N-terminal Residues Research Articles

BIG enhances Arg/N-degron pathway-mediated protein degradation to regulate Arabidopsis hypoxia responses and suberin deposition.

BIG/DARK OVEREXPRESSION OF CAB1/TRANSPORT INHIBITOR RESPONSE3 is a 0.5 MDa protein associated with multiple functions in Arabidopsis (Arabidopsis thaliana) signaling and development. However, the biochemical functions of BIG are unknown. We investigated a role for BIG in the Arg/N-degron pathways, in which substrate protein fate is influenced by the N-terminal residue. We crossed a big loss-of-function allele to 2 N-degron pathway E3 ligase mutants, proteolysis6 (prt6) and prt1, and examined the stability of protein substrates. Stability of model substrates was enhanced in prt6-1 big-2 and prt1-1 big-2 relative to the respective single mutants, and the abundance of the PRT6 physiological substrates, HYPOXIA-RESPONSIVE ERF2 (HRE2) and VERNALIZATION2 (VRN2), was similarly increased in prt6 big double mutants. Hypoxia marker expression was enhanced in prt6 big double mutants; this constitutive response required arginyl transferase activity and RAP-type Group VII ethylene response factor (ERFVII) transcription factors. Transcriptomic analysis of roots not only demonstrated increased expression of multiple hypoxia-responsive genes in the double mutant relative to prt6, but also revealed other roles for PRT6 and BIG, including regulation of suberin deposition through both ERFVII-dependent and independent mechanisms, respectively. Our results show that BIG acts together with PRT6 to regulate the hypoxia-response and broader processes in Arabidopsis.

Initial Steps in Methanobactin Biosynthesis: Substrate Binding by the Mixed-Valent Diiron Enzyme MbnBC.

The MbnBC enzyme complex converts cysteine residues in a peptide substrate, MbnA, to oxazolone/thioamide groups during the biosynthesis of copper chelator methanobactin (Mbn). MbnBC belongs to the mixed-valent diiron oxygenase (MVDO) family, of which members use an Fe(II)Fe(III) cofactor to react with dioxygen for substrate modification. Several crystal structures of the inactive Fe(III)Fe(III) form of MbnBC alone and in complex with MbnA have been reported, but a mechanistic understanding requires determination of the oxidation states of the crystallographically observed Fe ions in the catalytically active Fe(II)Fe(III) state, along with the site of MbnA binding. Here, we have used electron nuclear double resonance (ENDOR) spectroscopy to determine such structural and electronic properties of the active site, in particular, the mode of substrate binding to the MV state, information not accessible by X-ray crystallography alone. The oxidation states of the two Fe ions were determined by 15N ENDOR analysis. The presence and locations of both bridging and terminal exogenous solvent ligands were determined using 1H and 2H ENDOR. In addition, 2H ENDOR using an isotopically labeled MbnA substrate indicates that MbnA binds to the Fe(III) ion of the cluster via the sulfur atom of its N-terminal modifiable cysteine residue, with displacement of a coordinated solvent ligand as shown by complementary 1H ENDOR. These results, which underscore the utility of ENDOR in studying MVDOs, provide a molecular picture of the initial steps in Mbn biosynthesis.

Pharmacological Characterization of SDX-7320/Evexomostat: A Novel Methionine Aminopeptidase Type 2 Inhibitor with Anti-tumor and Anti-metastatic Activity.

Methionine aminopeptidase type 2 (METAP2) is a ubiquitous, evolutionarily conserved metalloprotease fundamental to protein biosynthesis which catalyzes removal of the N-terminal methionine residue from nascent polypeptides. METAP2 is an attractive target for cancer therapeutics based upon its over-expression in multiple human cancers, the importance of METAP2-specific substrates whose biological activity may be altered following METAP2 inhibition, and additionally, that METAP2 was identified as the target for the anti-angiogenic natural product, fumagillin. Irreversible inhibition of METAP2 using fumagillin analogues has established the anti-angiogenic and anti-tumor characteristics of these derivatives; however, their full clinical potential has not been realized due to a combination of poor drug-like properties and dose-limiting central nervous system (CNS) toxicity. This report describes the physicochemical and pharmacological characterization of SDX-7320 (evexomostat), a polymer-drug conjugate of the novel METAP2 inhibitor (METAP2i) SDX-7539. In vitro binding, enzyme, and cell-based assays demonstrated that SDX-7539 is a potent and selective METAP2 inhibitor. In utilizing a high molecular weight, water-soluble polymer to conjugate the novel fumagillol-derived, cathepsin-released, METAP2i SDX-7539, limitations observed with prior generation, small molecule fumagillol derivatives were ameliorated including reduced CNS exposure of the METAP2i, and prolonged half-life enabling convenient administration. Multiple xenograft and syngeneic cancer models were utilized to demonstrate the anti-tumor and anti-metastatic profile of SDX-7320. Unlike polymer-drug conjugates in general, reductions in small molecule-equivalent efficacious doses following polymer conjugation were observed. SDX-7320 has completed a phase I clinical safety study in patients with late-stage cancer and is currently being evaluated in multiple phase Ib/II clinical studies in patients with advanced solid tumors.

Biocontrol Potential of Sodin 5, Type 1 Ribosome-Inactivating Protein from Salsola soda L. Seeds.

Sodin 5 is a type 1 ribosome-inactivating protein isolated from the seeds of Salsola soda L., an edible halophytic plant that is widespread in southern Europe, close to the coast. This plant, known as 'agretti', is under consideration as a new potential crop on saline soils. Considering a possible defence role of sodin 5 in the plant, we report here its antifungal activity against different halophilic and halotolerant fungi. Our results show that sodin 5 at a concentration of 40 µg/mL (1.4 µM) was able to inhibit the growth of the fungi Trimmatostromma salinum (35.3%), Candida parapsilosis (24.4%), Rhodotorula mucilaginosa (18.2%), Aspergillus flavus (12.2%), and Aureobasidium melanogenum (9.1%). The inhibition observed after 72 h was concentration-dependent. On the other hand, very slight growth inhibition was observed in the fungus Hortaea werneckii (4.2%), which commonly inhabits salterns. In addition, sodin 5 showed a cytotoxic effect on the Sf9 insect cell line, decreasing the survival of these cells to 63% at 1.0 µg/mL (34.5 nM). Structural analysis of sodin 5 revealed that its N-terminal amino acid residue is blocked. Using mass spectrometry, sodin 5 was identified as a homologous to type 1 polynucleotide:adenosine glycosylases, commonly known as ribosome-inactivating proteins from the Amaranthaceae family. Twenty-three percent of its primary structure was determined, including the catalytic site.

Target-locked: A mechanism for disaggregase binding to aggregated proteins

ClpG is a novel autonomous disaggregase found in Pseudomonas aeruginosa that confers resistance to lethal heat stress. The mechanism by which ClpG specifically targets protein aggregates for disaggregation is unknown. In their recent work published in JBC, Katikaridis etal. (2023) identify an avidity-based mechanism by which four or more ClpG subunits, through specific N-terminal hydrophobic residues located on an exposed β-sheet loop, interact with multiple hydrophobic patches on an aggregated protein substrate. This study establishes a model for substrate binding to a prokaryotic disaggregase that should inform further investigations into other autonomous disaggregases.

Characterization of leucine aminopeptidase (LAP) activity in sweet pepper fruits during ripening and its inhibition by nitration and reducing events

Key messagePepper fruits contain two leucine aminopeptidase (LAP) genes which are differentially modulated during ripening and by nitric oxide. The LAP activity increases during ripening but is negatively modulated by nitration.Leucine aminopeptidase (LAP) is an essential metalloenzyme that cleaves N-terminal leucine residues from proteins but also metabolizes dipeptides and tripeptides. LAPs play a fundamental role in cell protein turnover and participate in physiological processes such as defense mechanisms against biotic and abiotic stresses, but little is known about their involvement in fruit physiology. This study aims to identify and characterize genes encoding LAP and evaluate their role during the ripening of pepper (Capsicum annuum L.) fruits and under a nitric oxide (NO)-enriched environment. Using a data-mining approach of the pepper plant genome and fruit transcriptome (RNA-seq), two LAP genes, designated CaLAP1 and CaLAP2, were identified. The time course expression analysis of these genes during different fruit ripening stages showed that whereas CaLAP1 decreased, CaLAP2 was upregulated. However, under an exogenous NO treatment of fruits, both genes were downregulated. On the contrary, it was shown that during fruit ripening LAP activity increased by 81%. An in vitro assay of the LAP activity in the presence of different modulating compounds including peroxynitrite (ONOO−), NO donors (S-nitrosoglutathione and nitrosocyteine), reducing agents such as reduced glutathione (GSH), l-cysteine (l-Cys), and cyanide triggered a differential response. Thus, peroxynitrite and reducing compounds provoked around 50% inhibition of the LAP activity in green immature fruits, whereas cyanide upregulated it 1.5 folds. To our knowledge, this is the first characterization of LAP in pepper fruits as well as of its regulation by diverse modulating compounds. Based on the capacity of LAP to metabolize dipeptides and tripeptides, it could be hypothesized that the LAP might be involved in the GSH recycling during the ripening process.

Site- and Stereoselective Glycomodification of Biomolecules through Carbohydrate-Promoted Pictet-Spengler Reaction.

Carbohydrates play pivotal roles in an array of essential biological processes and are consequently involved in many diseases. To meet the needs of glycobiology research, chemical enzymatic and non-enzymatic methods have been developed to generate glycoconjugates with well-defined structures. Herein, harnessing the unique properties of C6-oxidized glycans, we report a straightforward and robust strategy for site- and stereoselective glycomodification of biomolecules with N-terminal tryptophan residues by a carbohydrate-promoted Pictet-Spengler reaction, which is not adapted to typical aldehyde substrates under biocompatible conditions. This method reliably delivers highly homogeneous glycoconjugates with stable linkages and thus has great potential for functional modulation of peptides and proteins in glycobiology research. Moreover, this reaction can be performed at the glycosites of glycopeptides, glycoproteins and living-cell surfaces in a site-specific manner. Control experiments indicated that the protected α-O atom of aldehyde donors and free N-H bond of the tryptamine motif are crucial for this reaction. Mechanistic investigations demonstrated that the reaction exhibited a first-order dependence on both tryptophan and glycan, and deprotonation/rearomatization of the pentahydro-β-carbolinium ion intermediate might be the rate-determining step.

Two-Layer Inkjet-Printed Microwave Split-Ring Resonators for Detecting Analyte Binding to the Gold Surface.

This work focuses on demonstrating the working principle of inkjet-printed Au nanoparticle (NP) two-layer Gigahertz (2.6 GHz) microwave split-ring resonators (SRRs) as a novel platform for the detection of analytes on flexible substrates. In contrast to the standard fabrication of split-ring resonator biosensors using printed circuit board technology, which results in a seven-layer system, the resonators in this work were fabricated using a two-layer system. A ground plane is embedded in the SRR measurement setup. In this method, a microwave electromagnetic wave is coupled into the Au SRR via an inkjet-printed Cu-NP stripline that is photonically sintered. This coupling mechanism facilitates the detection of analytes by inducing resonance shifts in the SRR. In this study, the functionality of the printed sensors was demonstrated using two different Au functionalization processes, firstly, with HS-PEG7500-COOH, and, secondly, with protein G with an N-terminal cysteine residue. The sensing capabilities of the printed structures are shown by the attachment of biomolecules to the SRR and the measurement of the resulting resonance shift. The experiments show a clear shift of the resonance frequency in the range of 20-30 MHz for both approaches. These results demonstrate the functionality of the simplified printed two-layer microwave split-ring resonator for use as a biosensor.

Mechanisms of biased agonism by Gαi/o-biased stapled peptide agonists of the relaxin-3 receptor.

The neuropeptide relaxin-3 is composed of an A chain and a B chain held together by disulfide bonds, and it modulates functions such as anxiety and food intake by binding to and activating its cognate receptor RXFP3, mainly through the B chain. Biased ligands of RXFP3 would help to determine the molecular mechanisms underlying the activation of G proteins and β-arrestins downstream of RXFP3 that lead to such diverse functions. We showed that the i, i+4 stapled relaxin-3 B chains, 14s18 and d(1-7)14s18, were Gαi/o-biased agonists of RXFP3. These peptides did not induce recruitment of β-arrestin1/2 to RXFP3 by GPCR kinases (GRKs), in contrast to relaxin-3, which enabled the GRK2/3-mediated recruitment of β-arrestin1/2 to RXFP3. Relaxin-3 and the previously reported peptide 4 (an i, i+4 stapled relaxin-3 B chain) did not exhibit biased signaling. The staple linker of peptide 4 and parts of both the A chain and B chain of relaxin-3 interacted with extracellular loop 3 (ECL3) of RXFP3, moving it away from the binding pocket, suggesting that unbiased ligands promote a more open conformation of RXFP3. These findings highlight roles for the A chain and the N-terminal residues of the B chain of relaxin-3 in inducing conformational changes in RXFP3, which will help in designing selective biased ligands with improved therapeutic efficacy.

Evidence the Isc iron–sulfur cluster biogenesis machinery is the source of iron for [NiFe]-cofactor biosynthesis in Escherichia coli

[NiFe]-hydrogenases have a bimetallic NiFe(CN)2CO cofactor in their large, catalytic subunit. The 136 Da Fe(CN)2CO group of this cofactor is preassembled on a distinct HypC–HypD scaffold complex, but the intracellular source of the iron ion is unresolved. Native mass spectrometric analysis of HypCD complexes defined the [4Fe–4S] cluster associated with HypD and identified + 26 to 28 Da and + 136 Da modifications specifically associated with HypC. A HypCC2A variant without the essential conserved N-terminal cysteine residue dissociated from its complex with native HypD lacked all modifications. Native HypC dissociated from HypCD complexes isolated from Escherichia coli strains deleted for the iscS or iscU genes, encoding core components of the Isc iron–sulfur cluster biogenesis machinery, specifically lacked the + 136 Da modification, but this was retained on HypC from suf mutants. The presence or absence of the + 136 Da modification on the HypCD complex correlated with the hydrogenase enzyme activity profiles of the respective mutant strains. Notably, the [4Fe–4S] cluster on HypD was identified in all HypCD complexes analyzed. These results suggest that the iron of the Fe(CN)2CO group on HypCD derives from the Isc machinery, while either the Isc or the Suf machinery can deliver the [4Fe–4S] cluster to HypD.

Mapping Protein-Protein Interactions at Birth: Single-Particle Cryo-EM Analysis of a Ribosome-Nascent Globin Complex.

Interactions between ribosome-bound nascent chains (RNCs) and ribosomal components are critical to elucidate the mechanism of cotranslational protein folding. Nascent protein-ribosome contacts within the ribosomal exit tunnel were previously assessed mostly in the presence of C-terminal stalling sequences, yet little is known about contacts taking place in the absence of these strongly interacting motifs. Further, there is nearly no information about ribosomal proteins (r-proteins) interacting with nascent chains within the outer surface of the ribosome. Here, we combine chemical cross-linking, single-particle cryo-EM, and fluorescence anisotropy decays to determine the structural features of ribosome-bound apomyoglobin (apoMb). Within the ribosomal exit tunnel core, interactions are similar to those identified in previous reports. However, once the RNC enters the tunnel vestibule, it becomes more dynamic and interacts with ribosomal RNA (rRNA) and the L23 r-protein. Remarkably, on the outer surface of the ribosome, RNCs interact mainly with a highly conserved nonpolar patch of the L23 r-protein. RNCs also comprise a compact and dynamic N-terminal region lacking contact with the ribosome. In all, apoMb traverses the ribosome and interacts with it via its C-terminal region, while N-terminal residues sample conformational space and form a compact subdomain before the entire nascent protein sequence departs from the ribosome.

Formation and Properties of DNA Adducts Generated by Reactions of Abasic Sites with 1,2-Aminothiols Including Cysteamine, Cysteine Methyl Ester, and Peptides Containing N-Terminal Cysteine Residues.

The reaction of 1,2-aminothiol groups with aldehyde residues in aqueous solution generates thiazolidine products, and this process has been developed as a catalyst-free click reaction for bioconjugation. The work reported here characterized reactions of the biologically relevant 1,2-aminothiols including cysteamine, cysteine methyl ester, and peptides containing N-terminal cysteine residues with the aldehyde residue of apurinic/apyrimidinic (AP) sites in DNA oligomers. These 1,2-aminothiol-containing compounds rapidly generated adducts with AP sites in single-stranded and double-stranded DNA. NMR and MALDI-TOF-MS analyses provided evidence that the reaction generated a thiazolidine product. Conversion of an AP site to a thiazolidine-AP adduct protected against the rapid cleavage normally induced at AP sites by the endonuclease action of the enzyme APE1 and the AP-lyase activity of the biogenic amine spermine. In the presence of excess 1,2-aminothiols, the thiazolidine-AP adducts underwent slow strand cleavage via a β-lyase reaction that generated products with 1,2-aminothiol-modified sugar residues on the 3'-end of the strand break. In the absence of excess 1,2-aminothiols, the thiazolidine-AP adducts dissociated to release the parent AP-containing oligonucleotide. The properties of the thiazolidine-AP adducts described here mirror critical properties of SRAP proteins HMCES and YedK that capture AP sites in single-stranded regions of cellular DNA and protect them from cleavage.

Characterization of a LanC-free pathway for the formation of an ll-MeLan residue and an alloAviMeCys residue in the newly identified class V lanthipeptide triantimycins.

The thioether-connected bis-amino acid lanthionine (Lan) residues are class-defining residues of lanthipeptides. Typically, the cyclization step of lanthionine formation, which relies on the addition of a cysteine to an unsaturated dehydroamino acid, is directed either by a standalone cyclase LanC (class I) or by a cyclase domain (class II-IV). However, the pathways of characterized class V members often lack a known cyclase (domain), raising a question on the mechanism by which their multi-macrocycle systems are formed. Herein, we report a new RiPP gene cluster in Streptomyces TN 58, where it encodes the biosynthesis of 3 distinct class V lanthipeptides-termed triantimycins (TAMs). TAM A1∼A3 share an N-terminal ll-MeLan residue, and only TAM A1 contains an additional internal ll-Lan residue. TAM A1 also has a C-terminal (2S, 3R)-S-((Z)-2-aminovinyl)-3-methyl-d-cysteine (alloAviMeCys) residue, which is distinct from the previously reported (2S, 3S)-AviMeCys residue in other RiPPs. Gene deletion, heterologous coexpression, and structural elucidation demonstrated that the cyclization for an ll-MeLan formation occurs spontaneously and is independent of any known lanthionine cyclase. This study provides a new paradigm for lanthionine formation and facilitates genome mining and engineering efforts on RiPPs containing (Me)Lan and (allo)Avi(Me)Cys residues.

Designing and synthesizing peptide-based quorum sensing modulators.

Quorum sensing (QS) is a density-dependent bacterial communication system that uses small molecules as regulatory modulators. Synthetic changes to these molecules can up-or-down-regulate this system, leading to control of phenotypes, like competence and virulence factor production, that have implications in human health. In this chapter, a methodology for library design and screening of synthetic autoinducing peptides (AIPs) to uncover QS SARs is delineated. Additionally, procedures for the synthesis, purification and analysis of linear and cyclic AIPs are detailed. This includes solutions for potential synthetic challenges including diketopiperazine formation when using N-methyl amino acids and cyclization of peptides containing N-terminal cysteine residues. These procedures have and are currently being applied to develop potent QS modulators in Streptococcus pneumoniae, Bacillus cereus, Streptococcus gordonii and Lactiplantibacillus plantarum.

Elucidation of the CadA Protein 3D Structure and Affinity for Metals.

The mitigation of cadmium (Cd) pollution, a significant ecological threat, is of paramount importance. Pseudomonas aeruginosa harbors 2 Cd resistance genes, namely, cadR and cadA. Presently, our focus is on the identification and characterization of the cation-transporting P-type ATPase (cadA) in Pseudomonas aeruginosa BC15 through in silico methods. The CadA protein and its binding capacities remain poorly understood, with no available structural elucidation. The presence of the cadA gene in P aeruginosa was confirmed, showing a striking 99% sequence similarity with both P aeruginosa and P putida. Phylogenetic analysis unveiled the evolutionary relationship between CadA protein sequences from various Pseudomonas species. Physicochemical analysis demonstrated the stability of CadA, revealing a composition of 690 amino acids, a molecular weight of 73 352.85, and a predicted isoelectric point (PI) of 5.39. Swiss-Model homology modelling unveiled a 33.73% sequence homology with CopA (3J09), and the projected structure indicated that 89.3% of amino acid residues were situated favourably within the Ramachandran plot, signifying energetic stability. Notably, the study identified metal-binding sites in CadA, namely, H3, C30, C32, C35, H48, C89, and C106. Docking studies revealed a higher efficiency of Cd binding with CadA compared to other heavy metals. This underscores the crucial role of N-terminal cysteine residues in Cd removal. It is evident that CadA of P aeruginosa BC15 plays a crucial role in Cd tolerance, rendering it a potential microorganism for Cd toxicity bioremediation. The structural and functional elucidation of CadA, facilitated by this study, holds promise for advancing cost-effective strategies in the remediation of cadmium-contaminated environments.

Precision peptide theranostics: developing N- to C-terminus optimized theranostics targeting cholecystokinin-2 receptor.

Peptides are ideal for theranostic development as they afford rapid target accumulation, fast clearance from background tissue, and exhibit good tissue penetration. Previously, we developed a novel series of peptides that presented discreet folding propensity leading to an optimal candidate [68Ga]Ga-DOTA-GA1 ([D-Glu]6-Ala-Tyr-NMeGly-Trp-NMeNle-Asp-Nal-NH2) with 50 pM binding affinity against cholecystokinin-2 receptors (CCK2R). However, we were confronted with challenges of unfavorably high renal uptake. Methods: A structure activity relationship study was undertaken of the lead theranostic candidate. Prudent structural modifications were made to the peptide scaffold to evaluate the contributions of specific N-terminal residues to the overall biological activity. Optimal candidates were then evaluated in nude mice bearing transfected A431-CCK2 tumors, and their biodistribution was quantitated ex vivo. Results: We identified and confirmed that D-Glu3 to D-Ala3 substitution produced 2 optimal candidates, [68Ga]Ga-DOTA-GA12 and [68Ga]Ga-DOTA-GA13. These radiopeptides presented with high target/background ratios, enhanced tumor retention, excellent metabolic stability in plasma and mice organ homogenates, and a 4-fold reduction in renal uptake, significantly outperforming their non-alanine counterparts. Conclusions: Our study identified novel radiopharmaceutical candidates that target the CCK2R. Their high tumor uptake and reduced renal accumulation warrant clinical translation.

Structural Impact of N-terminal Pyroglutamate in an Amyloid-β(3-42) Fibril Probed by Solid-State NMR Spectroscopy.

Extracellular amyloid-β (Aβ) plaques, primarily formed by Aβ(1-40) and Aβ(1-42) fibrils, are a hallmark of Alzheimer's disease. The Aβ peptide can undergo a high variety of different post-translational modifications including formation of a pyroglutamate (pGlu, pE) at N-terminal Glu3 or Glu11 of truncated Aβ(3-x) or Aβ(11-x), respectively. Here we studied structural similarities and differences between pEAβ(3-42) and LS-shaped Aβ(1-42) fibrils grown under identical conditions (pH 2) using solid-state NMR spectroscopy. We show that the central region of pEAβ(3-42) fibrils including the turn region around V24 is almost identical to Aβ(1-42) showing similar β-strands also at the N-terminus. The missing N-terminal residues D1-A2 along with pE3 formation in pEAβ(3-42) preclude a salt bridge between K28-D1' as in Aβ(1-42) fibrils. G37 and G38 act as highly sensitive internal sensors for the modified N-terminus, which remains rigid over ~five pH units.

NAD<sup>+</sup>-dependent format dehydrogenase from the thermotolerant yeast <i>ogataea parapolymorpha</i>: properties and protein engineering of the <i>n</i>-terminal sequence

Previously, the gene of formate dehydrogenase (FDH, EC 1.2.1.2) from the thermotolerant methylotrophic yeast Ogataea parapolymorpha DL 1 (OpaFDH) was cloned in our laboratory. The recombinant enzyme with an additional glycine amino acid residue (OpaFDH_GK) was obtained in Escherichia coli cells in an active and soluble form with a yield of more than 1 g per liter of medium. In the present work, a detailed comparison of this enzyme with FDH from other sources was carried out. Among eukaryotic formate dehydrogenases, OpaFDH has the highest thermal stability. To elucidate the effect of the N-terminal residue on the properties of the enzyme, OpaFDH_K (identical to natural) and OpaFDH_AK variants containing an additional Ala residue at the N-terminus were also obtained. It was shown that the addition of an Ala residue to the N-terminus reduces the rate constant of thermal inactivation four times compared with the addition of a Gly residue. The addition of six more histidine residues to the N-terminus of OpaFDH_AK leads to an acceleration of purification, practically does not affect the kinetic parameters, but somewhat reduces the temperature stability, which, however, can be restored to the level of OpaFDH_AK by adding 0.5 M NaCl.

Application of N-Terminal Site-Specific Biotin and Digoxigenin Conjugates to Clinical Anti-drug Antibody Assay Development.

Biotin- and digoxigenin (DIG)-conjugated therapeutic drugs are critical reagents used for the development of anti-drug antibody (ADA) assays for the assessment of immunogenicity. The current practice of generating biotin and DIG conjugates is to label a therapeutic antibody with biotin or DIG via primary amine groups on lysine or N-terminal residues. This approach modifies lysine residues nonselectively, which can impact the ability of an ADA assay to detect those ADAs that recognize epitopes located at or near the modified lysine residue(s). The impact of the lysine modification is considered greater for therapeutic antibodies that have a limited number of lysine residues, such as the variable heavy domain of heavy chain (VHH) antibodies. In this paper, for the first time, we report the application of site-specifically conjugated biotin- and DIG-VHH reagents to clinical ADA assay development using a model molecule, VHHA. The site-specific conjugation of biotin or DIG to VHHA was achieved by using an optimized reductive alkylation approach, which enabled the majority of VHHA molecules labeled with biotin or DIG at the desirable N-terminus, thereby minimizing modification of the protein after labeling and reducing the possibility of missing detection of ADAs. Head-to-head comparison of biophysical characterization data revealed that the site-specific biotin and DIG conjugates demonstrated overall superior quality to biotin- and DIG-VHHA prepared using the conventional amine coupling method, and the performance of the ADA assay developed using site-specific biotin and DIG conjugates met all acceptance criteria. The approach described here can be applied to the production of other therapeutic-protein- or antibody-based critical reagents that are used to support ligand binding assays.

Abstract A142: Fragment based discovery of inhibitors of the eIF4E:eIF4G interaction

Abstract eIF4E is the rate limiting factor for cap-dependent protein translation, forming an essential part of the translation initiation complex, eIF4F and is a key signalling convergence point for the major oncogenic pathways MAPK and PI3K/AKT/MTOR. eIF4E has been shown to be overexpressed in cancer, which has been associated with poor prognosis and resistance to chemotherapeutics and targeted agents such as mTOR and MAPK inhibitors. eIF4E is therefore a promising target for cancer treatment but has so far been difficult to target with small molecules due to a lack of druggable pockets. Fragment based crystallographic screening is a powerful technique for probing the surface of proteins to identify potentially druggable binding sites. Here, we describe our fragment-based approach and hit optimisation campaign to generate potent compounds binding to eIF4E For the fragment screen, we generated an engineered form of eIF4E, where the N-terminal (residues 1-35) were removed and replaced a short, tethered peptide based on the canonical binding sequence of the eIF4E binding protein 4E-BP1, and screened 1400 fragments using a combination of Xray crystallography and NMR. Several low affinity (mM) fragment hits were identified, binding to both the mRNA cap- binding site and at an additional binding site of unknown functional relevance. Using a combination of biophysical and biochemical assays, subsequent rounds of iterative structure-based drug design of yielded potent lead molecules against both binding sites. For the cap-binding site, lead compounds of low nM potency were generated but due to the polar nature of the binding site, the molecules had very poor permeability properties and did not inhibit cell proliferation. For the non-canonical binding site, lead compounds with potency of 100nM alongside less potent enantiomers were generated which provided useful tools for probing the function of the binding site in vitro. We developed a MSD (Meso-scale discovery) assay to determine that the lead compound and not the inactive enantiomer could disrupt the eIF4E:eIF4G interaction in HeLa cell lysates with an IC50 of 1uM. Using a HeLa extract in vitro translation assay, we could also demonstrate that both the cap site and non-canonical site lead compounds could inhibit cap-dependent but not IRES driven protein translation. This work demonstrates the power of fragment screening to identify novel functional pockets on difficult to drug proteins such as eIF4E. Citation Format: Caroline J Richardson, Mladen Vincovic, Charlotte East, Nicola Wallis, George Ward, Andrew Woodhead. Fragment based discovery of inhibitors of the eIF4E:eIF4G interaction [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A142.