How Drugs for Heart and Metabolism Work with Proteins Related to Weight Loss and Heart Protection

Dulaglutide, a long-acting GLP-1R agonist, structurally mimics endogenous GLP-1 and binds both domains cooperatively. Structural studies have shown that GLP-1R agonists like dulaglutide interact with the ECD via α-helical motifs and hydrophobic residues, stabilizing the ligand-receptor complex. This dual-domain engagement is a hallmark of peptide-based agonists and facilitates conformational rearrangement essential for downstream signaling [13,42].

Cryo-electron microscopy and X-ray crystallographic data reveal that dulaglutide binds to GLP-1R in a conformation similar to the native GLP-1 peptide, engaging both the N-terminal extracellular domain (ECD) and the upper regions of the transmembrane helices (TMD). The crystal structure of GLP-1R in complex with a GLP-1 analogue (PDB ID: 5VAI) demonstrates key ligand-receptor interactions that stabilize the active conformation. Critical residues involved in the binding pocket include Glu364, which forms salt bridges with positively charged ligand residues; Arg190 and Tyr152, which contribute to hydrogen bonding; and Trp297 in the extracellular loop 2 (W297^ECL2), which provides hydrophobic contact that anchors the ligand. Additional supportive interactions include Ser32 and His212 in the ECD, which help secure the N-terminal α-helix of dulaglutide, while Ile196 and Phe230 within the TMD contribute to hydrophobic core formation. These residues together form a deep orthosteric pocket, locking the ligand in a bioactive conformation and aligning the receptor for downstream Gs protein coupling. Moreover, the C-terminal extension of dulaglutide, a covalently linked Fc fragment, enhances serum stability and resists degradation by dipeptidyl peptidase-4 (DPP-4), contributing to its prolonged receptor occupancy and pharmacodynamic action [13].

The specific protein–ligand interactions between dulaglutide and GLP-1R translate directly into its pharmacological efficacy and clinical outcomes. By maintaining high-affinity binding and inducing prolonged activation of GLP-1R, dulaglutide effectively enhances insulin secretion, inhibits glucagon release, and delays gastric emptying—mechanisms critical for glycemic control in type 2 diabetes mellitus (T2DM) [43]. Furthermore, dulaglutide-mediated GLP-1R activation promotes neuroprotective and cardioprotective effects, which have been linked to downstream signaling pathways including cAMP/PKA, AMPK, and PI3K-Akt [44,45]. The sustained receptor engagement enabled by these structural interactions also underpins dulaglutide's once-weekly dosing regimen and its durability in reducing β-cell stress without downregulating GLP-1R expression [42]. Thus, the structural compatibility between dulaglutide and GLP-1R ensures a therapeutic profile marked by efficacy, safety, and improved patient adherence.

Exenatide (Ex-4), a synthetic analog of exendin-4, binds to GLP-1R in a two-domain interaction mode. The C-terminal region of exenatide primarily engages the extracellular domain (ECD) of GLP-1R for high-affinity binding, while the N-terminal portion inserts into the transmembrane (TM) domain to induce receptor activation [13,46] The receptor itself is structured with a characteristic seven-transmembrane helical bundle (7TM), connected by extracellular and intracellular loops that accommodate both peptide and small-molecule ligands. Structural studies show that Trp-cage motifs on exenatide enhance its stability and modulate the conformational flexibility required for binding, while the TM6 region of the receptor functions as the principal signal transduction hub [46].

Cryo-electron microscopy (cryo-EM) and X-ray crystallography have provided detailed insights into the interaction between GLP-1R and exenatide. The structural configuration is well-documented in the Protein Data Bank under entries such as PDB: 6X18, which display the active-state GLP-1R bound to exenatide and related agonists [13,47]. These structures reveal that the ECD interacts with the α-helical region of exenatide, anchoring residues such as Tyr205, Glu364, and Trp33 (in the ECD), which contribute significantly to ligand recognition. Within the transmembrane bundle, residues Lys197, Tyr205, Gln210, Gln211, Trp214, Gln234, Thr298, Arg299, and Asn300 form a binding pocket that accommodates the N-terminal activation motif of the peptide (Figure 1) [46,47]. Moreover, the Trp25 residue in exenatide's Trp-cage motif aligns structurally with the receptor's extracellular loops, indicating a secondary stabilization role despite its minimal contribution to direct receptor engagement [46].

The structural interaction between exenatide and GLP-1R translates directly into therapeutic outcomes in metabolic and neurodegenerative diseases. By stabilizing GLP-1R in its active conformation, exenatide enhances glucose-stimulated insulin secretion, suppresses glucagon release, and slows gastric emptying—actions critical for managing type 2 diabetes mellitus [48,49]. The biased agonism of exenatide, favoring G protein signaling over β-arrestin recruitment, contributes to its beneficial effects on glucose metabolism while potentially reducing receptor desensitization and side effects such as nausea [47]. Furthermore, exenatide's ability to cross the blood–brain barrier and engage GLP-1Rs in the CNS confers neuroprotective properties, including reductions in oxidative stress and inflammation, making it a candidate for addressing central nervous system disorders like Parkinson's and Alzheimer's diseases [49]. Thus, understanding the precise molecular interactions between GLP-1R and exenatide not only elucidates the basis for its clinical efficacy but also informs the design of next-generation agonists with improved therapeutic profiles.

The GLP-1R adopts a conserved "secretin fold" in its ECD, stabilized by three disulfide bridges and key residues including Asp67, Trp72, and Pro86, which are essential for anchoring the C-terminal α-helix of GLP-1 [50]. Structural insights from cryo-EM and crystallography show that GLP-1 binds in a two-domain fashion: its C-terminal region interacts with the ECD while the N-terminal segment penetrates the transmembrane domain (TMD) to initiate receptor activation [51]. This bipartite engagement is similarly observed for dual GLP-1R/GCGR agonists such as MEDI0382 and SAR425899, which modify conserved motifs across both receptors to achieve dual activation [52].

High-resolution cryo-EM structures have revealed that both peptide (e.g., GLP-1, oxyntomodulin) and non-peptide agonists (e.g., PF 06882961) exploit overlapping receptor-binding surfaces, though their engagement strategies differ. Peptide agonists typically occupy the orthosteric site through extensive hydrophobic and hydrogen bonding interactions across ECL1, ECL2, and transmembrane helices 1 and 7 [53]. In contrast, non-peptide agonists like PF 06882961 use structured water networks and mimic the polar contacts of GLP-1, enabling them to trigger similar receptor conformational changes despite their smaller size. MEDI0382, a dual GLP-1R/GCGR agonist, engages the TM1-TM2 cleft with a lipid moiety that enhances its binding to GCGR, accounting for its higher potency and efficacy in metabolic regulation [52]. These distinct modes underscore the plasticity of class B1 GPCRs in accommodating diverse ligand chemotypes.

The structural nuances of GLP-1R and Glucagon ligand interactions have direct implications for therapeutic design. Biased agonism at GLP-1R, wherein ligands preferentially activate specific signaling pathways, can be tuned through targeted interactions at the extracellular surface and ECL regions, allowing for tailored pharmacodynamic profiles [51]. MEDI0382, by engaging both GLP-1R and GCGR, achieves superior metabolic effects—such as glycemic control and weight loss—by combining insulinotropic and thermogenic activities while mitigating glucagon-induced hyperglycemia [54,55]. These findings highlight the clinical promise of dual or multi-receptor agonists that leverage shared structural determinants while fine-tuning selectivity through engineered sequence variation [52]. Continued structural elucidation informs the rational design of next-generation metabolic therapeutics with improved efficacy and safety profiles.

Liraglutide, a long-acting GLP-1R agonist, binds to specific structural domains on the receptor to exert its therapeutic effects. Structural analyses revealed that the extracellular domain (ECD) of GLP-1R is critical for ligand recognition, with the C-terminal region of GLP-1 or its analogs, like liraglutide, binding deeply into a conserved hydrophobic pocket. When GLP-1 binds, it adopts an extended α-helical conformation from Thr13 to Val33, allowing precise interactions with receptor residues that differ slightly from those seen in antagonist-bound conformations [50,56].

Functionally, the binding mode of liraglutide to GLP-1R has been elucidated through structural studies that include crystallography data. The extracellular domain (ECD) of GLP-1R interacts directly with the C-terminal α-helical segment of liraglutide, a modification that enhances stability and resistance to enzymatic degradation. Specifically, the crystal structure of the GLP-1R ECD bound to GLP-1 (PDB ID: 3IOL) reveals that GLP-1 adopts a continuous α-helix from Thr13 to Val33, fitting into a hydrophobic groove formed by conserved residues on the receptor [50]. This interaction is critical for the initial high-affinity binding step, which is then followed by engagement of the ligand's N-terminal domain with the receptor's transmembrane core to trigger G-protein activation. The binding orientation of GLP-1 in 3IOL differs from that of Exendin-4, an agonist used in a prior structure (PDB ID: 3C5T), indicating subtle but important conformational distinctions that influence receptor activation and signaling efficiency [49].

From a therapeutic standpoint, the interaction between liraglutide and GLP-1R is crucial not only for its antidiabetic properties but also for its potential in treating conditions like nonalcoholic steatohepatitis (NASH) and obesity. For instance, liraglutide modulates hepatic inflammation and lipotoxicity via direct liver actions, independent of weight loss or improved glycemic control, likely through suppression of ceramide accumulation and alteration of gut microbiota [57]. These diverse outcomes are mediated by liraglutide's capacity to act on central and peripheral GLP-1R populations, particularly in glutamatergic neurons, where it modulates appetite and body weight without compromising glycemic benefits [58,59].

Structural studies have revealed that GLP-1 and albiglutide bind in a two-domain model: the C-terminal portion of the ligand engages the ECD while the N-terminal portion interacts with the transmembrane domain, particularly transmembrane helices and extracellular loops [50,60]. Cryo-EM structures show GLP-1R activation involves conformational rearrangements including a kink in TM6 to facilitate G protein coupling, emphasizing the importance of conserved motifs such as the secretin fold and disulfide-stabilized helices in signal transduction [60,61].

Albiglutide, a long-acting GLP-1R agonist, is engineered as a dimer of GLP-1 (7–36) molecules fused to recombinant human albumin, which confers extended half-life and resistance to enzymatic degradation. Albiglutide exhibits altered binding kinetics due to steric effects and slower dissociation from the receptor, yet it retains sufficient agonist efficacy to activate downstream signaling cascades, including PKA-mediated insulin release from pancreatic β-cells [62,63]. This slow-binding characteristic allows for once-weekly dosing and reduces peak plasma fluctuations, mitigating gastrointestinal side effects and improving patient adherence [64].

The therapeutic outcome of albiglutide's interaction with GLP-1R extends beyond glycemic control, reflecting the receptor's pleiotropic signaling potential. By stimulating glucose-dependent insulin secretion, suppressing glucagon release, and delaying gastric emptying, albiglutide contributes to improved HbA1c levels and modest weight reduction [65]. Clinical data support its efficacy, with significant HbA1c reductions over placebo at both 30 mg and 50 mg weekly doses [62]. Moreover, the cardioprotective and β-cell-preserving effects observed in preclinical and clinical studies underscore GLP-1R's central role as a therapeutic target in type 2 diabetes and its comorbidities [63,64].

Lixisenatide, an exendin-4-based GLP1R agonist, engages key structural elements such as the extracellular loop 2 (ECL2), TM helices 1, 2, 6, and 7, and the ECD's N-terminal region to trigger receptor activation [51,53]. Cryo-EM structures show that these peptide agonists are stabilized by a network of water-mediated hydrogen bonds that link the peptide's N-terminus to the receptor core, ensuring specificity and efficacy. Importantly, distinct residues in the ECD modulate biased agonism, allowing lixisenatide to differentially recruit downstream effectors [51].

Lixisenatide binds to the GLP-1 receptor (GLP1R) using a mechanism analogous to GLP-1, engaging both the extracellular domain (ECD) and transmembrane (TM) regions of the receptor. Cryo-EM structures of GLP1R in active states, such as PDB IDs 6B3J, 6X18, and 6LN2, which show peptide agonists interacting with lixisenatide via N-terminal contacts and inserting into the orthosteric TM cavity to stabilize the active conformation [53]. Notably, although lixisenatide shares the first 37 amino acids with exendin-4, it includes a unique six-lysine C-terminal extension, which contributes to altered receptor engagement and trafficking properties. Pharmacologically, lixisenatide induces robust receptor internalization but slower recycling to the membrane compared to exendin-4, likely due to subtle structural differences in receptor clustering and endosomal sorting [53]. These variations in binding and trafficking may contribute to differences in sustained insulin secretion and biased signaling profiles among GLP-1 analogs.

Lixisenatide's interactions with GLP1R not only underpin its glucose-lowering efficacy but also contribute to extrapancreatic effects such as neuroprotection and vascular modulation. It has demonstrated potent inhibition of postprandial glucagon and improved β-cell function while causing less gastrointestinal discomfort compared to longer-acting GLP1R agonists [66,67]. In neuronal tissues, lixisenatide exerts antioxidant and anti-inflammatory effects, partly via GLP1R-mediated upregulation of endothelial nitric oxide synthase (eNOS) and suppression of pro-apoptotic pathways, offering protection against ischemic injury [68,69].These findings underscore the therapeutic versatility of lixisenatide and its mechanistic reliance on GLP1R structural motifs to mediate both glycemic control and organ protection.

The glucagon-like peptide-1 receptor (GLP-1R) and amylin analog pramlintide engage distinct yet complementary structural and functional features that define their protein–ligand interactions and biological efficacy in metabolic disease therapy [70,71]. Pramlintide, an analog of the 37-amino acid hormone amylin, interacts predominantly with the calcitonin receptor (CALCR) complexed with receptor activity-modifying proteins (RAMPs) [72]. Structural features critical for pramlintide's activity include its engineered proline residues that reduce fibrillogenesis while preserving key agonistic motifs for receptor binding in the area postrema and nucleus of the solitary tract [73]. These differences in receptor-binding architecture underscore the unique but convergent signaling pathways activated by these agents.

Molecular and physiological studies have revealed that both GLP-1R agonists and pramlintide modulate central appetite-regulatory circuits, though through distinct mechanisms and structural interactions. The GLP-1 receptor has been extensively characterized, with crystallographic data available for multiple ligand-bound states. Notably, the cryo-EM structure of human GLP-1R bound to GLP-1 and Gs protein (PDB ID: 6X18) reveals a key interaction at the extracellular domain and transmembrane helices, stabilizing the active conformation of the receptor and initiating intracellular signaling cascades. Additionally, structures such as 6LN2 and 5VAI provide insight into small-molecule and peptide ligand interactions within the GLP1R binding pocket. Conversely, pramlintide binds to amylin receptors formed by heterodimerization of the calcitonin receptor (CTR) with RAMPs (particularly RAMP1, 2, or 3). This structure illustrates how peptide agonists interact with the extracellular domain of the receptor, inducing activation. Together, these structural insights reveal that while GLP-1R agonists activate through well-defined GPCR conformational shifts, pramlintide engages class B GPCRs with RAMP-modulated binding, offering complementary and synergistic potential in combination therapies [70,71,74].

Pramlintide (an amylin analog), although less widely used due to its injection burden, has demonstrated efficacy in reducing caloric intake and improving control over eating in obese individuals [74]. Notably, combination therapies leveraging both GLP-1 and pramlintide have shown synergistic effects in enhancing weight loss [72,75].

Semaglutide, a long-acting GLP-1 receptor (GLP-1R) agonist, exhibits high binding affinity to GLP-1R through conserved structural motifs common to class B G protein-coupled receptors. Structural analyses using cryo-electron microscopy (cryo-EM) reveal that semaglutide adopts a helical conformation similar to endogenous GLP-1 and binds within the orthosteric site of GLP-1R, engaging both the extracellular domain (ECD) and the transmembrane (TM) domain. Notably, semaglutide's synthetic modifications—such as an acylation at Lys26 with a C18 fatty diacid chain—contribute to its prolonged half-life and enhanced albumin binding [56,76]. These interactions involve key residues within TM1, TM2, and ECL2, stabilizing the active conformation of the receptor and facilitating Gs-protein coupling, as confirmed by structures at 2.5 Å resolution [76].

Semaglutide's binding mode has been extensively studied using structural and computational tools, anchored by the X-ray structure of the GLP-1R-ECD in complex with GLP-1 (PDB: 4ZGM). These structural insights reveal that semaglutide engages GLP-1R through both its N-terminal α-helix and modified C-terminal extension, mimicking endogenous GLP-1 interactions while introducing stabilizing modifications. Key binding residues within the receptor include Tyr148 and Thr149 in transmembrane helix 1 (TM1); Arg190 and Glu138 in TM2; Tyr205 in extracellular loop 1 (ECL1); Arg299 in TM5; Glu364 and Arg380 in TM6 and ECL3 [77]. Modeling suggests the formation of a stable interfacial electrostatic scaffold, composed of four salt bridges at the GLP-1R-ECD interface, which is predicted to improve receptor activation. These findings underscore the importance of receptor-ligand dynamics and have guided the rational design of next-generation GLP-1R agonists [47,77].

The biological outcomes of GLP-1R and semaglutide binding are multifaceted, with therapeutic implications extending beyond glycemic control. In Alzheimer's disease models, semaglutide binding to GLP-1R upregulates SIRT1 and GLUT4 expression in the hippocampus, enhancing glucose metabolism and reducing Aβ/tau pathology [78]. Similarly, in oncology, semaglutide reprograms tumor-associated macrophages via the GLP-1R/PPARG/ACSL1 pathway, promoting M1 polarization and suppressing tumor progression [79]. Clinically, semaglutide has been validated for its cardioprotective effects, reducing major adverse cardiovascular events in high-risk type 2 diabetes patients [80]. These therapeutic effects reflect the broad tissue expression of GLP-1R and highlight the clinical value of structurally informed ligand design [81].

Avexitide (exendin 9–39) is a potent antagonist of the glucagon-like peptide-1 receptor (GLP-1R), a class B G protein-coupled receptor (GPCR) involved in the regulation of glucose homeostasis. GLP-1R features two key structural domains: an extracellular domain (ECD) responsible for initial ligand binding and a transmembrane domain (TMD) that mediates downstream signaling. Avexitide binds primarily to the orthosteric site within the ECD [82,83]. Structural and functional analyses of class B GPCRs support a two-step activation mechanism—avexitide disrupts the first step, namely high-affinity binding to the ECD, thus blocking downstream metabolic signaling [83].

Detailed binding studies have identified key residues in the GLP-1R that interact with avexitide. These include residues in the N-terminal region such as Trp33, Tyr69, and His89, which are essential for ligand recognition at the ECD, as well as Glu128, Arg190, and Tyr205 within the transmembrane helices that contribute to allosteric conformational locking in the inactive state [82,83]. By occupying the ECD without engaging the transmembrane core, avexitide effectively stabilizes an inactive receptor conformation and disrupts endogenous peptide binding. Moreover, functional assays have shown that avexitide prevents the ligand-induced outward movement of transmembrane helix 6 (TM6), acting as a competitive antagonist at the orthosteric site [83,84].

The clinical relevance of avexitide's GLP-1R antagonism is underscored by its therapeutic use in treating postbariatric hypoglycemia (PBH), a condition characterized by excessive insulin secretion following gastric bypass surgery. By blocking GLP-1R, avexitide reduces postprandial insulin peaks and prevents hypoglycemia without significantly altering basal glucose levels [84]. In a randomized controlled trial, avexitide significantly raised glucose nadir and lowered insulin peaks during mixed-meal tolerance testing, leading to fewer symptomatic hypoglycemic episodes [84,85,86].

Activation of FGFR1 involves ligand-induced dimerization and transphosphorylation of tyrosine residues, especially within the activation loop and juxtamembrane regions. These phosphorylated tyrosines serve as docking sites for downstream signaling adaptors such as FRS2 and PLCγ [100]. Though fostamatinib is not a selective FGFR1 inhibitor, structural similarities among kinase domains suggest that off-target interactions may occur via these conserved motifs [101].

The binding of fostamatinib's active metabolite R406 to kinase domains has been structurally characterized in complex with spleen tyrosine kinase (SYK) and other kinases. Although no crystal structure exists for FGFR1 bound directly to R406, structural comparisons using PDB ID: 4V05 (FGFR1 bound to AZD4547, a selective inhibitor) and PDB ID: 4PUZ (SYK bound to R406) reveal significant conservation of the ATP-binding cleft across these kinases [100,102]. R406 binds in the DFG-out conformation of SYK, forming two key hydrogen bonds with hinge region residues and hydrophobic contacts with residues lining the ATP pocket. By analogy, R406 is predicted to form similar interactions with FGFR1's hinge region residues Met563 and Ala564, which correspond to the hydrogen bond acceptors seen in other kinase–R406 complexes. Additionally, critical FGFR1 residues involved in stabilizing Type II inhibitor binding include Lys514 (catalytic lysine), Glu531 (αC-helix), and Asp641–Phe642 (DFG motif), all of which contribute to ATP coordination and conformational dynamics [15].

Fostamatinib has been approved for immune thrombocytopenia due to its inhibitory effects on SYK-mediated Fc receptor signaling in macrophages [103]. However, broader kinase inhibition by R406 may impact pathways regulated by FGFR1, particularly in cancer, fibrosis, or tissue regeneration contexts [13]. Aberrant FGFR1 activity has been implicated in hematologic malignancies, and indirect suppression via multi-kinase inhibitors could contribute to therapeutic benefits [104]. Additionally, fostamatinib's interference with PI3K/AKT and MAPK pathways, also downstream of FGFR1, indicates potential for pathway-level crosstalk and synergy in oncology applications [105].

Erdafitinib is a potent, orally bioavailable, pan-FGFR tyrosine kinase inhibitor that targets fibroblast growth factor receptors 1 through 4 (FGFR1–4), with high affinity for FGFR1. The binding of erdafitinib to FGFR1 predominantly occurs at the intracellular tyrosine kinase domain, disrupting autophosphorylation and downstream signaling pathways such as PI3K-AKT and MAPK. Structural motifs essential for this interaction include the DFG (Asp-Phe-Gly) motif within the activation loop, which erdafitinib targets in its active (DFG-in) conformation. Erdafitinib inhibits FGFR1 by competing with ATP at the kinase domain, effectively suppressing ligand-mediated signaling involved in cell proliferation and survival [106,107].

Although a co-crystal structure of FGFR1 with erdafitinib has not been publicly resolved, comparative analysis with structurally similar FGFR inhibitors such as PDB ID: 4V05 allows inference of the binding mode. This inhibitor occupy the ATP-binding cleft of the FGFR1 tyrosine kinase domain, engaging key residues in the hinge region notably Ala564, Glu562, and Glu531 via hydrogen bonding. Erdafitinib, like 4V05, is presumed to bind the DFG-in conformation of FGFR1, interacting with residues in the P-loop (Gly485–Gly491), hinge region, and activation loop (Asp641–Phe642–Gly643). The central scaffold of erdafitinib is expected to form bidentate hydrogen bonds with the backbone of Ala564, a residue conserved across FGFR isoforms, anchoring the inhibitor in place. Hydrophobic contacts with residues such as Leu484, Val561, and Ala640 further stabilize the complex. These interactions facilitate effective kinase inhibition by preventing ATP binding and substrate phosphorylation, aligning with biochemical evidence from preclinical FGFR1 assays [108,109].

Erdafitinib has shown substantial clinical activity in tumors with FGFR1 alterations. In LUSC models, combining erdafitinib with STAT3 inhibitors enhances antitumor activity, supporting the importance of dual-pathway inhibition [110]. In the RAGNAR phase 2 study and the BLC2001 trial, erdafitinib elicited objective responses in patients with FGFR1–4-altered tumors, particularly urothelial carcinoma, demonstrating its efficacy in diverse histological settings [111,112]. Thus, the FGFR1–Erdafitinib interaction is not only structurally robust but also clinically significant, offering a promising strategy for treating FGFR-driven malignancies [106,107].

Futibatinib is a structurally novel, covalent inhibitor of FGFR1–4 that displays high affinity for key structural motifs within the ATP-binding pocket of these receptor tyrosine kinases. The drug forms an irreversible bond specifically with a conserved cysteine residue (Cys491 in FGFR2) located in the glycine-rich P-loop of the kinase domain, a region critical for ATP binding and catalysis. The glycine-rich loop's conformational flexibility is thought to facilitate this binding mode, allowing futibatinib to trap multiple FGFR conformers [90,113].

Crystallographic and biochemical studies have elucidated the specific amino acid interactions involved in futibatinib binding to FGFRs. The covalent bond is formed with a conserved cysteine residue located within the P-loop of the kinase domain—specifically Cys488 in FGFR1 and Cys491 in FGFR2 [90,113]. This cysteine lies adjacent to the ATP-binding cleft and is part of a flexible glycine-rich loop that accommodates the acrylamide warhead of futibatinib. In addition to covalent bonding, futibatinib engages in key non-covalent interactions with residues in the hinge region, such as Ala564 and Glu565 in FGFR1, which stabilize the ligand within the active site. Hydrogen bonds with backbone atoms in the kinase hinge region, as well as van der Waals contacts with hydrophobic residues like Leu484 and Val561, further contribute to the high binding affinity and specificity [113,114].

Clinically, the irreversible inhibition of FGFR signaling by futibatinib translates into significant therapeutic activity and a favorable safety profile in patients with FGFR-driven tumors. Phase I–II trials demonstrated meaningful responses in intrahepatic cholangiocarcinoma (iCCA) patients harboring FGFR2 fusions, with an objective response rate of 42% and a median duration of response of 9.7 months. Hyperphosphatemia, an on-target effect related to renal FGFR inhibition, was the most common adverse event but was manageable with dietary and pharmacologic interventions. These findings underscore the biological and therapeutic relevance of futibatinib's protein–ligand interaction features, supporting its clinical use in FGFR-aberrant malignancies [114,115,116].

FGFR1 contains three extracellular immunoglobulin-like (Ig) domains, with the heparin-binding domain localized between IgII and IgIII, a region rich in positively charged residues (lysines and arginines), which facilitates electrostatic interactions with heparin [117]. Structural modeling and mutational analyses have highlighted key residues such as Lys126, Arg121, and Asn28 in FGFR2 that directly interact with sulfated groups of heparin, enabling the formation of a stable complex with FGFR1 [118]. The ternary complex formed between FGFR1, FGF2, and heparin exhibits an electrostatic structure, with the negatively charged heparin nestled between the positively charged surfaces of FGFR2 and FGFR1 [119].

Several structural models and crystallographic studies have clarified the molecular details of the FGFR1–heparin interaction. PDB homology models and partial complexes have been constructed based on structures such as PDB ID: 1FQ9, which shows FGFR2 and FGFR1 bound to a heparin-derived hexasaccharide [118]. In these models, key positively charged residues on FGFR2—Lys27, Arg121, Lys126, Lys136, and Asn102—mediate electrostatic and hydrogen bond interactions with sulfated saccharide groups [118]. The FGFR1 ectodomain, particularly the IgII–IgIII linker region, contributes to heparin binding via conserved basic residues including Lys160, Arg163, and Lys168 [119]. The electrostatic model proposes that heparin is stabilized between two FGF2–FGFR1 complexes, enabling dimerization and receptor activation. Molecular modeling has further supported a 2:2:1 stoichiometry (2 FGFR1s: 2 FGF2s: 1 heparin), consistent with binding data and mutagenesis studies [118]. Despite the absence of a full PDB-resolved ternary structure, these data strongly support a mechanism in which heparin acts as a structural scaffold, orienting the proteins to facilitate receptor dimerization and signal initiation [120,121].

The FGFR1–heparin interaction is fundamental to effective FGF-mediated signal transduction and is implicated in numerous physiological and pathological processes, including cell proliferation, differentiation, angiogenesis, and cancer progression [117,121]. Blocking this interaction, such as with the IMB-R1 antibody that specifically targets the heparin-binding domain of FGFR1, has been shown to inhibit FGF2-induced cell growth and induce apoptosis in cancer cells [117]. The ability of heparin to facilitate dimerization and receptor activation supports its role as a crucial coreceptor in FGF signaling [119,122]. Therefore, understanding the structural basis of FGFR1–heparin interactions not only advances our knowledge of receptor biology but also opens avenues for targeted cancer therapies.

Infigratinib is a potent, ATP-competitive tyrosine kinase inhibitor with selectivity for FGFR1–3. It targets the highly conserved ATP-binding cleft within the intracellular kinase domain of FGFRs, crucial for receptor autophosphorylation and activation. Structurally, this interaction is mediated by infigratinib's ability to bind key residues within the catalytic cleft of the tyrosine kinase domain, stabilizing the inactive conformation of FGFR and preventing downstream signaling [123]. Molecular docking and crystal structures deposited in the Protein Data Bank (e.g., PDB ID: 4V05) show that infigratinib forms hydrogen bonds with the hinge region (including Ala564 in FGFR1), hydrophobic interactions with residues such as Val561 and Leu630, and a critical interaction with Asp641 at the DFG motif of the activation loop [124]. These interactions underline the high binding affinity and selectivity of infigratinib for FGFR isoforms over other kinases.

The binding of infigratinib to FGFR1 has been extensively validated in various tumor models. Particularly in gliomas, activating point mutations in FGFR1 confer constitutive activation of the kinase and ligand-independent signaling. Infigratinib retains potent binding to these altered receptors, effectively suppressing their downstream oncogenic signaling [125]. The structural positioning of these mutations within the kinase domain preserves the binding cleft architecture, allowing infigratinib to maintain its affinity [126].

Clinically, the inhibition of FGFR signaling by infigratinib has profound implications in diseases. In multiple cancers, including glioblastoma and cholangiocarcinoma, FGFR fusions and mutations contribute to aberrant cell proliferation, survival, and angiogenesis [124]. Infigratinib suppresses these pathways, notably the MAPK, PI3K-AKT, and PLCγ signaling axes, by blocking receptor autophosphorylation. However, its efficacy may be modulated by tumor-specific co-mutations or pathway redundancies. For instance, concurrent mutations in PI3K pathway genes may necessitate combination therapies [126]. Moreover, in developmental biology, off-target effects of infigratinib at high doses highlight the relevance of FGFRs in normal morphogenesis and the need for careful dose titration in pediatric applications [123]. Thus, understanding the molecular underpinnings of FGFR–infigratinib interaction informs both precision oncology and safety profiling.

Lenvatinib is a type V ATP-competitive inhibitor that binds to the kinase domain of FGFR1, stabilizing it in the DFG-out inactive conformation, thus preventing downstream signaling. Structural insights from PDB entry 5A46, which features FGFR1 bound to lenvatinib, reveal a conserved mode of interaction within the ATP-binding cleft. Critical interactions involve the hinge region residues Glu562 and Ala564, which form hydrogen bonds with the backbone of the inhibitor, anchoring it in place. The gatekeeper residue Val561, along with Leu484, Ala640, and Phe642, shapes the hydrophobic pocket that accommodates the aromatic core of lenvatinib. Additionally, Asp641 and Tyr653/Tyr654 within the activation loop contribute to conformational stability and are crucial for kinase activity modulation [127,128,129].

From a therapeutic perspective, lenvatinib's inhibition of FGFR1 not only suppresses tumor growth but also improves the immune landscape in HCC. It reduces regulatory T cell (Treg) infiltration and PD-L1 expression by targeting FGFR1/FGFR4, enhancing the efficacy of immune checkpoint blockade with anti-PD-1 agents [130,131]. The combination of lenvatinib with agents like oxysophocarpine has shown synergistic effects by further downregulating FGFR1 expression and associated downstream signals [132]. This highlights the clinical relevance of FGFR1 inhibition in overcoming resistance mechanisms and improving patient outcomes, particularly in advanced HCC where FGFR1 overexpression correlates with poor prognosis and treatment failure [133].

Nintedanib is a small molecule that targets multiple receptor tyrosine kinases, including FGFR1, by competitively binding to the ATP-binding site of the receptor. FGFR1 is composed of an extracellular ligand-binding domain, a transmembrane domain, and an intracellular tyrosine kinase domain, which is the primary target for nintedanib. The ATP-binding pocket within this intracellular domain is crucial for receptor activation, and structural motifs such as the DFG (Asp-Phe-Gly) motif and hinge region residues are commonly involved in ligand binding. Although specific crystallographic structures of the FGFR1-nintedanib complex are limited, modeling and structural comparisons suggest nintedanib stabilizes the inactive conformation of FGFR1 by interacting with residues within the ATP-binding cleft, mimicking ATP hydrogen bonds and hydrophobic contacts [134,135].

Experimental data and in vitro studies indicate that key amino acid residues contributing to the binding of nintedanib include those located in the kinase domain of FGFR1 Lys514, Glu531, Val561, Glu562, and Asp641. These residues are critical for ATP coordination and contribute to the formation of hydrogen bonds and van der Waals interactions with nintedanib indolinone core and adjacent functional groups. Crystallographic modeling based on the FGFR1 tyrosine kinase domain structure PDB ID: 3C4F supports this interaction, showing that nintedanib occupies the ATP-binding cleft and stabilizes the DFG-in conformation. This type I binding mode effectively inhibits kinase activation and disrupts downstream signaling cascades such as MAPK/ERK [136,137,138].

Therapeutically, these interactions have significant implications for the treatment of diseases such as idiopathic pulmonary fibrosis and cancers like non-small cell lung cancer and esophagogastric adenocarcinoma. Nintedanib inhibition of FGFR1, along with VEGFR and PDGFR, results in suppression of angiogenesis, fibroblast proliferation, and tumor growth. In cancer, FGFR1 amplification is associated with aggressive behavior and resistance to therapy, making FGFR1 a critical target. The ability of nintedanib to maintain FGFR1 signaling suppression, even in resistant tumor subclones, offers a clinical benefit by delaying progression and overcoming resistance mechanisms, particularly when used in combination therapies [134,135,139].

The interaction between fibroblast growth factor receptor 1 (FGFR1) and palifermin, a recombinant truncated form of keratinocyte growth factor (KGF/FGF7), is mediated through specific structural motifs on the extracellular domain of FGFR1. Binding primarily involves the immunoglobulin-like domains D2 and D3, as well as the D2–D3 linker region, which collectively determine ligand specificity and affinity [140]. These domains are both necessary and sufficient for ligand binding, whereas domain D1 and the D1–D2 linker act as autoinhibitory elements that modulate receptor activation. Structural studies have also highlighted the role of the alternatively spliced C′–E loop in domain III, which enhances the flexibility and specificity of FGF binding. This loop is particularly relevant in accommodating ligands like FGF1, which is known for its broad receptor promiscuity [140,141].

Crystallographic and biochemical analyses of FGFR–ligand complexes, including structures such as the FGFR3c–FGF1 complex (PDB: 1RY7), show that receptor activation requires not only ligand binding but also co-factors like heparin or heparan sulfate, which stabilize the formation of a ternary FGF–FGFR–heparin complex [142]. This interaction leads to receptor dimerization and subsequent transphosphorylation of intracellular tyrosine residues, initiating downstream signaling cascades. Key amino acid residues in the D2 and D3 domains of FGFR1—particularly Asn173, Glu176, and Lys160 in domain D2, and His254, Asn262, and Lys271 in domain D3—have been identified as essential contact points for FGF1 and related ligands [140]. Additionally, residues within the alternatively spliced C′–E loop, such as Pro252 and Ser254, contribute to the specificity of binding. Although palifermin binds with high specificity to FGFR2b via a similar interface, it has been observed to interact with FGFR1 under specific conditions, especially when FGFR1 is overexpressed or when cofactors modulate receptor conformation. Its reduced receptor promiscuity compared to FGF1 helps limit off-target activation, contributing to its safety profile in therapeutic applications [143].

Functionally, the FGFR1–palifermin interaction plays a significant role in epithelial tissue regeneration and cytoprotection. Palifermin activates several intracellular signaling pathways, including MAPK/ERK and PI3K/Akt, leading to enhanced epithelial cell survival, proliferation, and DNA repair [143]. Clinically, this translates into measurable therapeutic benefits. For instance, in patients with metastatic colorectal cancer undergoing fluorouracil-based chemotherapy, palifermin significantly reduced the incidence and severity of oral mucositis, thus improving tolerability and reducing chemotherapy dose modifications [144]. These effects validate the biological relevance of FGFR1–palifermin signaling and support its continued use and investigation in both oncologic supportive care and regenerative medicine [141,143].

Pemigatinib, a potent and selective inhibitor, interacts primarily with FGFR1-3 by targeting the ATP-binding site, leading to inhibition of receptor autophosphorylation and downstream signaling. Pemigatinib's molecular binding involves conserved residues in the kinase domain and exploits the DFG-in active conformation of FGFR1, which differentiates it from type II inhibitors like ponatinib that bind to a DFG-out inactive conformation [108,145].

In terms of binding characteristics, crystal structural data and pharmacologic evaluations reveal that pemigatinib forms hydrogen bonds and hydrophobic contacts with specific residues such as Ala564, Glu531, and Phe642 within the kinase domain of FGFR1 [108]. These interactions stabilize the ligand within the ATP pocket and block the kinase activity by preventing ATP access. Structural insights into these interactions can be modeled using the crystal structure of FGFR1 bound to the selective inhibitor AZD4547, available in the Protein Data Bank under the accession code PDB: 4RWI, which shares a binding pocket of Leu484, Phe489, Lys514, Ile545, and Leu630 and highlights the critical residues in the ATP-binding cleft (Figure 2). Pemigatinib exhibits high selectivity for FGFR1–3, with IC₅₀ values in 0.4–1 nM indicating that subtle differences in the amino acid environment surrounding the ATP-binding site contribute to binding specificity [146].

Biologically, the interaction between pemigatinib and FGFR1 has significant therapeutic implications. By inhibiting FGFR1 signaling, pemigatinib attenuates tumor cell proliferation, angiogenesis, and resistance mechanisms in FGFR-driven malignancies, including cholangiocarcinoma and urothelial cancers [147]. Pemigatinib's selective engagement with FGFR delineates a pathway-specific oncologic intervention and offers a platform for precision oncology based on FGFR genotyping [148,149,150].

Ponatinib is a type II multikinase inhibitor that demonstrates strong binding affinity toward fibroblast growth factor receptor 1 (FGFR1), targeting its inactive DFG-out conformation. The binding pocket exploited by ponatinib includes the adenine region, a hydrophobic back pocket adjacent to the gatekeeper residue (Val561 in FGFR1), and a pocket created by the DFG-out conformation of the activation loop. Ponatinib's rigid acetylene linker and trifluoromethylphenyl moiety facilitate van der Waals interactions and hydrogen bonding, securing the molecule within the ATP-binding cleft [108]. X-ray crystallography studies (PDB: 4V04) confirm that ponatinib binds FGFR1 in this DFG-out conformation, stabilizing an inactive state of the kinase and enabling high potency across FGFR isoforms [108,151].

At the amino acid level, key FGFR1 residues involved in ponatinib binding include the gatekeeper residue Val561, and other hydrophobic spine components such as Leu530, Met537, His544, and Phe565. These residues contribute to a tightly packed hydrophobic environment critical for type II inhibitor binding. Additionally, ponatinib forms hydrogen bonds with residues in the hinge region, notably Ala564, reinforcing its stable interaction [108]. This is clearly visualized in the crystal structure of the FGFR1–ponatinib complex (PDB ID: 4V04), where ponatinib occupies the ATP-binding cleft and extends into the hydrophobic pocket formed by the DFG-out conformation [151,152].

These interactions have critical implications for biological function and therapeutic efficacy. In models of FGFR1-driven malignancies such as 8p11 myeloproliferative syndrome (EMS) and FGFR1-fusion positive leukemias, ponatinib effectively suppresses downstream signaling via pathways such as PLCγ, STAT5, ERK1/2, and AKT, leading to reduced proliferation and apoptosis induction in vitro and in vivo [153,154]. Furthermore, in non-small cell lung cancer (NSCLC) cells overexpressing FGFR1, ponatinib demonstrated potent antiproliferative effects, suggesting therapeutic applicability in FGFR1-addicted solid tumors [155]. These preclinical outcomes underscore ponatinib's capacity to act against both native and fusion-activated FGFR1 variants, providing a strong rationale for its continued investigation and clinical deployment in FGFR1-driven pathologies [156].

The interaction between fibroblast growth factor receptor 1 (FGFR1) and the selective RET inhibitor pralsetinib exemplifies the cross-target promiscuity often observed in kinase-targeting therapies. Although pralsetinib was developed to inhibit RET-driven oncogenic signaling, its inhibitory activity extends to several other kinases, including FGFR1, TRK, VEGFR2, and PDGFRβ, due to structural similarities in the ATP-binding pockets [157,158]. FGFR1 contains a conserved tyrosine kinase domain that undergoes autophosphorylation upon ligand-induced dimerization, triggering downstream signaling cascades. These include the MAPK, PI3K-AKT, and PLCγ pathways, which are central to cell survival and proliferation [124].

Structurally, pralsetinib interacts with the FGFR1 kinase domain through key motifs near the ATP-binding cleft. These include the hinge region, gatekeeper residue, and DFG motif, which regulate kinase activity [108]. In FGFR1, critical amino acid residues involved in pralsetinib or related FGFR inhibitor binding include the gatekeeper residue Val561, hinge residue Glu562, and the conserved DFG motif residues Asp641–Phe643. These structural elements are essential for ATP binding and inhibitor selectivity. Relevant structural insights are drawn from PDB entry 4V05, which depicts FGFR1 in complex with the type II inhibitor ponatinib and illustrates a similar DFG-out conformation that could potentially be adopted by pralsetinib in off-target FGFR1 binding [108,124].

Clinically, the off-target inhibition of FGFR1 by pralsetinib may contribute to both therapeutic outcomes and adverse effects in RET-altered cancers. While RET fusions are the primary target in NSCLC and thyroid cancer, concurrent FGFR aberrations can exist, especially in aggressive tumors [159]. FGFR1 inhibition may provide additive or synergistic anticancer effects, particularly in tumors with FGFR1 amplification or dependency. However, this multi-target profile also introduces risks, such as hypertension and hepatotoxicity, potentially exacerbated by FGFR1's role in vascular homeostasis [157].

Regorafenib, a type II multikinase inhibitor, preferentially binds FGFR1 in its DFG-out conformation, exploiting the hydrophobic back pocket adjacent to the ATP-binding site, a characteristic binding mode for type II inhibitors [128]. Structural analyses reveal that key motifs such as the gatekeeper residue (Val561 in FGFR1) and the molecular interaction network (involving residues Glu562, Asn546, and Lys638) contribute to the ligand selectivity and kinase inhibition, highlighting the importance of these domains in inhibitor design [13].

Although no crystallographic structure of an FGFR1–regorafenib complex has been resolved to date, mechanistic and structural insights suggest that regorafenib similarly targets the DFG-out conformation of the activation loop and interacts with key residues within the hydrophobic pocket adjacent to the ATP-binding site. These include the Phe residue of the DFG motif, Val561, and catalytic residues in the hinge region such as Glu562 and Lys638. Regorafenib's broad-spectrum kinase inhibition includes potent activity against FGFR1 (IC₅₀ ≈ 202 nM) [98,160]. Through stabilization of the inactive conformation of FGFR1, regorafenib is presumed to effectively inhibit autophosphorylation and downstream effector activation, contributing to its therapeutic efficacy.

Therapeutically, regorafenib's interaction with FGFR1 is highly relevant in tumors where FGFR signaling drives oncogenesis or resistance to VEGF-targeted therapies. For instance, in colorectal and gastric cancers with FGFR2 amplification, regorafenib was shown to effectively inhibit receptor phosphorylation and downstream signaling, leading to reduced tumor proliferation and induced apoptosis in preclinical models [161]. Moreover, regorafenib has demonstrated survival benefits in hepatocellular carcinoma patients progressing on sorafenib, suggesting that FGFR inhibition plays a key role in its mechanism of action [162]. By targeting both angiogenic and oncogenic pathways, including FGFR1, regorafenib contributes to tumor microenvironment modulation and offers a therapeutic advantage in malignancies with activated FGFR1-2 signaling [160].

Romiplostim, although not a natural FGF ligand, acts on a separate receptor—the thrombopoietin receptor (MPL)—to stimulate megakaryocyte proliferation and platelet production [163]. However, FGFR1 remains central in mediating thrombopoiesis through the FGF1/FGFR1/PI3K/Akt/NF-κB axis, as demonstrated in models of radiation-induced thrombocytopenia where activation of FGFR1 upregulated this entire pathway, promoting platelet recovery [164].

At the structural level, crystallographic studies have elucidated the FGF2–FGFR1 interaction, with the dimeric complex structure resolved at 2.8 Å (PDB ID: 1CVS). The FGF2 ligand binds across both D2 and D3 domains of FGFR1, engaging residues within the β-sheets and the linker region between these domains [102,165]. FGFR1 residues Tyr653 and Tyr654 are key autophosphorylation sites, and their activation promotes downstream signaling. In contrast, romiplostim binds specifically to the MPL receptor using a peptibody scaffold composed of engineered thrombopoietin mimetic domains fused to an IgG Fc region [166]. Although romiplostim does not interact directly with FGFR1, cross-talk between FGFR1-driven and TPO/MPL-driven pathways has significant role, particularly in overlapping roles in megakaryocyte maturation and platelet production.

Romiplostim, by mimicking TPO and activating MPL, has shown high efficacy in treating chronic immune thrombocytopenia (ITP), with durable platelet responses and a favorable safety profile [163,166]. Meanwhile, activation of FGFR1, either endogenously by FGF1 or via pharmacological agents such as methylophiopogonanone A (MO-A), supports an alternative or complementary thrombopoietic strategy, especially in radiation-induced thrombocytopenia models [164,167].

Selpercatinib is a highly selective small-molecule RET kinase inhibitor, yet it also exhibits limited off-target inhibition of FGFR1 at higher concentrations. FGFR1 is a receptor tyrosine kinase whose activation loop features a conserved Asp–Phe–Gly (DFG) motif, a crucial element in kinase regulation [168]. Selpercatinib is primarily designed for RET, its limited inhibition of FGFR1 suggests potential off-target activity through similar structural engagement of the activation loop, though at significantly reduced potency [169].

Crystallographic and biochemical analyses have shown that FGFR1 binds ponatinib at its ATP-binding cleft in the DFG-out conformation. In this context, key residues involved in inhibitor interaction include Val561, Ala564, and Tyr563, which stabilize the ponatinib–FGFR1 complex. Although selpercatinib is not a high-affinity binder of FGFR1, these conserved residues in the ATP-binding site might still facilitate weak interactions under high drug concentrations. The Protein Data Bank (PDB) entry associated with the FGFR1–ponatinib complex is 4V05, which captures the kinase in its inactive, DFG-out conformation stabilized by inhibitor binding [168]. The interaction with the activation loop, particularly Asp641 and the pH-sensitive ionization of this residue, contributes to the conformational equilibrium and the binding kinetics of type II inhibitors. This structural motif is critical for selective targeting and is likely involved in any off-target effects seen with selpercatinib when binding FGFR1 [170].

Dysregulation or overexpression of FGFR1 has been implicated in oncogenesis, particularly in cancers such as breast and lung carcinoma [171]. The limited but measurable inhibitory activity of selpercatinib on FGFR1 may contribute to off-target therapeutic or toxicological effects. However, its high selectivity for RET helps to minimize unintended inhibition of FGFR family members [172]. Clinically, selpercatinib's success in treating RET-altered thyroid and non-small cell lung cancers is attributed to its potent and sustained inhibition of RET signaling [173,174]. Its therapeutic efficacy is reinforced by favorable pharmacokinetics and a manageable toxicity profile, distinguishing it from multikinase inhibitors that indiscriminately inhibit kinases such as FGFR1 and VEGFR2 [169].

Sorafenib, a multi-kinase inhibitor, interacts directly and indirectly with FGFR1 but exhibits limited specificity [175]. The binding mode of sorafenib, while not primarily optimized for FGFR1, can still impact this receptor via downstream off-target inhibition. Notably, sorafenib targets the phosphorylation of FGFR1, indirectly affecting downstream signaling cascades such as the MAPK/ERK, PI3K/AKT, and STAT3 pathways [176]. Structural analyses of FGFR1 reveal that key amino acid residues in the ATP-binding pocket (PDB IDs: 2FGI, 3GQL, 3C4F) such as Ala564, Glu562, and Cys488 are critical for ligand interaction and receptor activation [175]. Among them, Cys488, located within the glycine-rich loop of the kinase domain, is particularly notable as a target for covalent inhibitors due to its strategic position and low conservation across kinases. In the context of tumor angiogenesis, DJ-1 has been identified as a key secreted factor that promotes FGFR1 activation in endothelial cells by enhancing phosphorylation at these residues, thereby contributing to sorafenib resistance in hepatocellular carcinoma (HCC) [176]. Moreover, in malignant pleural mesothelioma tumor-initiating cells, the antiproliferative effect of sorafenib was associated with inhibition of FGFR1 phosphorylation at the kinase domain, leading to suppression of ERK and STAT3 activation following bFGF stimulation [177].

From a therapeutic perspective, FGFR1 overexpression and its downstream effector FRS2α correlate with reduced progression-free survival in patients treated with sorafenib, suggesting a role in intrinsic resistance mechanisms [178]. In hepatocellular carcinoma, FGFR1 activation promotes tumor-initiating cell enrichment and angiogenesis, contributing to acquired sorafenib resistance. Experimental models have shown that blocking FGFR signaling, either directly or via co-factors like DJ-1, can restore sorafenib sensitivity and suppress tumor growth [176,178]. These results emphasize the functional importance of FGFR1 in mediating therapeutic outcomes and advocate for the co-targeting of FGFR1 in cancers where resistance to sorafenib limits treatment efficacy.

Tirzepatide is a synthetic 39-amino acid peptide designed to act as a dual agonist for both the glucose-dependent insulinotropic polypeptide receptor (GIPR) and the glucagon-like peptide-1 receptor (GLP1R). The GLP1R, a class B G protein-coupled receptor, contains two key domains essential for ligand binding: the extracellular N-terminal domain (NTD) and the transmembrane domain (TMD). The activation of GLP1R by peptide ligands, such as GLP-1 or tirzepatide, follows a two-domain model. In this model, the C-terminal region of the ligand initially interacts with the NTD, positioning the N-terminal domain of the ligand to engage deeply within the TMD, triggering receptor activation and downstream signaling [,]. Tirzepatide utilizes this structural framework to bind both incretin receptors, leveraging structural motifs from native GIP and exendin-4 to optimize binding efficacy and duration of action []. ^{179 180 181}

The high-resolution cryo-EM structure of GLP1R bound to small-molecule agonists has illuminated key residues involved in ligand recognition and receptor activation. Based on Protein Data Bank entry PDB: 7RBT, and associated cryo-EM data, critical residues mediating binding include Glu138 and Leu141 in transmembrane domain 1 (TM1), Lys197 and Leu201 in TM2, Phe230 and Met233 in TM3, Thr298 in extracellular loop 2 (ECL2), and Arg380, Phe381, Leu384 and Phe385 in TM7 (). Additionally, essential interactions in the N-terminal domain (NTD) are formed with Leu32, Trp33, Val36, and Gln37 []. Tirzepatide's engineered hybrid sequence enables it to interact with both the NTD and TMDs, stabilizing the receptor in its active conformation and maximizing downstream cAMP-mediated signal transduction [,]. Figure 3 ^{13 172 179}

The dual receptor activity of tirzepatide results in enhanced insulinotropic effects, superior glycemic control, and greater weight reduction compared to GLP-1 receptor agonists alone. Clinical trials, particularly the SURPASS program, demonstrate tirzepatide's dose-dependent superiority in lowering HbA1c and body weight [,]. Beyond metabolic improvements, tirzepatide has been associated with reductions in all-cause mortality and adverse cardiovascular and renal events compared to GLP-1R []. These therapeutic benefits are believed to stem from the synergistic activation of GLP1R and GIPR, with the latter contributing to enhanced β-cell function, insulin sensitivity, and central appetite regulation []. Thus, the structural and functional engagement of tirzepatide with GLP1R plays a pivotal role in its therapeutic success in type 2 diabetes and associated comorbidities. ^{182 183 180 181}

Tirzepatide is a dual incretin receptor agonist that interacts with both the glucose-dependent insulinotropic polypeptide receptor (GIPR) and the glucagon-like peptide-1 receptor (GLP-1R), contributing to its therapeutic effects in type 2 diabetes (T2D) and obesity (). Structurally, key binding domains include the N-terminal α-helix of tirzepatide that penetrates the transmembrane domain of both GIPR and GLP-1R. Cryo-EM studies show that tirzepatide mimics GIP in its interaction with GIPR, while displaying weaker interactions with GLP-1R due to diminished contact between its N-terminal Tyr1 and the GLP-1R core. This results in biased agonism at GLP-1R with reduced β-arrestin recruitment and receptor internalization [,]. These interactions enable tirzepatide to signal preferentially through cAMP generation while maintaining a long-acting profile via acylation with a C20 fatty diacid that binds albumin, enhancing its pharmacokinetics [,]. Figure 4 ^{184 185 186 187}

The structural determinants of tirzepatide's binding were resolved through cryo-electron microscopy (cryo-EM) models—PDB: 7RBT for the GIPR–tirzepatide complex and GLP-1R–tirzepatide complex—revealing specific and differential residue-level interactions [,]. In the GIPR complex, tirzepatide residues Tyr1, Phe6, and Asp9 interact with GIPR residues Tyr1, Glu3, Tyr145, Arg183, and Ser381, forming stable hydrogen bonds and hydrophobic contacts within transmembrane helices 2, 3, and 5. These interactions create a compact and high-affinity ligand-receptor conformation, promoting efficient receptor activation. Conversely, at the GLP-1R, the same tirzepatide residues Tyr1, Phe6, and Asp9 show reduced interaction strength with GLP-1R residues Leu141, Glu364, and Lys197, leading to a more transient and weaker receptor engagement. This results in reduced receptor internalization and a signaling bias favoring cAMP generation over β-arrestin recruitment, ultimately leading to less desensitization. These structural nuances account for tirzepatide's stronger and more stable activation of GIPR, while preserving a more tolerable signaling profile at GLP-1R, contributing to its superior metabolic efficacy and reduced gastrointestinal adverse effects compared to GLP-1 monotherapy. ^{184 187}

These protein–ligand interactions have critical implications for biological function and therapeutic outcomes. The dual activation of GIPR and GLP-1R enhances insulin secretion, improves insulin sensitivity and reduces body weight. Tirzepatide's biased signaling at GLP-1R avoids rapid receptor desensitization, allowing sustained insulinotropic action []. Clinically, this translates to superior glycemic control and weight loss compared to selective GLP-1R agonists [,]. Moreover, tirzepatide's engagement with GIPR in adipose and pancreatic tissue contributes to enhanced metabolic flexibility and β-cell preservation, highlighting its advantage as a multi-target agent for obesity [,]. ^{185 181 188 186 189}