K_B}}{1 + \alpha \frac{[B]}{K_B}} where KAK_A and KBK_B are the equilibrium dissociation constants of the agonist and modulator (B), respectively, and α\alpha is the allosteric cooperativity factor (α>1\alpha > 1 for PAMs, increasing affinity; α<1\alpha < 1 for NAMs, decreasing affinity; α=1\alpha = 1 for neutral effects). Dose-ratio analyses, adapted from classical Schild plots for allosteric contexts, quantify these interactions by plotting log⁡(dose ratio−1)\log(\text{dose ratio} - 1) against log⁡[modulator]\log[\text{modulator}], where slopes deviating from unity indicate non-competitive allosteric behavior rather than orthosteric antagonism. In experimental assays, such as radioligand competition binding studies, allosteric modulators demonstrate their affinity effects through parallel shifts in agonist saturation curves. For instance, PAMs cause leftward shifts in the binding isotherm of a labeled agonist (e.g., the PAM JNJ-46281222 increases glutamate affinity ~5-fold at mGlu2 by reducing the dissociation rate constant), while maintaining the maximum number of binding sites (B_{\max}), confirming selective modulation of binding strength without altering receptor density. NAMs produce analogous rightward shifts, often with similar parallelism in binding assays. A representative receptor-specific example involves PAMs of the metabotropic glutamate receptor subtype 2 (mGluR2), which enhance glutamate affinity (e.g., via α\alpha values >1 in ternary complex analyses), thereby potentiating synaptic glutamate signaling and eliciting anxiolytic-like effects in models of anxiety without the associated with orthosteric .

Effects on Agonist Efficacy

Allosteric modulators influence by altering the maximal functional response (E_max) elicited by orthosteric at their receptors, without necessarily affecting binding affinity. Positive allosteric modulators (PAMs) enhance by stabilizing the active receptor conformation, thereby increasing the proportion of receptors that can transduce a signal upon binding, which raises the observed E_max in dose-response curves. This effect arises from the modulator's ability to increase the of the receptor in the active state, promoting more efficient to downstream effectors such as G proteins or ion channels. In contrast, negative allosteric modulators (NAMs) reduce by favoring inactive or less responsive receptor states, lowering E_max and potentially converting full into partial ones, even at saturating concentrations. A subset of PAMs, known as ago-PAMs, exhibit partial intrinsic activity in the absence of orthosteric , directly eliciting a submaximal response while also potentiating when co-applied. This dual action allows ago-PAMs to activate receptors independently but with lower efficiency than orthosteric , while enhancing the E_max of co-administered through allosteric stabilization of active conformations. The extended operational model of allosterically modulated (OMAM) quantifies this allosteric modulation of via a cooperativity factor β representing the modulator's effect on operational , distinct from affinity modulation by α. In this framework, the apparent maximal response in the presence of the modulator is given by: Emax⁡′=βτAEmax⁡βτA+1E_{\max}^{\prime} = \frac{\beta \tau_A E_{\max}}{\beta \tau_A + 1} where τ_A is the operational efficacy of the agonist A, E_{\max} is the system maximum, and β scales efficacy (β > 1 for PAMs enhancing efficacy; β < 1 for NAMs reducing it). This model distinguishes efficacy effects from affinity changes, as shifts in E_max occur independently of alterations in the agonist's EC_50. Functional assays commonly demonstrate these efficacy modulations through changes in peak response amplitude without parallel shifts in agonist potency. For instance, in cAMP accumulation assays for G protein-coupled receptors, PAMs increase the maximal cAMP production induced by agonists like glutamate at mGluR5, reflecting enhanced G protein coupling efficiency. Similarly, electrophysiological recordings in ligand-gated ion channels reveal amplified current amplitudes; benzodiazepines, as PAMs at GABA_A receptors, boost the maximal chloride conductance evoked by GABA, enhancing inhibitory neurotransmission without altering GABA's EC_50, which underlies their sedative effects. These assays highlight how efficacy modulation fine-tunes receptor signaling ceilings, offering a mechanistic basis for therapeutic selectivity.

Therapeutic Relevance

Advantages in Drug Design

Allosteric modulators offer enhanced subtype selectivity in compared to orthosteric ligands, as allosteric binding sites are often less conserved across receptor subtypes, allowing for more precise targeting. For instance, positive allosteric modulators (PAMs) of the (mAChR) demonstrate high selectivity over M1 and M3 subtypes, enabling potential treatment of without activating peripheral muscarinic receptors that contribute to side effects like salivation or . This selectivity arises from structural differences in allosteric pockets, which vary more significantly between subtypes than the highly conserved orthosteric sites. A key advantage is the reduced risk of side effects due to the inherent "ceiling effect" of allosteric modulation, where the response saturates at physiological levels of the endogenous , preventing excessive activation and overdose toxicity. Unlike orthosteric agonists, which can drive signaling beyond natural tones and lead to desensitization or toxicity, allosteric modulators preserve the spatiotemporal patterns of endogenous signaling, maintaining physiological control. This property is particularly beneficial for G protein-coupled receptors (GPCRs) in the , where overactivation can cause adverse effects. Allosteric modulators can overcome resistance to orthosteric drugs caused by at the , as they bind to distinct sites unaffected by such alterations, restoring or enhancing therapeutic efficacy. In cancer, for example, allosteric inhibitors of kinases like BCR-ABL1 have been combined with orthosteric inhibitors to resensitize resistant cells by modulating allosteric pathways that compensate for . This approach is also relevant for viral receptors, where confer drug resistance without impacting allosteric sites. Recent advances in the 2020s have improved the drug-like properties of allosteric ligands, particularly their , enabling better (CNS) penetration for neurological indications. For example, the M1 mAChR PAM VU0467319 exhibits enhanced brain exposure and favorable oral compared to earlier analogs, advancing it into clinical trials for and while minimizing adverse events. These optimizations, including reduced molecular weight and improved , facilitate the development of CNS-penetrant PAMs with superior therapeutic windows.

Clinical Applications and Examples

Allosteric modulators have found significant clinical utility in central nervous system (CNS) disorders, particularly through positive allosteric modulation (PAM) of ionotropic receptors. Benzodiazepines, such as diazepam and lorazepam, act as PAMs at the benzodiazepine site on GABA_A receptors, enhancing GABA-mediated inhibitory neurotransmission to alleviate symptoms of anxiety disorders. These agents have been a cornerstone of anxiolytic therapy since the 1960s, with their efficacy stemming from increased chloride influx and neuronal hyperpolarization without directly activating the receptor. Beyond CNS applications, allosteric modulators target endocrine pathways for metabolic disorders. , a PAM of the calcium-sensing receptor (CaSR), was approved by the FDA in 2004 for treating in patients with on dialysis, where it sensitizes the receptor to extracellular calcium, thereby reducing secretion and serum calcium levels. Investigational negative allosteric modulators (NAMs) have been explored for neurodegenerative conditions. Basimglurant (RO4917523), a selective mGluR5 NAM, advanced to Phase II trials in the 2010s for and depression, showing limited efficacy that led to discontinuation of the program; it has also been investigated in preclinical models for targeting amyloid-beta pathology. As of 2025, it is in Phase II/III trials for . Emerging applications span and . For , allosteric modulators of acid-sensing ion channels (), such as heteroarylguanidines targeting ASIC1a and ASIC3, demonstrate analgesic potential by inhibiting proton-gated sodium influx in preclinical models of inflammatory and . In , allosteric NAMs for mutant (EGFR), like EAI045 and JBJ-09-063, address resistance in non-small cell by stabilizing inactive conformations of T790M and C797S mutants, showing efficacy in combination with orthosteric inhibitors in preclinical and early clinical studies. Recent advancements include neurosteroid-based PAMs for mood disorders. (SAGE-217), a PAM, was approved by the FDA in 2023 and the EMA in 2025 for and completed Phase III trials for , with approval pending as of November 2025, offering rapid antidepressant effects through enhanced inhibitory signaling comparable to intravenous brexanolone.

Challenges in Development

Selectivity and Safety Concerns

One major challenge in developing allosteric modulators is selectivity, arising from the conservation of allosteric binding sites across related protein families, which can promote polypharmacology and off-target effects. For instance, allosteric sites in G protein-coupled receptors (GPCRs), a common target for such modulators, exhibit structural similarities that allow ligands to bind multiple receptor subtypes or even unrelated proteins, potentially leading to unintended pharmacological actions. In the case of positive allosteric modulators (PAMs) of GABA_A receptors, such as benzodiazepines, off-target binding to non-α1 subtypes or other ion channels can exacerbate beyond therapeutic levels, contributing to adverse events like excessive drowsiness or . Safety concerns with negative allosteric modulators (NAMs) include the risk of inverse agonism, where these compounds not only block activity but also suppress constitutive receptor signaling, potentially altering receptor expression levels over time. Although inverse agonism typically leads to compensatory upregulation in constitutively active systems, Additionally, probe dependence—the phenomenon where an allosteric modulator's effect varies depending on the orthosteric used—can result in unpredictable outcomes, as the modulator may enhance or inhibit signaling differently across endogenous ligands or physiological conditions, complicating translation from assays to clinical efficacy and safety. Toxicological profiles of early allosteric modulators highlight specific risks, such as observed with certain mGluR5 NAMs developed in the 2010s. For example, acetylene-based compounds like raseglurant (RO4917523) were withdrawn from clinical development due to elevated liver levels and hepatic in patients during long-term use. To mitigate cardiac risks, hERG channel assays are routinely employed in preclinical screening of allosteric modulators, as inhibition of this can prolong the and increase susceptibility, a concern for many small-molecule modulators regardless of mechanism. Regulatory oversight emphasizes evaluating abuse potential for allosteric modulators targeting receptors, given their capacity to enhance or alter endogenous effects. The FDA's 2017 guidance on assessing abuse potential requires comprehensive preclinical and clinical studies for such compounds, including those acting allosterically, to identify risks of dependence or misuse similar to orthosteric s; updated emphases in subsequent reviews underscore monitoring for biased signaling that might retain rewarding properties.

Strategies for Discovery and Optimization

The discovery of allosteric modulators relies on advanced screening methods that enable the identification of compounds interacting with non-orthosteric sites. High-throughput assays utilizing bioluminescent resonance energy transfer (BRET) have emerged as a powerful tool for detecting allosteric effects in real-time, particularly for G protein-coupled receptors (GPCRs) and ion channels, by monitoring conformational changes or protein-protein interactions without the interference of orthosteric ligands. For instance, NanoBRET platforms facilitate the screening of modulators that disrupt or enhance interactions like those between and downstream effectors, allowing for the prioritization of selective hits in cellular contexts. Complementing these experimental approaches, AI-driven has accelerated site prediction since 2021, leveraging models like to predict allosteric pockets in proteins lacking experimental structures, thereby guiding docking simulations for novel modulators. This integration has proven effective in identifying potential allosteric sites on GPCRs, where AI-generated models rival cryo-EM data in hit discovery campaigns. Optimization of lead allosteric modulators emphasizes structure-based , incorporating high-resolution cryo-EM structures to refine binding poses and enhance potency. Iterative docking and simulations against cryo-EM-derived allosteric pockets, such as those in the , have yielded positive allosteric modulators with improved selectivity by targeting extrahelical sites. Similarly, cryo-EM has illuminated modulator-bound conformations in Class A, B, and C GPCRs, enabling the of compounds that stabilize specific states while minimizing off-target effects. To further boost specificity, the development of bitopic ligands—molecules that simultaneously engage orthosteric and allosteric sites—has gained traction, as these dual-acting agents amplify affinity and subtype selectivity in targets like GPCRs. For example, bitopic modulators of the CB2 receptor demonstrate enhanced functional bias, reducing unwanted signaling pathways compared to single-site ligands. Looking ahead, strategies increasingly target allosteric modulation in historically undruggable proteins, exemplified by the 2021 FDA approval of , a negative allosteric modulator (NAM) that covalently binds the switch-II pocket of G12C to lock it in an inactive GDP-bound state, offering a paradigm for applications. Pharmacogenomics further supports personalized approaches by identifying genetic variants in GPCR allosteric sites that influence modulator efficacy, allowing tailored dosing to optimize therapeutic responses and mitigate adverse effects. As of 2025, emerging trends focus on biased allosteric modulators that selectively activate pathway-specific signaling, particularly in , where PAR2 NAMs promote anti-tumor immune responses by repressing immunosuppressive pathways without broad receptor antagonism.

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