Ternary Complex Research Articles

Co(II), Ni(II), and Cu(II) Ternary Complexes With 2,6‐Pyridinedicarboxylic Acid and 2,2′‐Bipyridyl: Synthesis, Characterization, DNA Binding, Photo‐Cleavage, and Antimicrobial Studies

ABSTRACTThe interaction of 2,6‐pyridinedicarboxylicacid (PDA) and 2,2′‐bipyridyl (Bipy) with Co(II), Ni(II), and Cu(II) ions produced new ternary complexes [M (PDA)(Bipy)(H2O)] (M = Co(II), Ni(II), and Cu(II)), which were studied by elemental, spectral (FT‐IR, Mass, UV–Vis), and thermal analysis, and their interaction with Ct‐DNA was thoroughly studied using various methods, including absorption spectroscopy, fluorescence spectroscopy, and viscosity measurements. Spectrophotometric titration and viscosity measurements indicated that all the complexes bind to DNA via the groove mode of binding. The salt‐dependent binding of ternary metal complexes to Ct‐DNA has been studied by a UV–Vis spectrophotometric titration experiment. Furthermore, photocleavage studies were used to examine how the metal complexes interacted with the pBR322 DNA, and the results proved that these compounds have obvious photo cleavage properties. The biological effects were studied by antibacterial activity.

Design of Murine Double Minute 2 Proteolysis Targeting Chimera Degraders with a Built-In Tumor-Targeting Ability.

Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules to induce the proteasomal degradation of target proteins. Currently, there are no tumor-targeting PROTACs for modulating oncogenic murine double minute 2 (MDM2). AS1411 is a tumor-targeting aptamer that specifically recognizes nucleolin (NCL) overexpressed on the surface of tumor cells. We recently repurposed AS1411 as an MDM2 recruiter since it could form an NCL-bridged ternary complex with MDM2. In this study, we design a PROTAC molecule AS1411-VH032 via conjugating AS1411 with a recruiter of von Hippel-Lindau (VHL) ligase VH032. AS1411-VH032 facilitates tumor-selective degradation of MDM2, leading to tumor shrinkage with no detectable toxicity. Besides being a molecular target, MDM2 also serves as an E3 ligase harnessed by PROTACs. Thus, we developed an AS1411-based homo-PROTAC homoAS1411, which induces tumor-specific suicide degradation of MDM2 and prevents tumor progression without causing side effects. Both AS1411-VH032 and homoAS1411 are promising MDM2 degraders with built-in tumor-targeting ability, which balances the antitumor efficacy with a favorable safety profile.

Speciation studies of binary and ternary complexes formed with fe(II) ion 2,6-pyridinedicarboxylic acid and several amino acids

The chemical speciation of ternary systems involving Fe(II), 2,6-pyridinedicarboxylic acid (H2Dipi), and various amino acids (glycine, β-alanine, α-alanine, leucine, methionine, lysine, phenylalanine, threonine, serine, proline, valine, and histidine) was investigated in this work. Potentiometric titrations were conducted for each system in aqueous solution at 25 °C using 1 mol dm−3 NaCl solution as the ionic medium. The resulting data were analyzed using the least-squares program LETAGROP. Additionally, the binary system of Fe(II)-2,6-pyridinedicarboxylic acid in aqueous solution at 25 °C with NaCl 1.0 mol dm−3 as the ionic medium was also examined. Species distribution diagrams were generated, and the relative stability parameter Δlog K was calculated.

Structures of a DNA Polymerase Caught while Incorporating Responsive Dual-Functional Nucleotide Probes.

Functionalizing nucleic acids using DNA polymerases is essential in biophysical and biotechnology applications. This study focuses on understanding how DNA polymerases recognize and incorporate nucleotides with diverse chemical modifications, aiming to develop advanced nucleotide probes. We present the crystal structures of ternary complexes of Thermus aquaticus DNA polymerase (KlenTaq) with C5-heterocycle-modified environment-sensitive 2'-deoxyuridine-5'-triphosphate (dUTP) probes. These nucleotides include SedUTP, BFdUTP and FBFdUTP, which bear selenophene, benzofuran and fluorobenzofuran, respectively, at the C5 position of uracil, and exhibit high conformational sensitivity. SedUTP and FBFdUTP serve as dual-app probes, combining a fluorophore with X-ray anomalous scattering Se or 19F NMR labels. Our study reveals that the size of the heterocycle influences how DNA polymerase families A and B incorporate these modified nucleotides during single nucleotide incorporation and primer extension reactions. Remarkably, FBFdUTP's responsiveness enabled real-time monitoring of the binary complex formation and polymerase activity through fluorescence and 19F NMR. Comparative analysis of incorporation profiles, fluorescence, 19F NMR data, and crystal structures of ternary complexes highlights the enzyme's plasticity. Key insights are provided into the role of gatekeeper amino acids (Arg660 and Arg587) in accommodating and processing these modified substrates, offering a structural basis for next-generation nucleotide probe development.

Structures of a DNA Polymerase Caught while Incorporating Responsive Dual‐Functional Nucleotide Probes

Functionalizing nucleic acids using DNA polymerases is essential in biophysical and biotechnology applications. This study focuses on understanding how DNA polymerases recognize and incorporate nucleotides with diverse chemical modifications, aiming to develop advanced nucleotide probes. We present the crystal structures of ternary complexes of Thermus aquaticus DNA polymerase (KlenTaq) with C5‐heterocycle‐modified environment‐sensitive 2′‐deoxyuridine‐5′‐triphosphate (dUTP) probes. These nucleotides include SedUTP, BFdUTP and FBFdUTP, which bear selenophene, benzofuran and fluorobenzofuran, respectively, at the C5 position of uracil, and exhibit high conformational sensitivity. SedUTP and FBFdUTP serve as dual‐app probes, combining a fluorophore with X‐ray anomalous scattering Se or 19F NMR labels. Our study reveals that the size of the heterocycle influences how DNA polymerase families A and B incorporate these modified nucleotides during single nucleotide incorporation and primer extension reactions. Remarkably, FBFdUTP's responsiveness enabled real‐time monitoring of the binary complex formation and polymerase activity through fluorescence and 19F NMR. Comparative analysis of incorporation profiles, fluorescence, 19F NMR data, and crystal structures of ternary complexes highlights the enzyme's plasticity. Key insights are provided into the role of gatekeeper amino acids (Arg660 and Arg587) in accommodating and processing these modified substrates, offering a structural basis for next‐generation nucleotide probe development.

Leveraging Covalency to Stabilize Ternary Complex Formation For Cell-Cell "Induced Proximity".

Recent advances in the field of translational chemical biology use diverse "proximity-inducing" synthetic modalities to elicit new modes of "event driven" pharmacology. These include mechanisms of targeted protein degradation and immune clearance of pathogenic cells. Heterobifunctional "chimeric" compounds like Proteolysis TArgeting Chimeras (PROTACs) and Antibody Recruiting Molecules (ARMs) leverage these mechanisms, respectively. Both systems function through the formation of reversible "ternary" or higher-order biomolecular complexes. Critical to function are key parameters, such as bifunctional molecule affinity for endogenous proteins, target residence time, and turnover. To probe the mechanism and enhance function, covalent chemical approaches have been developed to kinetically stabilize ternary complexes. These include electrophilic PROTACs and Covalent Immune Recruiters (CIRs), the latter designed to uniquely enforce cell-cell induced proximity. Inducing cell-cell proximity is associated with key challenges arising from a combination of steric and/or mechanical based destabilizing forces on the ternary complex. These factors can attenuate the formation of ternary complexes driven by high affinity bifunctional/proximity inducing molecules. This Account describes initial efforts in our lab to address these challenges using the CIR strategy in antibody recruitment or receptor engineered T cell model systems of cell-cell induced proximity. ARMs form ternary complexes with serum antibodies and surface protein antigens on tumor cells that subsequently engage immune cells via Fc receptors. Binding and clustering of Fc receptors trigger immune cell killing of the tumor cell. We applied the CIR strategy to convert ARMs to covalent chimeras, which "irreversibly" recruit serum antibodies to tumor cells. These covalent chimeras leverage electrophile preorganization and kinetic effective molarity to achieve fast and selective covalent engagement of the target ternary complex protein, e.g., serum antibody. Importantly, covalent engagement can proceed via diverse binding site amino acids beyond cysteine. Covalent chimeras demonstrated striking functional enhancements compared to noncovalent ARM analogs in functional immune assays. We revealed this enhancement was in fact due to the increased kinetic stability and not concentration, of ternary complexes. This finding was recapitulated using analogous CIR modalities that integrate peptidic or carbohydrate binding ligands with Sulfur(VI) Fluoride Exchange (SuFEx) electrophiles to induce cell-cell proximity. Mechanistic studies in a distinct model system that uses T cells engineered with receptors that recognize covalent chimeras or ARMs, revealed covalent receptor engagement uniquely enforces downstream activation signaling. Finally, this Account discusses potential challenges and future directions for adapting and optimizing covalent chimeric/bifunctional molecules for diverse applications in cell-cell induced proximity.

Ternary π-π Stacking Complexes by Allosteric Regulation in Multilayer Nanographenes.

Construction of π-π stacking supramolecular complexes with more than two different components is challenging due to the weak and directionless nature of dispersion interactions. Here, we report ternary complexes of a ditopic nanographene tetraimide (1), α-substituted phthalocyanine (Pc), and polyaromatic hydrocarbons (PAHs) in solution and the crystalline state via allosteric regulation. Binding of one Pc gives rise to significant distortion and conformational changes in 1 that in turn lead to the inhibition of the second binding of Pc. The conformational changes associated with first binding allowed an allosteric binding of a third component (PAHs) to form ternary complexes in solution. 1H NMR titration revealed moderately high thermodynamic stability for the ternary complexes in CDCl3. Competition between allosterically regulated ternary complexes ([Pc·1·PAH]) and 1:2 stoichiometric binary complexes of 1 with PAHs ([PAH·1·PAH]) was elucidated. Further, the selective formation of ternary complexes in solution led to the generation of ternary cocrystals from a 1:1:1 mixture of three components in solution. Our work shows that large π-conjugated nanographenes designed with allosteric recognition sites allow the construction of multilayer ternary complexes in solution and the solid state even with dispersive π-π interactions.

Targeting oncogenic transcriptional factor c-myc by oligonucleotide PROTAC for the treatment of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death, but effective therapeutic strategies are limited. Transcriptional factor c-Myc plays an oncogenic role in tumorigenesis and is an attractive target for HCC treatment. However, targeted therapy against c-Myc remains challenging. Herein, by conjugating VH032 with an optimized DNA sequence that recognized c-Myc complex, we discovered oligonucleotide-based proteolysis targeting chimeras (PROTACs), termed as MP-16 and MP-17, which effectively induced degradation of c-Myc. Mechanically, MP-16 or MP-17 directly interacted with c-Myc complex to form VHL/PROTAC/c-Myc ternary complex, and triggered c-Myc degradation by recruiting ubiquitin-proteasome system, suppressing cell proliferation of HCC cells. In mice model, MP-16 or MP-17 significantly inhibited HCC tumor growth and exhibited promising drug safety. This work provided novel oligonucleotide PROTACs that degraded c-Myc, giving a new lead structure for HCC therapy.

Ultra-High Adsorption Capacity of Calcium-Iron Layered Double Hydroxides for HEDP Removal through Phase Transition Processes.

Antiscalant disposal in reverse osmosis concentrate (ROC) treatment is a significant obstacle in desalination. This study investigated the adsorption performance of LDHs for removing 1-hydroxyethylidene-1,1-diphosphonic acid (HEDP). CaFe-LDH presented a specific adsorption behavior and ultrahigh adsorption capacity for HEDP, with a maximum adsorption capacity of 335.7 mg P/g (1116.5 mg HEDP/g) at pH 7.0. X-ray diffraction (XRD) demonstrated that HEDP adsorption induced a structural transformation of CaFe-LDH from a layered configuration to a highly ordered structure, leading to a noticeable phase transition. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) and Raman spectroscopy further confirmed that two distinct binding modes of HEDP, relating to chelation with Ca2+ and adsorption on Fe3+ simultaneously, are connected by phosphonic acid groups (-C-PO(OH)2), forming the CaFe-HEDP complex. X-ray fluorescence (XRF) and X-ray photoelectron spectroscopy (XPS) analyses revealed that the CaFe-HEDP ternary complex exhibits a highly ordered arrangement in an oxygen-bridged framework. The construction of an oxygen-coordinated framework contributes to the incorporation of more HEDP into CaFe-LDH, leading to a well-aligned lattice in the new phase. These findings provide valuable insights into developing novel LDH-based adsorbents for removing phosphorus-containing antiscalants, establishing a sustainable approach to ROC management, and potential environmental risk reduction.

The Second Methylation in Psilocybin Biosynthesis Is Enabled by a Hydrogen Bonding Network Extending into the Secondary Sphere Surrounding the Methyltransferase Active Site.

The Psilocybe cubensis SAM-dependent methyltransferase, PsiM, catalyzes the last step in the biosynthesis of psilocybin. Likely evolved from monomethylating RNA methyltransferases, PsiM acquired a key amino acid exchange in the secondary sphere of the active site, M247N, which is responsible for its capacity to dimethylate. Two variants, PsiMN247M and PsiMN247A, were generated to further examine the role of Asn247 for mono- and dimethylation in PsiM. Herein, we present the kinetic profiles of both variants and crystal structures at resolutions between 0.9 and 1.0 Å. Each variant was crystallized as a ternary complex with the non-methylated acceptor substrate, norbaeocystin and S-adenosyl-l-homocysteine, and in a second complex with the cofactor analog, sinefungin, and the monomethylated substrate, baeocystin. Consistent with the inability of the variants to catalyze a second methyl transfer, these structures reveal catalytically non-productive conformations and a high level of disorder of the methylamine group of baeocystin. Additionally, both variants exhibit destabilization in the β5-β7 sheets and a conserved β-turn of the core Rossmann fold, causing 20-fold reduced substrate binding and 2-fold lower catalytic efficiency even with norbaeocystin.

Design of a Cereblon construct for crystallographic and biophysical studies of protein degraders

The ubiquitin E3 ligase cereblon (CRBN) is the target of therapeutic drugs thalidomide and lenalidomide and is recruited by most targeted protein degraders (PROTACs and molecular glues) in clinical development. Biophysical and structural investigation of CRBN has been limited by current constructs that either require co-expression with the adaptor DDB1 or inadequately represent full-length protein, with high-resolution structures of degrader ternary complexes remaining rare. We present the design of CRBNmidi, a construct that readily expresses from E. coli with high yields as soluble, stable protein without DDB1. We benchmark CRBNmidi for wild-type functionality through a suite of biophysical techniques and solve high-resolution co-crystal structures of its binary and ternary complexes with degraders. We qualify CRBNmidi as an enabling tool to accelerate structure-based discovery of the next generation of CRBN based therapeutics.

Emerging Role of Natural Topoisomerase Inhibitors as Anticancer agents

Abstract: Topoisomerases I and II are the functionally two forms of DNA topoisomerase. In anticancer research, novel anticancer chemotherapeutical capable of blocking topoisomerase enzymes have been discovered. Most commonly, topoisomerase causes replication fork arrest and doublestrand breaks, and this is how a clinically successful topoisomerase-targeting anticancer medicines work. Unfortunately, this novel mechanism of action has been linked to the development of secondary malignancies as well as cardiotoxicity. The specific binding locations and mechanisms of topoisomerase poisons have been identified by studying the structures of topoisomerase-drug-DNA ternary complexes. Recent breakthroughs in science have revealed that isoform-specific human topoisomerase II poison could be created as safer anticancer drug molecules. It may also be able to develop catalytic inhibitors of topoisomerases by focusing on their inactive conformations. In addition to this, the discovery of new bacterial topoisomerase inhibitor molecules and regulatory proteins could lead to the discovery of new human topoisomerase inhibitors. As a result, biologists, organic chemists, and medicinal chemists worldwide have been identifying, designing, synthesizing, and testing a variety of novel topoisomerase-targeting bioactive compounds. This review focused on topoisomerase inhibitors, their mechanisms of action, and different types of topoisomerase inhibitors that have been developed during the last ten years.

Principles of paralog-specific targeted protein degradation engaging the C-degron E3 KLHDC2

PROTAC® (proteolysis-targeting chimera) molecules induce proximity between an E3 ligase and protein-of-interest (POI) to target the POI for ubiquitin-mediated degradation. Cooperative E3-PROTAC-POI complexes have potential to achieve neo-substrate selectivity beyond that established by POI binding to the ligand alone. Here, we extend the collection of ubiquitin ligases employable for cooperative ternary complex formation to include the C-degron E3 KLHDC2. Ligands were identified that engage the C-degron binding site in KLHDC2, subjected to structure-based improvement, and linked to JQ1 for BET-family neo-substrate recruitment. Consideration of the exit vector emanating from the ligand engaged in KLHDC2’s U-shaped degron-binding pocket enabled generation of SJ46421, which drives formation of a remarkably cooperative, paralog-selective ternary complex with BRD3BD2. Meanwhile, screening pro-drug variants enabled surmounting cell permeability limitations imposed by acidic moieties resembling the KLHDC2-binding C-degron. Selectivity for BRD3 compared to other BET-family members is further manifested in ubiquitylation in vitro, and prodrug version SJ46420-mediated degradation in cells. Selectivity is also achieved for the ubiquitin ligase, overcoming E3 auto-inhibition to engage KLHDC2, but not the related KLHDC1, KLHDC3, or KLHDC10 E3s. In sum, our study establishes neo-substrate-specific targeted protein degradation via KLHDC2, and provides a framework for developing selective PROTAC protein degraders employing C-degron E3 ligases.

Mechanism of degrader-targeted protein ubiquitinability.

Small-molecule degraders of disease-driving proteins offer a clinically proven modality with enhanced therapeutic efficacy and potential to tackle previously undrugged targets. Stable and long-lived degrader-mediated ternary complexes drive fast and profound target degradation; however, the mechanisms by which they affect target ubiquitination remain elusive. Here, we show cryo-EM structures of the VHL Cullin 2 RING E3 ligase with the degrader MZ1 directing target protein Brd4BD2 toward UBE2R1-ubiquitin, and Lys456 at optimal positioning for nucleophilic attack. In vitro ubiquitination and mass spectrometry illuminate a patch of favorably ubiquitinable lysines on one face of Brd4BD2, with cellular degradation and ubiquitinomics confirming the importance of Lys456 and nearby Lys368/Lys445, identifying the "ubiquitination zone." Our results demonstrate the proficiency of MZ1 in positioning the substrate for catalysis, the favorability of Brd4BD2 for ubiquitination by UBE2R1, and the flexibility of CRL2 for capturing suboptimal lysines. We propose a model for ubiquitinability of degrader-recruited targets, providing a mechanistic blueprint for further rational drug design.

Structure-Activity Relationship of Potent, Selective, and Orally Bioavailable Molecular Glue Degraders of CK1α.

The original molecular glue degraders (thalidomide, lenalidomide, and pomalidomide) are known to bind to cereblon (CRBN) and alter its surface to induce recruitment, ubiquitination, and degradation of therapeutically valuable neosubstrates (IKZF1, IKZF3, and CK1α). With the aim of understanding and modulating neosubstrate specificity, we recently reported the discovery of SJ3149 (4), a selective and potent molecular glue degrader of CK1α, that is active in multiple cancer cell lines. Herein, we describe the medicinal chemistry efforts that resulted in the discovery of SJ3149 as well as other potent and selective CK1α degraders. We report kinetic profiling and parameters of CK1α degradation, ternary complex, antiproliferative effects, in vitro ADME data, and in vivo pharmacokinetic studies with demonstrated oral bioavailability.

Preparation of Antimicrobial Agents: From Interpolyelectrolyte Complexes to Silver-Containing Metal–Polymer Complexes and Nanocomposites

In order to control pathogenic microorganisms, three polymer compositions were prepared and tested. First, a water-soluble positively charged polycomplex was synthesized via the electrostatic binding of anionic polyacrylic acid to an excess of polyethylenimine to enhance the biocidal activity of the polycation. Second, an aqueous solution of AgNO3 was added to the polycomplex, thus forming a ternary polycation-polyanion-Ag1+ complex with an additional antimicrobial effect. Third, the resulting ternary complex was subjected to UV irradiation, which ensured the conversion of Ag1+ ions into Ag nanoparticles ranging in size mainly from 10 to 20 nm. Aqueous solutions of the polymer compositions were added to suspensions of the Gram-positive bacteria S. aureus and the Gram-negative bacteria P. aeruginosa, with the following main results: (a) Upon the addition of the binary polycomplex, 30% or more of the cells survived after 20 h. (b) The ternary complex killed S. aureus bacteria but was ineffective against P. aeruginosa bacteria. (c) When the ternary complex with Ag nanoparticles was added, the percentage of surviving cells of both types did not exceed 0.03%. The obtained results are valuable for the development of antibacterial formulations.

Disentangling the Complexity in Protein Complexes Using Complementary Isotope-Labeling and Multiple-Receiver NMR Spectroscopy.

Intrinsically disordered proteins are abundant in eukaryotic systems, but they remain largely elusive pharmacological targets. NMR spectroscopy proved to be a suitable method to study these proteins and their interaction with one another or with drug candidates. Although NMR can give atomistic information about these interplays, molecular complexity due to severe spectral overlap, limited sample stability, and quantity remain an issue and hamper widespread applications. Here, we propose an approach to simultaneously map protein-protein binding sites onto two interacting partners by employing a complementary isotope-labeling strategy and a multiple receiver NMR detection scheme. With one partner being 15N,2H labeled and the interacting one being 13C,1H-labeled, we exploited proton and carbon detection to obtain clean and easily readable information. The method is illustrated with an application to the 50 kDa ternary protein complex formed between the prominent oncogenic transcription factor complex Myc/MAX and the tumor suppressor BRCA1.

GCRV-encoded circRNA circ_20 forms a ternary complex with BIP and PERK to delay virus replication by inhibiting the PERK-eIF2α pathway

Viral circRNAs play important roles in host-virus interactions. Previous reports showed that grass carp reovirus (GCRV) encodes 32 circRNAs, and circ_20 from the negative strand of GCRV genome segment 7 has the potential to regulate GCRV replication. However, the regulatory mechanism of circ_20 on GCRV remains unknown. In this study, circ_20 was further validated, and circ_20 negatively regulated ERS, the PERK pathway, and ROS production in GCRV-infected cells. Furthermore, circ_20 inhibited the PERK pathway by forming a ternary complex with BIP and PERK, resulting in delaying GCRV replication. RNA pull-down results indicated that the 51–102 nt region of circ_20 interacts with BIP, while the 451–502 and 514–565 nt regions interact with PERK. After the deletion of these interaction regions, the ability of circ_20 to promote BIP-PERK interaction decreases, leading to a decrease in the ability to inhibit GCRV proliferation. These findings uncovered new insights into the complex interplay between viruses and host cells and provided a novel understanding of the significance of viral circRNAs in virus-host interactions.

Molecular Basis of the Recognition of the Active Rab8a by Optineurin

Optineurin (OPTN), a multifunctional adaptor protein in mammals, plays critical roles in many cellular processes, such as vesicular trafficking and autophagy. Notably, mutations in optineurin are directly associated with many human diseases, such as amyotrophic lateral sclerosis (ALS). OPTN can specifically recognize Rab8a and the GTPase-activating protein TBC1D17, and facilitate the inactivation of Rab8a mediated by TBC1D17, but with poorly understood mechanism. Here, using biochemical and structural approaches, we systematically characterize the interaction between OPTN and Rab8a, revealing that OPTN selectively recognizes the GTP-bound active Rab8a through its leucine-zipper domain (LZD). The determined crystal structure of OPTN LZD in complex with the active Rab8a not only elucidates the detailed binding mechanism of OPTN with Rab8a but also uncovers a unique binding mode of Rab8a with its effectors. Furthermore, we demonstrate that the central coiled-coil domain of OPTN and the active Rab8a can simultaneously interact with the TBC domain of TBC1D17 to form a ternary complex. Finally, based on the OPTN LZD/Rab8a complex structure and relevant biochemical analyses, we also evaluate several known ALS-associated mutations found in the LZD of OPTN. Collectively, our findings provide mechanistic insights into the interaction of OPTN with Rab8a, expanding our understanding of the binding modes of Rab8a with its effectors and the potential etiology of diseases caused by OPTN mutations.

8015 Growth Hormone Axis Dysfunction in PMM2-CDG: Exploring the Role of Dynamic Tests for the Management of Short Stature in Two Pediatric Cases

Abstract Disclosure: G. Del Medico: None. E. Procopio: None. L. Ferri: None. A. Barbato: None. A. Morrone: None. S. Stagi: None. PMM2-CDG is the most prevalent congenital disorder of glycosylation (CDG) and is caused by defective phosphomannomutase2 (PMM2) activity. According to the 2019 guidelines, approximately half of PMM2-CDG patients exhibit short stature and low serum levels of insulin-like growth factor-1 (IGF-1), IGF binding protein 3 (IGFBP3), and acid-labile subunit (ALS). Despite this, there is no expert consensus on the endocrinological management of this condition. We investigated the growth hormone (GH) axis in two 12-year-old female patients with PMM2-CDG. The first patient was compound heterozygote for the most common PMM2 gene variants in Italy, p.(Leu32Arg) and p.(Arg141His). She presented with short stature (< 2 Standard Deviation Score, SDS) and low serum levels of IGF-1 and IGF-BP3 (< 2.5° for age and sex). Bone age was significantly delayed. Tanner stage was prepubertal. Somatotropic response to GH stimulation testing (Levodopa) was normal. After the IGF-1 generation test with recombinant GH, serum IGF-1 increased by 115%. The second patient was compound heterozygote for the known PMM2 pathogenetic variant p.(Ala108Val) and the novel p.(Thr171Asnfs*11) variant (only case described in literature). She presented short stature (< 2 SDS) with near-to-normal IGF-1 and IGF-BP3 serum levels (2.5° for age and sex). Bone age was slightly delayed. Tanner stage was prepubertal. She also exhibited severe obesity, hypertriglyceridemia, and hepatic steatosis. Her somatotropic response to two GH stimulation tests (Levodopa and Arginine) was deficient, probably because of her significant overweight. IGF-1 serum levels didn’t increase after the IGF-1 generation test. Hypoglycosylation in PMM2-CDG impairs the IGF1 system on multiple levels. It destabilizes the ternary complex formed by IGF-1 and glycoproteins IGFBP3 and ALS, therefore reducing the half-life of circulating IGF-1. Hypoglycosylation also reduces IGF1 production by impairing its precursor proIGF-1Ea and its cellular secretion. As a result, PMM2-CDG patients have GH insensitivity, with normal GH production but low levels of IGF-1. However, there is a lack of information in the literature on the use of dynamic tests and hormonal therapies (GH and rhIGF-1) in CDG. The IGF-1 generation test in our patients showed partial GH sensitivity in the first patient and GH insensitivity in the second patient. These observations suggest variability. Some PMM2-CDG patients may benefit from an extended trial with GH therapy, while for GH-insensitive patients a trial with recombinant IGF-1 might be considered. This is one of the first reports that explores the potential role of dynamic tests in PMM2-CDG. As the biochemical effects of hypoglycosylation on the GH-IGF1 pathway are becoming clearer more clinical research is needed to reach a consensus on the management and treatment of short stature in these patients. Presentation: 6/3/2024