Large Pocket Research Articles

Unusual neck swellings: a case series of a new Pandora’s box

The neck is an important region in the body containing the greatest number of vital structures traversing it. The variety of structures also means that many varied neck masses may present in this region. We described a series of eight such cases of large and rare neck swellings. These include cystic swellings, benign tumours as well as malignant secondary nodal metastasis. We also describe for the first time a rare case of a large air pocket in the neck. Thus, this series serves to highlight the amazing capability of the neck to throw up a wide range of complex and rare swellings - earning its name as a new Pandora’s box.

Structural basis for substrate flexibility of the O-methyltransferase MpaG' involved in mycophenolic acid biosynthesis.

MpaG' is an S-adenosyl-L-methionine (SAM)-dependent methyltransferase involved in the compartmentalized biosynthesis of mycophenolic acid (MPA), a first-line immunosuppressive drug for organ transplantations and autoimmune diseases. MpaG' catalyzes the 5-O-methylation of three precursors in MPA biosynthesis including demethylmycophenolic acid (DMMPA), 4-farnesyl-3,5-dihydroxy-6-methylphthalide (FDHMP), and an intermediate containing three fewer carbon atoms compared to FDHMP (FDHMP-3C) with different catalytic efficiencies. Here, we report the crystal structures of S-adenosyl-L-homocysteine (SAH)/DMMPA-bound MpaG', SAH/FDHMP-3C-bound MpaG', and SAH/FDHMP-bound MpaG' to understand the catalytic mechanism of MpaG'and structural basis for its substrate flexibility. Structural and biochemical analyses reveal that MpaG' utilizes the catalytic dyad H306-E362 to deprotonate the C5 hydroxyl group of the substrates for the following methylation. The three substrates with differently modified farnesyl moieties are well accommodated in a large semi-open substrate binding pocket with the orientation of their phthalide moiety almost identical. Based on the structure-directed mutagenesis, a single mutant MpaG'Q267A is engineered with significantly improved catalytic efficiency for all three substrates. This study expands the mechanistic understanding and the pocket engineering strategy for O-methyltransferases involved in fungal natural product biosynthesis. Our research also highlights the potential of O-methyltransferases to modify diverse substrates by protein design and engineering.

Discovery of the 1-naphthylamine biodegradation pathway reveals a broad-substrate-spectrum enzyme catalyzing 1-naphthylamine glutamylation.

1-Naphthylamine (1NA), which is harmful to human and aquatic animals, has been used widely in the manufacturing of dyes, pesticides, and rubber antioxidants. Nevertheless, little is known about its environmental behavior and no bacteria have been reported to use it as the growth substrate. Herein, we describe a pathway for 1NA degradation in the isolate Pseudomonas sp. strain JS3066, determine the structure and mechanism of the enzyme NpaA1 that catalyzes the initial reaction, and reveal how the pathway evolved. From genetic and enzymatic analysis, a five gene-cluster encoding a dioxygenase system was determined to be responsible for the initial steps in 1NA degradation through glutamylation of 1NA. The γ-glutamylated 1NA was subsequently oxidized to 1,2-dihydroxynaphthalene which was further degraded by the well-established pathway of naphthalene degradation via catechol. A glutamine synthetase-like (GS-like) enzyme (NpaA1) initiates 1NA glutamylation, and this enzyme exhibits a broad substrate selectivity toward a variety of anilines and naphthylamine derivatives. Structural analysis revealed that the aromatic residues in the 1NA entry tunnel and the V201 site in the large substrate-binding pocket significantly influence NpaA1's substrate preferences. The findings enhance understanding of degrading polycyclic aromatic amines, and will also enable the application of bioremediation at naphthylamine contaminated sites.

Status and Depth of Mastoid Antrum and the Pattern of Mastoid Pneumatization in Cases of Large, Subtotal Perforations and Posterosuperior Retraction Pockets (PSRP).

The present study aimed to evaluate the pneumatization status of the mastoid air cells in general with the depth and status of mastoid antrum in particular, in patients of chronic otitis media (COM). This is an observational cross-sectional study in sample size of 60 participants with large, subtotal perforation and posterosuperior retraction pocket (PSRP). Mean age with large central and subtotal perforation combined was 35.78 years, compared to 32.13 years in PSRP. Granulations seen in antrum in 71.6% and cholesteatoma observed in 15%. Mean depth of the mastoid antrum was 17.27 mm. A well pneumatized mastoid in 21.6%, diploic 28.3% and sclerotic in 50% was noted in this study. Chronic otitis media affects temporal bone pneumatization, particularly the mastoid antrum. Our findings indicate that the depth of the mastoid antrum is slightly greater in cholesteatoma cases due to bone erosion. As early intervention enhances postoperative quality of life by preserving or restoring auditory function, understanding the mastoid pneumatization and antrum depth is crucial for managing chronic otitis media effectively.

MicroED structure of the C11 cysteine protease clostripain

Clostripain secreted from Clostridium histolyticum is the founding member of the C11 family of Clan CD cysteine peptidases, which is an important group of peptidases secreted by numerous bacteria. Clostripain is an arginine-specific endopeptidase. Because of its efficacy as a cysteine peptidase, it is widely used in laboratory settings. Despite its importance the structure of clostripain remains unsolved. Here we describe the first structure of an active form of C. histolyticum clostripain determined at 2.5 Å resolution using microcrystal electron diffraction (MicroED). The structure was determined from a single nanocrystal after focused ion beam milling. The structure of clostripain shows a typical Clan CD α/β/α sandwich architecture and the Cys231/His176 catalytic dyad in the active site. It has a large electronegative substrate binding pocket showing its ability to accommodate large and diverse substrates. A loop in the heavy chain formed between residues 452 and 457 is potentially important for substrate binding. In conclusion, this result demonstrates the importance of MicroED to determine the unknown structure of macromolecules such as clostripain, which can be further used as a platform to study substrate binding and design of potential inhibitors against this class of peptidases.

Increasing Multienzyme Cascade Efficiency and Stability of MOF via Partitioning Immobilization.

Enhancing the stability of multienzyme cascade reactions in metal-organic frameworks (MOFs) is a challenging task in the fields of biotechnology and chemistry. However, addressing this challenge could yield far-reaching benefits across the application range in the biomedical, food, and environmental sectors. In this study, multienzyme partitioning immobilization that sequentially immobilizes cascade enzymes with hierarchical MOFs is proposed to reduce substrate diffusion resistance. Conversion results of ginsenosides indicate that this strategy improves the cascade efficiency up to 1.26 times. The substrate diffusion model is used to investigate the dual-interenzyme mass transfer behavior of substrates in the restricted domain space and evaluate the substrate channeling effect under partitioning immobilization. Molecular docking and kinetic simulations reveal that the MOFs effectively limit the conformational changes of cascade enzymes at high temperatures and in organic solvents while maintaining a large pocket of active centers. This phenomenon increased efficient substrate docking to the enzyme molecules, further optimizing cascade efficiency. The results of the immobilization of GOX and horseradish peroxidase as model enzymes indicate that the partitioned MOF immobilization strategy could be used for universal adaptation of cascade enzymes.

Experimental investigation of storm sewer geyser using a large-scale setup

The storm sewer geyser is a process where an air–water mixture violently erupts from a manhole. Despite the low hydrostatic pressure, violent eruptions can achieve a height of tens of meters above the ground. This current study experimentally investigates large-scale violent geysers using a large air pocket inserted from a pressurized air tank. The total length of the pipe system is approximately 88 m with a 0.1572 m diameter pipe. This large-scale experiment facilitates the investigation of spontaneous geyser eruptions. This study identifies the role of air–water volume ratio and coefficient of pressure (ratio of absolute initial static pressure to initial dynamic pressure) on the geyser intensity using eruption images and pressure plots. A total of 116 cases are tested, in which the volume ratio is parametrically increased from 0 to 1.1 under various operating conditions. A geyser score is defined to quantify the geyser eruption nature based on visual observations. The key findings are as follows: first, a sharp transition in geyser intensity is observed at the critical volume ratio of 0.5, and pre-transition and post-transition intensity exhibit a linear relationship with the volume ratio; and second, the critical volume ratio linearly varies with the coefficient of pressure.

Drugs Form Ternary Complexes with Human Liver Fatty Acid Binding Protein 1 (FABP1) and FABP1 Binding Alters Drug Metabolism.

Liver fatty acid binding protein 1 (FABP1) binds diverse endogenous lipids and is highly expressed in the human liver. Binding to FABP1 alters the metabolism and homeostasis of endogenous lipids in the liver. Drugs have also been shown to bind to rat FABP1, but limited data are available for human FABP1 (hFABP1). FABP1 has a large binding pocket, and up to two fatty acids can bind to FABP1 simultaneously. We hypothesized that drug binding to hFABP1 results in formation of ternary complexes and that FABP1 binding alters drug metabolism. To test these hypotheses, native protein mass spectrometry (MS) and fluorescent 11-(dansylamino)undecanoic acid (DAUDA) displacement assays were used to characterize drug binding to hFABP1, and diclofenac oxidation by cytochrome P450 2C9 (CYP2C9) was studied in the presence and absence of hFABP1. DAUDA binding to hFABP1 involved high (Kd,1 = 0.2 μM) and low (Kd,2 > 10 μM) affinity binding sites. Nine drugs bound to hFABP1 with equilibrium dissociation constant (Kd) values ranging from 1 to 20 μM. None of the tested drugs completely displaced DAUDA from hFABP1, and fluorescence spectra showed evidence of ternary complex formation. Formation of DAUDA-hFABP1-diclofenac ternary complex was verified with native MS. Docking predicted diclofenac binding in the portal region of FABP1 with DAUDA in the binding cavity. The catalytic rate constant of diclofenac hydroxylation by CYP2C9 was decreased by ∼50% (P < 0.01) in the presence of FABP1. Together, these results suggest that drugs form ternary complexes with hFABP1 and that hFABP1 binding in the liver will alter drug metabolism and clearance. SIGNIFICANCE STATEMENT: Many commonly prescribed drugs bind fatty acid binding protein 1 (FABP1), forming ternary complexes with FABP1 and the fluorescent fatty acid 11-(dansylamino)undecanoic acid. These findings suggest that drugs will bind to apo-FABP1 and fatty acid-bound FABP1 in the human liver. The high expression of FABP1 in the liver, together with drug binding to FABP1, may alter drug disposition processes in vivo.

Control of phosphodiesterase activity in the regulator of biofilm dispersal RbdA from Pseudomonas aeruginosa.

The switch between planktonic and biofilm lifestyle correlates with intracellular concentration of the second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). While bacteria possess cyclase and phosphodiesterase enzymes to catalyse formation or hydrolysis of c-di-GMP, both enzymatic domains often occur in a single protein. It is tacitly assumed that one of the two enzymatic activities is dominant, and that additional domains and protein interactions enable responses to environmental conditions and control activity. Here we report the structure of the phosphodiesterase domain of the membrane protein RbdA (regulator of biofilm dispersal) in a dimeric, activated state and show that phosphodiesterase activity is controlled by the linked cyclase. The phosphodiesterase region around helices α5/α6 forms the dimer interface, providing a rationale for activation, as this region was seen in contact with the cyclase domain in an auto-inhibited structure previously described. Kinetic analysis supports this model, as the activity of the phosphodiesterase alone is lower when linked to the cyclase. Analysis of a computed model of the RbdA periplasmatic domain reveals an all-helical architecture with a large binding pocket that could accommodate putative ligands. Unravelling the regulatory circuits in multi-domain phosphodiesterases like RbdA is important to develop strategies to manipulate or disperse bacterial biofilms.

Ionic liquid modification reshapes the substrate pockets of lipase to boost its stability and activity in vitamin E succinate synthesis.

The relative low stability, reusability and activity of enzymes made the industrial production of vitamin E succinate (VES) can only be performed with complex processes and high cost using chemical methods. To address these issues, in the present study, an ionic liquids (ILs) modification strategy was developed to improve the activity and stability of lipases in VES synthesis. The results showed that the [1-butyl-3-methyl imidazole] [N-acetyl-l-proline] ILs modified Candida rugosa lipase (CRL) has the highest modification degree (48.28%), activity (774 U g-1 ), thermostability and solvent tolerance in three selected modifiers. Additionally, after reaction condition optimization, the highest yield of VES can be improved to 95.18% at 45 °C for 15 h, which was significantly improved compared to some previous studies. In the present study, a high-efficiency VES synthesis strategy was successfully developed via modification of lipase. Moreover, the mechanism by which ILs modification can enhance the activity and stability of lipase was investigated via both experimental and computational-aided methods. Molecular dynamics simulation suggested that ILs modification changed the geometry of Phe344 from flat to upright, which significantly reshaped and enhanced the size of substrate binding pocket of CRL. It is also agreement with our circular dichroism and fluorescence spectroscopy results, which suggested that the modification changed the secondary structure of CRL to a certain extent. The larger pocket also endowed the suitable binding pose of succinate, which made the hydrogen bonds between succinate and active site Ser209 become stronger, and thus improving the yield of VES. © 2023 Society of Chemical Industry.

TRNA shape is an identity element for an archaeal pyrrolysyl-tRNA synthetase from the human gut.

Protein translation is orchestrated through tRNA aminoacylation and ribosomal elongation. Among the highly conserved structure of tRNAs, they have distinguishing features which promote interaction with their cognate aminoacyl tRNA synthetase (aaRS). These key features are referred to as identity elements. In our study, we investigated the tRNA:aaRS pair that installs the 22nd amino acid, pyrrolysine (tRNAPyl:PylRS). Pyrrolysyl-tRNA synthetases (PylRSs) are naturally encoded in some archaeal and bacterial genomes to acylate tRNAPyl with pyrrolysine. Their large amino acid binding pocket and poor recognition of the tRNA anticodon have been instrumental in incorporating >200 noncanonical amino acids. PylRS enzymes can be divided into three classes based on their genomic structure. Two classes contain both an N-terminal and C-terminal domain, however the third class (ΔpylSn) lacks the N-terminal domain. In this study we explored the tRNA identity elements for a ΔpylSn tRNAPyl from Candidatus Methanomethylophilus alvus which drives the orthogonality seen with its cognate PylRS (MaPylRS). From aminoacylation and translation assays we identified five key elements in ΔpylSn tRNAPyl necessary for MaPylRS activity. The absence of a base (position 8) and a G-U wobble pair (G28:U42) were found to affect the high-resolution structure of the tRNA, while molecular dynamic simulations led us to acknowledge the rigidity imparted from the G-C base pairs (G3:C70 and G5:C68).

Characterization and structure-based protein engineering of a regiospecific saponin acetyltransferase from Astragalus membranaceus

Acetylation contributes to the bioactivity of numerous medicinally important natural products. However, little is known about the acetylation on sugar moieties. Here we report a saponin acetyltransferase from Astragalus membranaceus. AmAT7-3 is discovered through a stepwise gene mining approach and characterized as the xylose C3′/C4′-O-acetyltransferse of astragaloside IV (1). To elucidate its catalytic mechanism, complex crystal structures of AmAT7-3/1 and AmAT7-3A310G/1 are obtained, which reveal a large active pocket decided by a specific sequence AADAG. Combining with QM/MM computation, the regiospecificity of AmAT7-3 is determined by sugar positioning modulated by surrounding amino acids including #A310 and #L290. Furthermore, a small mutant library is built using semi-rational design, where variants A310G and A310W are found to catalyze specific C3′-O and C4′-O acetylation, respectively. AmAT7-3 and its variants are also employed to acetylate other bioactive saponins. This work expands the understanding of saponin acetyltransferases, and provide efficient catalytic tools for saponin acetylation.

QM/MM and MM MD Simulations on Enzymatic Degradation of the Nerve Agent VR by Phosphotriesterase.

V-type nerve agents are hardly degraded by phosphotriesterase (PTE). Interestingly, the PTE variant of BHR-73MNW can effectively improve the hydrolytic efficiency of VR, especially for its Sp-enantiomer. Here, the whole enzymatic degradation of both Sp and Rp enantiomers of VR by the wild-type PTE and its variant BHR-73MNW was investigated by quantum mechanics/molecular mechanics (QM/MM) calculations and MM molecular dynamics simulations. Present results indicate that the degradation of VR can be initiated by the nucleophilic attack of the bridging OH- and the zinc-bound water molecule. The QM/MM-predicted energy barriers for the hydrolytic process of Sp-VR are 19.8 kcal mol-1 by the variant with water as a nucleophile and 22.0 kcal mol-1 by the wild-type PTE with OH- as a nucleophile, and corresponding degraded products are bound to the dinuclear metal site in monodentate and bidentate coordination modes, respectively. The variant effectively increases the volume of the large pocket, allowing more water molecules to enter the active pocket and resulting in the improvement of the degradation efficiency of Sp-VR. The hydrolysis of Rp-VR is triggered only by the hydroxide with an energy span of 20.6 kcal mol-1 for the wild-type PTE and 20.7 kcal mol-1 for the variant BHR-73-MNW PTE. Such mechanistic insights into the stereoselective degradation of VR by PTE and the role of water may inspire further studies to improve the catalytic efficiency of PTE toward the detoxification of nerve agents.

Extended Scope and Understanding of Zinc-Dependent Alcohol Dehydrogenases for Reduction of Cyclic α-Diketones.

Alcohol dehydrogenases (ADH) are important tools for generating chiral α-hydroxyketones. Previously, only the ADH of Thauera aromatica was known to convert cyclic α-diketones with appropriate preference. Here, we extend the spectrum of suitable enzymes by three alcohol dehydrogenases from Citrifermentans bemidjiense (CibADH), Deferrisoma camini (DecADH), and Thauera phenylacetica (ThpADH). Of these, DecADH is characterized by very high thermostability; CibADH and ThpADH convert α-halogenated cyclohexanones with increased activity. Otherwise, however, the substrate spectrum of all four ADHs is highly conserved. Structural considerations led to the conclusion that conversion of diketones requires not only the expansion of the active site into a large binding pocket, but also the circumferential modification of almost all amino acid residues that form the first shell of the binding pocket. The constellation appears to be overall highly specific for the relative positioning of the carbonyl functions and the size of the C-ring.

Identification and evaluation of potential inhibitor molecules against TcyA from Candidatus Liberibacter asiaticus

Of the two putative amino acid binding periplasmic receptors of ABC transporter family in Candidatus Liberibacter asiaticus (CLas), cystine binding receptor (CLasTcyA) has been shown to mainly express in phloem of citrus plant and is a target for inhibitor development. The crystal structure of CLasTcyA in complex with substrates has been reported earlier. The present work reports the identification and evaluation of potential candidates for their inhibitory potential against CLasTcyA. Among many compounds, selected through virtual screening, and MD simulation, pimozide, clidinium, sulfasalazine and folic acid showed significantly higher affinities and stability in complex with CLasTcyA. The SPR studies with CLasTcyA revealed significantly higher binding affinities for pimozide and clidinium (Kd, 2.73 nM and 70 nM, respectively) as compared to cystine (Kd, 1.26 µM). The higher binding affinities could be attributed to significantly increased number of interactions in the binding pocket as evident from the crystal structures of CLasTcyA in complex with pimozide and clidinium as compared to cystine. The CLasTcyA possess relatively large binding pocket where bulkier inhibitors fit quite well. In planta studies, carried out to assess the effect of inhibitors on HLB infected Mosambi plants, showed significant reduction in CLas titre in plants treated with inhibitors as compared to control plants. The results showed that pimozide exhibited higher efficiency as compared to clidinium in reducing CLas titre in treated plants. Our results showed that the inhibitor development against critical proteins like CLasTcyA can be an important strategy in management of HLB.

Design of Heme Enzymes with a Tunable Substrate Binding Pocket Adjacent to an Open Metal Coordination Site.

The catalytic versatility of pentacoordinated iron is highlighted by the broad range of natural and engineered activities of heme enzymes such as cytochrome P450s, which position a porphyrin cofactor coordinating a central iron atom below an open substrate binding pocket. This catalytic prowess has inspired efforts to design de novo helical bundle scaffolds that bind porphyrin cofactors. However, such designs lack the large open substrate binding pocket of P450s, and hence, the range of chemical transformations accessible is limited. Here, with the goal of combining the advantages of the P450 catalytic site geometry with the almost unlimited customizability of de novo protein design, we design a high-affinity heme-binding protein, dnHEM1, with an axial histidine ligand, a vacant coordination site for generating reactive intermediates, and a tunable distal pocket for substrate binding. A 1.6 Å X-ray crystal structure of dnHEM1 reveals excellent agreement to the design model with key features programmed as intended. The incorporation of distal pocket substitutions converted dnHEM1 into a proficient peroxidase with a stable neutral ferryl intermediate. In parallel, dnHEM1 was redesigned to generate enantiocomplementary carbene transferases for styrene cyclopropanation (up to 93% isolated yield, 5000 turnovers, 97:3 e.r.) by reconfiguring the distal pocket to accommodate calculated transition state models. Our approach now enables the custom design of enzymes containing cofactors adjacent to binding pockets with an almost unlimited variety of shapes and functionalities.

A large conserved family of small-molecule carboxyl methyltransferases identified from microorganisms

Small-molecule carboxyl methyltransferases (CbMTs) constitute a small proportion of the reported methyltransferases, but they have received extensive attention due to their important physiological functions. Most of the small-molecule CbMTs isolated to date originate from plants and are members of the SABATH family. In this study, we identified a type of CbMT (OPCMT) from a group of Mycobacteria, which has a distinct catalytic mechanism from the SABATH methyltransferases. The enzyme contains a large hydrophobic substrate-binding pocket (~400Å3) and utilizes two conserved residues, Thr20 and Try194, to retain the substrate in a favorable orientation for catalytic transmethylation. The OPCMT_like MTs have a broad substrate scope and can accept diverse carboxylic acids enabling efficient production of methyl esters. They are widely (more than 10,000) distributed in microorganisms, including several well-known pathogens, whereas no related genes are found in humans. Invivo experiments implied that the OPCMT_like MTs was indispensable for M. neoaurum, suggesting that these proteins have important physiological functions.

Structure of WNT inhibitor adenomatosis polyposis coli down-regulated 1 (APCDD1), a cell-surface lipid-binding protein

Diverse extracellular proteins negatively regulate WNT signaling. One such regulator is adenomatosis polyposis coli down-regulated 1 (APCDD1), a conserved single-span transmembrane protein. In response to WNT signaling in a variety of tissues, APCDD1 transcripts are highly up-regulated. We have determined the three-dimensional structure of the extracellular domain of APCDD1, and this structure reveals an unusual architecture consisting of two closely apposed β-barrel domains (ABD1 and ABD2). ABD2, but not ABD1, has a large hydrophobic pocket that accommodates a bound lipid. The APCDD1 ECD can also bind to WNT7A, presumably via its covalently bound palmitoleate, a modification that is common to all WNTs and is essential for signaling. This work suggests that APCDD1 functions as a negative feedback regulator by titrating WNT ligands at the surface of responding cells.

Abstract 2717: A potent and highly tumor-selective KRAS inactivator for tumor targeting

Abstract Cancer driver mutations in the RAS-RAF-MEK-ERK pathway occur in 46% of all human cancers. This has inspired the successful development of many small molecule inhibitors of BRAF and MEK. However, development of RAS small molecule inhibitors has not been successful in large part due to the lack of a sufficiently large and deep hydrophobic pocket in RAS proteins to allow for small molecule binding. Notably, in this regard, many bacterial pathogens have evolved potent protein toxins to disrupt the RAS-RAF-MEK-ERK pathway. Fortunately, these naturally designed potent toxins, such as anthrax toxin, Fortunately, these potent, naturally designed toxins, such as anthrax toxin and DUF5, can be structurally modified to achieve high tumor specificity. DUF5 (amino acids Glu3581-Gln4089) is a toxin effector domain of MARTX (a multifunctional-autoprocessing repeats-in-toxin) from Vibrio vulnificus. DUF5 was recently identified to be a specific and potent endopeptidase that cleaves and inactivates RAS. To address the above critical unmet medical needs, in this work, we have generated an anthrax toxin/DUF5-based, highly tumor-selective RAS inactivator and evaluated its anti-tumor activity in a variety of tumor models. This tumor-selective RAS inactivator can specifically bind to the major anthrax toxin receptor CMG2 on tumor cells and tumor stromal cells, and strictly relies on the simultaneous presence of two distinct tumor-associated proteases, MMPs and urokinase, to gain entry into tumor cells and tumor stromal cells to proteolytically inactivate RAS signaling, achieving potent selective targeting. Our data show that our tumor-selective RAS inactivator displays potent cytotoxicity to cancer cells whose survival depends on oncogenic RAS signaling. Our engineered toxin displayed potent in vivo antitumor activity to a range of tumors with oncogenic RAS mutations in syngeneic and human xenograft mouse tumor models. Therefore, we have developed a tumor-selective, safe, and highly potent RAS inactivator that has high potential for targeting tumors with oncogenic RAS mutations. Citation Format: Shihui Liu, Zehua Zuo, Jie Liu. A potent and highly tumor-selective KRAS inactivator for tumor targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2717.

Bioluminescent imaging systems boosting near-infrared signals in mammalian cells.

Bioluminescence (BL) is broadly used as an optical readout in bioassays and molecular imaging. In this study, the near-infrared (NIR) BL imaging systems were developed. The system was harnessed by prototype copepod luciferases, artificial luciferase 30 (ALuc30) and its miniaturized version picALuc, and were characterized with 17 kinds of coelenterazine (CTZ) analogues carrying bulky functional groups or cyanine 5 (Cy5). They were analyzed of BL spectral peaks and enzymatic kinetics, and explained with computational modeling. The results showed that (1) the picALuc-based system surprisingly boosts the BL intensities predominantly in the red and NIR region with its specific CTZ analogues; (2) both ALuc30- and picALuc-based systems develop unique through-bond energy transfer (TBET)-driven spectral bands in the NIR region with a Cy5-conjugated CTZ analogue (Cy5-CTZ); and (3) according to the computational modeling, the miniaturized version, picALuc, has a large binding pocket, which can accommodate CTZ analogues containing bulky functional groups and thus allowing NIR BL. This study is an important addition to the BL imaging toolbox with respect to the development of orthogonal NIR reporter systems applicable to physiological samples, together with the understanding of the BL-emitting chemistry of marine luciferases.