Bioorganic & Medicinal Chemistry Letters

We have developed and characterized a potent and cell active tau tubulin kinase 1 and 2 (TTBK1 and TTBK2) inhibitor, 13. Compound 13 demonstrates in-cell, kinome-wide selectivity, and potently inhibits both TTBK1 and TTBK2. As part of our medicinal chemistry campaign, we also identified a structurally similar negative control, compound 5, which lacks in-cell affinity for TTBK1 and TTBK2. Based on their substrates, which include TDP-43, tau, and tubulin, TTBK1 and TTBK2 inhibition has been pursued as a therapeutic approach for Alzheimers disease, frontotemporal lobe dementia, and amyotrophic lateral sclerosis. TTBK2 is also an effector of ciliogenesis, acting in concert with CEP164, CP110, and CEP83 to initiate the biogenesis of primary cilia. The development of selective chemical tools for these kinases facilitates investigation into TTBK1/2-mediated pathways and potential disease-altering ramifications linked to their pharmacological perturbation.

The use of covalent warheads targeting the catalytic cysteine has been a cornerstone in coronavirus main protease (Mpro) inhibitor development, where various electrophilic motifs have been used including aldehydes, nitriles, ketoamides, and hydroxymethyl ketones (HMKs). Recent efforts have been mostly centered around nitrile warheads, given the success of compounds like Nirmatrelvir and Ensitrelvir in the clinic. However, finding and advancing alternative chemotypes with differentiating chemical and pharmacological profiles is essential for future pandemic preparedness. Among such alternatives, HMKs hold special interest because they balance reduced intrinsic electrophilicity with an excellent selectivity profile. Nevertheless, early HMK-based compounds, such as the clinical-stage Mpro inhibitor PF-00835231, suffered from poor oral bioavailability and therefore required intravenous administration, with or without prodrug derivatization of the hydroxyl group. Here, we describe our efforts in advancing the HMK field via the discovery of mCMX110, a lead that has superior potency, increased unbound exposure in vivo, and favorable oral bioavailability in preclinical studies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/725542v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@abe1c9org.highwire.dtl.DTLVardef@746a08org.highwire.dtl.DTLVardef@dd5861org.highwire.dtl.DTLVardef@1d572c7_HPS_FORMAT_FIGEXP M_FIG C_FIG

Ligand optimization is central to drug discovery as hundreds of analogs might be designed and synthesized between an initial hit and a therapeutic candidate. The efficiency of this process is unclear, at least partly because there is no random background for optimization against which to compare. Such a random background might emerge from synthetically accessible but otherwise systematic random small substitutions across starting ligands, measuring likelihood of achieving a substantial improvement in affinity/potency or other property by any single perturbation. Recent literature and ligand-affinity/potency databases suggest that perhaps 10% of analogs with minor modifications improve upon a parents potency substantially (by [≥]10-fold), but this number is clouded by reporting bias, intentional improvement, and inter-group reproducibility. To begin to establish a background expectation for ligand optimization, we comprehensively and systematically modified 18 lead molecules across six targets with single atom changes; 257 compounds were synthesized. Unexpectedly, 11.2% of these random small perturbation analogs improved potency by [≥]10-fold over their parents. Conversely, these more potent analogs typically had worse in vitro pharmacokinetics (e.g. reduced metabolic stability, lower plasma free fraction). While it was possible to find analogs where the potency increase compensated for inferior exposure and half-life, resulting in more potent compounds in vivo, overall a frustrated landscape for ligand optimization is revealed. This study begins to establish a background expectation for ligand potency optimization and offers a simple strategy to do so. It also begins to quantify the challenges confronting the field in moving beyond in vitro potency.

Homeodomain-interacting protein kinase 4 (HIPK4) remains an understudied member of the dark kinome. While genetic knockout studies suggest roles for HIPK4 in spermiogenesis and cutaneous squamous cell carcinoma, whether these cellular functions can be recapitulated by pharmacological inhibition remains to be determined. However, such investigations have been hampered by a lack of high-quality chemical tools. To address this, we employed a rational design strategy utilizing macrocyclization of a bosutinib-based scaffold. Systematic optimization led to the discovery of AZ137 (28e), a potent and selective HIPK4 inhibitor (IC50 = 11 nM; cellular EC50 = 76 nM). AZ137 exhibits exceptional selectivity across three comprehensive orthogonal panels, high solubility, and no detectable cytotoxicity. Its cellular activity was confirmed in cell-based assays of HIPK4-dependent F-actin remodeling. Together with a negative control compound, this probe set provides a foundational framework for the validating HIPK4 as a therapeutic target and a high-quality resource to elucidate its roles in normal physiology and disease. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/720179v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@12438borg.highwire.dtl.DTLVardef@11083beorg.highwire.dtl.DTLVardef@1395fb4org.highwire.dtl.DTLVardef@1ba3db8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Protein tyrosine kinases are important regulators of cell signaling, and aberrant kinase activity contributes to many human diseases, including cancers. All protein tyrosine kinases share a highly-conserved ATP binding pocket but diverge in their substrate binding sites in order to mediate distinct signaling events. Many potent and efficacious ATP-competitive tyrosine kinase inhibitors have been developed, however it remains challenging to achieve on-target selectivity across different kinases and target specific disease mutants, given the high degree of conservation in the ATP-binding pocket. By contrast, the variable substrate-binding site offers an opportunity for selective inhibition, provided molecules can be targeted to this site. Here, we present a modular strategy to design selective, peptide-based covalent inhibitors of tyrosine kinases with a distinct binding mode from existing ATP-competitive inhibitors. Using Src kinase as a model system, we demonstrate that Src-selective reactivity can be achieved by first designing an optimized substrate peptide and then strategically positioning an electrophile on the peptide to target a non-conserved cysteine on the kinase. We show that substrate-derived covalent peptides can inhibit kinase activity, bind simultaneously with an ATP-competitive inhibitor, and even inhibit the activity of kinases bearing a common drug resistance mutation. We further explore the application of this approach to develop an inhibitor of the cancer-relevant fibroblast growth factor receptor 1 kinase that shows selectivity for an oncogenic mutant over the wild-type enzyme. Our modular strategy to generate selective covalent peptides targeting protein tyrosine kinases provides a promising framework for future chemical probe and drug development efforts.

Lung cancer remains the leading cause of cancer-related deaths worldwide, with cisplatin as the primary chemotherapy despite its limitations. Organotin(IV) dithiocarbamates have emerged as promising anticancer agents due to their potent cytotoxicity and stability. This study reports the successful synthesis of four novel organotin(IV) dithiocarbamates: dimethyltin(IV) N-methyl-N-benzyldithiocarbamate (DioSn-1), diphenyltin(IV) N-methyl-N-benzyldithiocarbamate (DioSn-2), triphenyltin(IV) N-methyl-N-benzyldithiocarbamate (TriSn-3), and triphenyltin(IV) N-ethyl-N-benzyldithiocarbamate (TriSn-4). Their cytotoxicity against A549 lung carcinoma cells was evaluated via MTT assay, while Annexin V-FITC/PI staining determined the mode of cell death. DioSn-2, TriSn-3, and TriSn-4 exhibited potent cytotoxicity (IC: 0.52-1.86 M), whereas DioSn-1 was inactive (IC > 50 M). Apoptotic features such as cell shrinkage and membrane blebbing were observed, with apoptosis rates ranging from 58% to 91%. DioSn-2 was the most selective (SI = 6.45) and induced early DNA damage within 30 minutes, followed by mitochondrial depolarization and excessive ROS generation. Caspase-9 activation exceeded caspase-8, confirming intrinsic apoptosis. NAC treatment reduced apoptosis by 52%, highlighting oxidative stress as a key cytotoxic mechanism. These findings suggest DioSn-2 as a promising alternative to cisplatin for lung cancer therapy.

The isolation of cellulose nanofibrils (CNFs), a promising precursor for sustainable and high-performance materials, has relied on chemically intensive, energy-demanding processes. As these processes were originally designed for the isolation of CNFs from wood, we herein show that the intrinsic ultrastructure of non-structural plant cells provides unique opportunities, namely direct access to loosely organized cellulose nanonetworks. We demonstrate that this loose nanofibrillar tissue can be transformed into CNFs with sizes down to elementary nanofibrils ([~]4 nm) at high yields (reaching [~]32%) under exceptionally mild hydrothermal conditions. Three distinct plants were evaluated and the physicochemical properties of the obtained nanonetworks and corresponding CNFs were thoroughly studied, including the hydrodynamics of the resulting gels. Films prepared from the obtained CNFs showed similar performance to those obtained from conventionally isolated wood-based CNFs. Overall, this study demonstrates that CNFs can be obtained through low-intensity, hazard-free, processes from widely available biomass. Thus, this approach offers a unique shift in the range of opportunities to produce CNFs facilitating the integration of their use into the food supply chain, biomedical applications, and other regulatory-constrained applications.

Serotonergic psychedelics such as N,N-dimethyltryptamine (DMT) and 4-phosphoryloxy-N,N-dimethyltryptamine (psilocybin) show therapeutic promise for psychiatric and neurodegenerative disorders but may be limited by liabilities from serotonin (5-HT)-2A mediated psychoactive effects and potential cardiotoxicity via 5-HT2B activation. To address these limitations, we designed and synthesized 2-halogenated derivatives of DMT and psilacetin to reduce 5-HT2A/5-HT2B activity while retaining engagement of therapeutically relevant targets, particularly 5-HT6, 5-HT2C, and 5-HT1B. This study demonstrated that 2-position halogenation decreased affinities, potencies, and efficacies at 5-HT2A and 5-HT1A receptors while preserving potent 5-HT6 agonism, especially for 2-Br-psilocin. The analogues exhibited reduced affinities at 5-HT2B and hERG ion channels, suggesting safer cardiac valve and cardiotoxic profiles. In C57BL/6J mice, 2-Br-psilacetin did not induce the head-twitch response and attenuated 2,5 dimethoxy-4-iodoamphetamine (DOI)-induced head-twitch behavior, suggesting a reduced potential for inducing psychedelic effects. Behavioral assays further revealed improvements in stress-induced affective measures and hippocampus-independent cued learning at intermediate doses. These findings identify 2-halogenated tryptamines as polypharmacological serotonergic ligands with reduced psychoactivity and cardiac valve and toxic liabilities, supporting their potential as next-generation psychedelic-inspired therapeutics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/718915v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@17975a5org.highwire.dtl.DTLVardef@11ae1f1org.highwire.dtl.DTLVardef@1e7a00aorg.highwire.dtl.DTLVardef@1bbfcc8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Large-conductance calcium-activated potassium (BKCa) channels are ubiquitously expressed in mammalian cells and regulate electrical activity, intracellular calcium signaling, and cell survival. Although BKCa dysfunction has been linked to multiple diseases, the number of selective channel modulators is limited. In this study, we characterize dibenzoylmethane (DBM), a plant-derived compound isolated from Hottonia palustris, as a novel inhibitor of BKCa channel activity in both plasma membrane and mitochondrial BKCa. Electrophysiological recordings revealed that DBM lowers the open probability of BKCa channels in a concentration-dependent fashion and markedly reduces mean open time, leading to a pronounced flickering behavior - hallmarks of pore-targeted blockade. Competition experiments demonstrated that DBM antagonizes the effect of paxilline, a high-affinity pore-binding inhibitor, suggesting overlapping binding sites. Molecular dynamics simulations further supported this hypothesis, showing that several DBM molecules can block the pore by employing {pi}-{pi} interactions with each other and pore residues. On top of the pore, the carbonyl groups of DBM block the nearest potassium ion in the selectivity filter. The presence of DBM induces the removal of water molecules from the pore. To assess the structural requirements for activity, we tested three DBM analogs: phenyl-1,3-butanedione (PBD), trans-chalcone (T-Ch), and (E)-1,3-diphenylprop-2-en-1-ol (DPE). T-Ch and DPE inhibited BKCa channels with comparable efficacy to DBM, whereas PBD was significantly less potent. These results indicate that diphenyl substitution and structural rigidity are critical determinants of inhibitory activity. Our findings position DBM and its analogs as promising chemical scaffolds for the development of selective BKCa channel modulators with potential pharmacological applications.

Human Neutrophil Elastase (HNE) plays a vital role in several inflammatory diseases, however its role in the tumour microenvironment and the potential in cancer treatment is still unrevealed. Considering the potential of {beta}-lactams as HNE inhibitors, the present work describes the development of a synthetic strategy to obtain two different types (Type I and Type II) of quenched activity-based probes (qABPs), using a {beta}-lactam ring as a warhead and BODIPY-FL as a fluorophore. The two types differ in mechanism and relative position between the fluorophore and the quencher moiety. The qABPs synthesized presented IC50 values against HNE lower than 0.5 {micro}M, and high selectivity compared with homologous serine hydrolases. Type II qABPs showed a more efficient turn-on mechanism, and selectively targeted HNE in different cell lysates. The qABP 22 was internalized in U937 cells and in human neutrophils and successfully targeted HNE in both.

The rise of new, rapidly mutating viruses presents increasing challenges for drug developers. Traditional methods, such as high-throughput screening and drug repurposing against mutagenic viral targets, have recently shown their limitations. Our current rational molecular engineering approach offers a sustainable solution by targeting viral ion channels, which generally have low mutation rates. First, extending the amantadine molecule led to the development of new compounds that better match the alternating hydrophobic and hydrophilic patterns of the inner walls of ion channels--a common feature across many viruses. Then, simplifying the structure yielded a cyclohexylamine-based minimalist scaffold that effectively blocks the ion channel and demonstrates improved antiviral activity compared to well-known agents such as amantadine and arterolane. SARS-CoV-2 variants served as test systems in laboratory experiments. The new molecular scaffolds presented here provide a strong foundation for designing potent, broad-spectrum viral ion channel blockers.

Despite the development of vaccines and antivirals, coronavirus disease 2019 (COVID-19) continues to affect populations worldwide. Given the high mutation rate of the SARS-CoV-2 virus and reports of drug resistance, there is a continued need for new therapeutic options. SARS-CoV-2 main protease (Mpro) is essential for viral replication and is a conserved target among coronaviruses. Most known Mpro inhibitors target the active site, although allosteric sites have already been identified. In this study, we conducted a virtual screening of 2,060 compounds targeting an allosteric site of SARS-CoV-2 Mpro. From this screen, 41 computational hits and analogs were selected and evaluated using biochemical assays against SARS-CoV-2 Mpro. Among them, compound 25, a semicarbazone, demonstrated a half-maximal inhibitory concentration (IC50) of 99 M. Additionally, two thiosemicarbazone analogs (compounds 50 and 51) inhibited SARS-CoV-2 Mpro with IC50 values of 61 M and 70 M. Biochemical assays suggest that these compounds act as noncovalent competitive inhibitors of SARS-CoV-2 Mpro. Molecular dynamics simulations revealed that compound 25 is unstable at the allosteric site of SARS-CoV-2 Mpro but forms stable and favorable interactions at the active site, supporting its potential as a competitive inhibitor, a finding subsequently confirmed by biochemical assays. Our structure-based computational and biochemical approach identified semicarbazone and thiosemicarbazone scaffolds as promising candidates for the development of reversible SARS-CoV-2 Mpro inhibitors.

Fungal pathogens are an escalating global public health concern, particularly in the context of invasive and opportunistic infections. Cryptococcosis, primarily caused by Cryptococcus neoformans var. grubii, can manifest as acute, subacute, or chronic disease, affecting multiple organs and frequently leading to life-threatening meningitis in immunocompromised individuals. Given the limited antifungal therapeutic strategies and the emergence of resistance and toxicity-related constraints, the development of novel anti-cryptococcal agents remains an urgent priority. In this study, a library of innovative hybrids (5a-f) based on the 3-hydroxypyridin-4(1H)-one scaffold was developed. Their antimicrobial activity was evaluated towards a panel of clinically relevant Gram-positive (methicillin-resistant Staphylococcus aureus - MRSA) and Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii), as well as fungal species Candida albicans and Cryptococcus neoformans var. grubbi. Cytotoxicity was assessed in HEK293 and HepG2 cell lines, and haemolytic profile was determined to evaluate safety. In addition, iron-chelating capacity and lipophilic properties were also investigated. All compounds formed stable complexes with iron(III) and were non-toxic at concentrations up to 25 M. Lipophilicity studies showed that compounds in series 1 (5a-c) exhibited lower lipophilicity than those in Series 2 (5d-f), mainly due to the regioisomeric position of the hydroxyl group on the 2-methyl-4-pyridone scaffold; specifically, the C3-substitution pattern in Series 2 that enhances the hydrophobic character compared to the C5-substitution in Series 1. Fluorination further increased lipophilicity in both series. Notably, compounds 5c-5f emerged as potent, selective, and non-toxic antifungal agents against Cryptococcus neoformans var. grubii (MIC < 16 {micro}g/mL; CC50 > 32 {micro}g/mL; HC10 > 32 {micro}g/mL). Their distinct structural features appear to play a key role in antifungal selectivity, supporting the potential of these 3-hydroxypyridin-4(1H)-one-based hybrids as promising approach for the development of novel therapeutics for cryptococcal meningitis.

Aminopyridines, including 4-aminopyridine (4AP), 3,4-diaminopyridine, and [18F]3-fluoro-4-aminopyridine, are voltage-gated potassium (KV) channel blockers used clinically to enhance conduction in neurological disorders and to image demyelination by PET. Developing new aminopyridines may yield improved therapeutics or imaging agents. Here, we characterized the physicochemical properties (pKa, log D), KV channel-blocking activity, toxicity (LD50), and pharmacokinetics of a novel compound, 4-methyl-3-aminopyridine (4Me3AP). 4Me3AP was less basic and more lipophilic than 4AP and showed greater blocking potency across multiple KV channels expressed in Xenopus oocytes. In mice, 4Me3AP exhibited lower acute toxicity (LD50= 29.3 mg/kg) than 4AP (LD50= 12.7 mg/kg) and a longer plasma half-life. These findings indicate that 4Me3AP is a potent KV channel blocker with favorable pharmacological properties, supporting its potential for symptomatic treatment of demyelinating diseases.

Poly(ADP-ribose) polymerase 1 (PARP1) is a central mediator of DNA damage repair and an established therapeutic target in homologous recombination-deficient cancers. Radiolabelled PARP inhibitors provide a strategy to deliver cytotoxic radiation directly to tumour DNA by exploiting PARP overexpression and trapping at sites of DNA damage. Here, we describe the design, radiosynthesis, and in vitro evaluation of [123I]Italia, a talazoparib-derived Auger electron-emitting agent for PARP-targeted radionuclide therapy. Stereochemically pure [123I]Italia, (8S,9R)-5-fluoro-8-(4-(iodo-123I)phenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-2,7,8,9-tetrahydro-3H-pyrido[4,3,2-de]phthalazin-3-one was synthesised in one step via copper-mediated iodo-deboronation, achieving activity yields >80% and molar activities >6.2 {+/-} 3.1 GBq/{micro}mol (n=8). UPLC analysis confirmed radiochemical purity >97%. Italia exhibited potent PARP1 inhibition (IC50 0.48 nM) and in silico predicted binding affinity comparable to talazoparib. In a panel of PARP-expressing cancer cell lines, [123I]Italia demonstrated highest uptake at 60 min, PARP-selective uptake, predominant nuclear localisation (up to 60% of added activity) and chromatin association consistent with PARP trapping (up to 15% of total activity recorded). Uptake was reduced more than 50-fold by addition of an excess of any PARP inhibitor (e.g. olaparib, talazoparib, and rucaparib) and in PARP1 knockout cells, confirming target specificity. Clonogenic assays showed a marked, added activity-dependent reduction in survival of PARP-expressing cells following a brief one-hour exposure, whereas PARP1-deficient cells were resistant. Collectively, these findings identify [123I]Italia as a promising PARP-targeted Auger electron-emitting theranostic candidate that warrants further in vivo evaluation.

The chikungunya virus (CHIKV) outbreak imposes a significant burden on healthcare systems and raises an urgent need for effective antiviral therapies. So far there are no specific drugs against CHIKV. A CHIKV macrodomain is critical for virulence and counteracts the host immune response, representing a promising antiviral drug target. Here, we describe small molecule inhibitors targeting the CHIKV macrodomain. Compound 1 (MDOLL-0273) was identified through a high-throughput screening using a fluorescence resonance energy transfer (FRET)-based assay, and its inhibitory activity was validated through multiple orthogonal assays. Compound 1 has a dual thiobarbiturate-indole scaffold and exhibits an IC50 of 8.9 {micro}M. X-ray crystallography revealed that the inhibitor occupies an adenine binding site of the macrodomain and extends into a novel cryptic pocket. Notably, the inhibitor shows high selectivity for the CHIKV macrodomain over a panel of human and viral ADP-ribosyl binding and hydrolyzing proteins. Structure-activity relationship studies and medicinal chemistry efforts provide a promising starting point for further hit optimization.

Pathological neovascularization in the eye is a significant contributor to vision loss in diseases such as age-related macular degeneration (AMD) and diabetic retinopathy. While anti-VEGF biologics are effective, they require repeated intravitreal injections and carry procedural risks. Here, we report a novel principle for designing photoactivatable VEGFR2 inhibitors, along with two examples, EYE1090 and EYE1118, engineered from the sunitinib and vorolanib scaffolds, respectively. Azido-functionalization in these molecules enables light-triggered receptor binding while preserving potent inhibition in the dark. Both compounds exhibit significantly enhanced activity upon exposure to green light capable of reaching the retina, also in the elderly. In vitro, the compounds robustly inhibited angiogenesis and endothelial migration that was further potentiated by light. In vivo, orally administered EYE1090 and EYE1118 suppressed VEGF-induced retinal leakage and reduced lesion size in a mouse model of choroidal neovascularization at doses tenfold lower than parental compounds. Photoactivation also influenced compound biodistribution, suggesting light-guided targeting. Acute toxicity studies revealed no hepatotoxicity. This strategy exploits the natural light-focusing anatomy of the eye to locally activate systemically administered drugs, thereby reducing therapeutic doses and systemic exposure. Our findings introduce a light-targeted pharmacological approach for treating retinal diseases using photoactivable kinase inhibitors.

TEA domain (TEAD) proteins bind co-activator Yes-associated protein (YAP) to regulate the expression of target genes of the Hippo pathway. The TEAD*YAP protein-protein interaction is not druggable, but TEADs possess a unique and deep palmitate pocket with a highly conserved cysteine located outside the TEAD*YAP protein-protein interaction interface. Here, we screen a fragment library of acrylamide electrophiles and identify a fragment that forms an adduct with the conserved palmitate pocket cysteine and inhibits TEAD4 binding to YAP. Synthesis of a focused set of derivatives and time- and concentration-dependent studies with four TEADs provide reaction rates and binding constants. Co-crystal structures of fragments bound to TEAD2 and TEAD3 reveal reaction at the conserved palmitate pocket cysteine but also at another less conserved cysteine located in the palmitate pocket of TEAD2 closer to the TEAD*YAP interface. These fragments provide a starting point for the development of allosteric acrylamide small-molecule covalent TEAD*YAP inhibitors.

Arylmalonate decarboxylase (AMDase) stereoselectively converts disubstituted malonates to chiral carboxylic acids, but its substrate spectrum is very limited regarding the size of the smaller substituent. Inspired by the observation that (S)-selective AMDase variants also convert larger substrates, we unlocked the synthesis of the (R)-enantiomers of -aryl and -alkenyl n-butanoic and n-pentanoic acids, respectively, in exquisite enantiopurity.

The glucose-dependent insulinotropic polypeptide receptor (GIPR) is an attractive therapeutic target for metabolic disorders, with GIPR antagonism emerging as a promising strategy for obesity and type 2 diabetes. However, developing functional antibodies against GPCRs remains challenging due to their complex architecture and conformational dynamics. Here, we employed AlfaBodY, an iterative active learning platform integrating structural and sequence information, to in silico design human anti-GIPR antibodies. Through four rounds of optimization, we generated antibodies with high binding affinities. Lead candidates AB106-131 (KD 1.2 nM) and AB106-156 (KD 1.7 nM) exhibited 7 to 10-fold higher affinity than 2G10 (KD 12 nM) while maintaining comparable antagonistic activity in a cAMP reporter assay (IC50 4[~]5 nM). In diet-induced obese mice, AB106-156 alone induced weight loss comparable to that of semaglutide ([~] -15%), while preserving lean mass and achieving sustained weight control after treatment withdrawal. Co-administration with the GLP-1 receptor agonist semaglutide produced synergistic weight reduction (-25.4%) and markedly attenuated the fat-mass rebound observed with semaglutide alone. Our results demonstrate that AI-driven design can generate potent anti-GIPR antibodies with favourable in vivo efficacy, supporting further development of GIPR antagonist for obesity and related metabolic disorders. The AlfaBodY platform enables faster development of more efficacious biologic drugs.