Transition State Analogue Research Articles

Development of an immobilized Mycobacterium tuberculosis purine nucleoside phosphorylase platform for ligand fishing and inhibition assays

Purine nucleoside phosphorylase (PNP) from Mycobacterium tuberculosis (MtPNP) plays a crucial role in purine metabolism, making it an attractive target for developing new tuberculosis treatments. In this study, we developed a ligand screening platform using MtPNP covalently immobilized on magnetic particles (MtPNP-MPs). The immobilization process achieved a high enzyme loading and preserved the enzyme catalytic activity, enabling its use in both activity and affinity-based screening assays. The activity of MtPNP-MPs was monitored by quantifying hypoxanthine released from inosine phosphorolysis, and kinetic studies revealed Michaelis-Menten behavior for inosine and inorganic phosphate substrates, with KM values comparable to those of free MtPNP. A proof-of-concept inhibitor study using the transition state analog DI4G demonstrated the platform capability for recognizing and characterizing inhibitors, yielding an IC50 value of 91.4 nM and a competitive inhibition mechanism with a Ki of 69.2 nM. Furthermore, the MtPNP-MPs exhibited high stability, retaining over 80 % of their activity after six months of storage and more than 90 % after five consecutive reaction cycles, highlighting their potential for reuse in high-throughput assays. We optimized key parameters for ligand fishing assay, including the amount of MtPNP-MPs, incubation time, and elution conditions. While higher organic solvent concentrations and longer elution times improved ligand isolation, these conditions also reduced enzyme activity. This trade-off between ligand isolation yield and enzyme reusability suggests that elution conditions should be tailored based on the ligand binding strength. Overall, this study establishes the MtPNP-MPs platform as a versatile tool for ligand identification and inhibitor characterization, with promising applications in the screening of complex libraries, such as natural products, for bioactive compounds.

Synthesis at the Interface of Chemistry and Biology.

ConspectusChemical synthesis as a tool to control the structure and properties of matter is at the heart of chemistry─from the synthesis of fine chemicals and polymers to drugs and solid-state materials. But as the field evolves to tackle larger and larger molecules and molecular complexes, the traditional tools of synthetic chemistry become limiting. In contrast, Mother Nature has developed very different strategies to create the macromolecules and molecular systems that make up the living cell. Our focus has been to ask whether we can use the synthetic strategies and machinery of Mother Nature, together with modern chemical tools, to create new macromolecules, and even whole organisms with properties not existing in nature. One such example involves reprogramming the complex, multicomponent machinery of ribosomal protein synthesis to add new building blocks to the genetic code, overcoming a billion-year constraint on the chemical nature of proteins. This methodology exploits the concept of bioorthogonality to add unique codons, tRNAs and aminoacyl-tRNA synthetases to cells to encode amino acids with physical, chemical and biological properties not found in nature. As a result, we can make precise changes to the structures of proteins, much like those made by chemists to small molecules and beyond those possible by biological approaches alone. This technology has made it possible to probe protein structure and function in vitro and in vivo in ways heretofore not possible, and to make therapeutic proteins with enhanced pharmacology. A second example involves exploiting the molecular diversity of the humoral immune system together with synthetic transition state analogues to make catalytic antibodies, and then expanding this diversity-based strategy (new to chemists at the time) to drug discovery and materials science. This work ushered in a new nature-inspired synthetic strategy in which large libraries of natural or synthetic molecules are designed and then rationally selected or screened for new function, increasing the efficiency by which we can explore chemical space for new physical, chemical and biological properties. A final example is the use of large chemical libraries, robotics and high throughput phenotypic cellular screens to identify small synthetic molecules that can be used to probe and manipulate the complex biology of the cell, exemplified by druglike molecules that control cell fate. This approach provides new insights into complex biology that complements genomic approaches and can lead to new drugs that act by novel mechanisms of action, for example to selectively regenerate tissues. These and other advances have been made possible by using our knowledge of molecular structure and reactivity hand in hand with our understanding of and ability to manipulate the complex machinery of living cells, opening a new frontier in synthesis. This Account overviews the work in my lab and with our collaborators, from our early days to the present, that revolves around this central theme.

Molecular mechanism of thiocyanate dehydrogenase at atomic resolution

Some sulfur-oxidizing bacteria playing an important role in global geochemical cycles utilize thiocyanate as the sole source of energy and nitrogen. In these bacteria the process of thiocyanate into cyanate conversion is mediated by thiocyanate dehydrogenases — a recently discovered family of copper-containing enzymes with the three‑copper active site unique among the other copper proteins. To get a deeper insight into the structure and molecular mechanism of action of thiocyanate dehydrogenases we isolated, purified, and comprehensively characterized an enzyme from the bacterium Pelomicrobium methylotrophicum. High-resolution crystal structures of the thiocyanate dehydrogenase in the free state and in the complexes with the transition state analog, thiourea, and the closest substrate analog, selenocyanate, unveiled the fine details of molecular events occurring at the enzyme active site. During the reaction thiocyanate dehydrogenase undergoes profound conformational change that affects the position of the constituent copper ions and results in the activation of the attacking water molecule. The structure of the enzyme complex with the selenium atom bridged in-between two copper ions was obtained representing an important transient intermediate. Structures of the complexes with inhibitors supplemented with quantum chemical calculations clarify the role of copper ions and refine molecular mechanism of catalysis by thiocyanate dehydrogenase.

Biocatalytic Synthesis of Ruxolitinib Intermediate via Engineered Imine Reductase.

An enzyme catalyzed strategy for the synthesis of a chiral hydrazine from 3-cyclopentyl-3-oxopropanenitrile 5 and hydrazine hydrate 2 is presented. An imine reductase (IRED) from Streptosporangium roseum was identified to catalyze the reaction between 3-cyclopentyl-3-oxopropanenitrile 5 and hydrazine hydrate 2 to produce trace amounts of (R)-3-cyclopentyl-3-hydrazineylpropanenitrile 4. We employed a 2-fold approach to optimize the catalytic performance of this enzyme. First, a transition state analogue (TSA) model was constructed to illuminate the enzyme-substrate interactions. Subsequently, the Enzyme_design and Funclib methods were utilized to predict mutants for experimental evaluation. Through three rounds of site-directed mutagenesis, site saturation mutagenesis, and combinatorial mutagenesis, we obtained mutant M6 with a yield of 98% and an enantiomeric excess (ee) of 99%. This study presents an effective method for constructing a hydrazine derivative via IRED-catalyzed reductive amination of ketone and hydrazine. Furthermore, it provides a general approach for constructing suitable enzymes, starting from nonreactive enzymes and gradually enhancing their catalytic activity through active site modifications.

Elucidating the conformational change of dengue envelope protein using the Markov state model

ABSTRACT Dengue virus, an arbovirus of genus flavivirus, is one of the most prevalent infectious disease causing organisms in the tropical environment leading to numerous deaths every year. The envelope protein plays a crucial role in the viral genome entry into the host cell. This is achieved through structure and oligomeric status change in the dengue envelope protein. The pre-fusion and post-fusion states’ structures of the dengue envelope protein are known although the pathway and intermediate structures involved in this process have remained in the dark till date. In this study, we have used targeted molecular dynamics (MD) simulation followed by conventional MD simulation and the Markov state model analysis to elucidate this pathway of change in conformation from the pre-fusion state to the post-fusion state. Intermediate conformations which may in the future serve as therapeutic targets have also been deduced from the Markov state model. This pathway determination may pave the way for the design of transition state analog inhibitors which prevent the dengue virus envelope protein from changing its conformation and thus establishing infection. The results obtained could lead to development of novel drugs to combat the viral infection.

Tubulin code eraser CCP5 binds branch glutamates by substrate deformation.

Microtubule function is modulated by the tubulin code, diverse posttranslational modifications that are altered dynamically by writer and eraser enzymes1. Glutamylation-the addition of branched (isopeptide-linked) glutamate chains-is the most evolutionarily widespread tubulin modification2. It is introduced by tubulin tyrosine ligase-like enzymes and erased by carboxypeptidases of the cytosolic carboxypeptidase (CCP) family1. Glutamylation homeostasis, achieved through the balance of writers and erasers, is critical for normal cell function3-9, and mutations in CCPs lead to human disease10-13. Here we report cryo-electron microscopy structures of the glutamylation eraser CCP5 in complex with the microtubule, and X-ray structures in complex with transition-state analogues. Combined with NMR analysis, these analyses show that CCP5 deforms the tubulin main chain into a unique turn that enables lock-and-key recognition of the branch glutamate in a cationic pocket that is unique to CCP family proteins. CCP5 binding of the sequences flanking the branch point primarily through peptide backbone atoms enables processing of diverse tubulin isotypes and non-tubulin substrates. Unexpectedly, CCP5 exhibits inefficient processing of an abundant β-tubulin isotype in the brain. This work provides an atomistic view into glutamate branch recognition and resolution, and sheds light on homeostasis of the tubulin glutamylation syntax.

Small-Molecule Allosteric Inhibitors of Human Aspartate Transcarbamoylase Suppress Proliferation of Bone Osteosarcoma Epithelial Cells.

Aspartate transcarbamoylase (ATC) is the first committed step in de novo pyrimidine biosynthesis in eukaryotes and plants. A potent transition state analog of human ATCase (PALA) has previously been assessed in clinical trials for the treatment of cancer, but was ultimately unsuccessful. Additionally, inhibition of this pathway has been proposed to be a target to suppress cell proliferation in E. coli, the malarial parasite and tuberculosis. In this manuscript we screened a 70-member library of ATC inhibitors developed against the malarial and tubercular ATCases for inhibitors of the human ATC. Four compounds showed low nanomolar inhibition (IC50 30-120 nM) in an in vitro activity assay. These compounds significantly outperform PALA, which has a triphasic inhibition response under identical conditions, in which significant activity remains at PALA concentrations above 10 μM. Evidence for a druggable allosteric pocket in human ATC is provided by both in vitro enzyme kinetic, homology modeling and in silico docking. These compounds also suppress the proliferation of U2OS osteoblastoma cells by promoting cell cycle arrest in G0/G1 phase. This report provides the first evidence for an allosteric pocket in human ATC, which greatly enhances its druggability and demonstrates the potential of this series in cancer therapy.

1H, 13C, and 15N backbone and methyl group resonance assignments of ricin toxin A subunit

Ricin is a potent plant toxin that targets the eukaryotic ribosome by depurinating an adenine from the sarcin-ricin loop (SRL), a highly conserved stem-loop of the rRNA. As a category-B agent for bioterrorism it is a prime target for therapeutic intervention with antibodies and enzyme blocking inhibitors since no effective therapy exists for ricin. Ricin toxin A subunit (RTA) depurinates the SRL by binding to the P-stalk proteins at a remote site. Stimulation of the N-glycosidase activity of RTA by the P-stalk proteins has been studied extensively by biochemical methods and by X-ray crystallography. The current understanding of RTA’s depurination mechanism relies exclusively on X-ray structures of the enzyme in the free state and complexed with transition state analogues. To date we have sparse evidence of conformational dynamics and allosteric regulation of RTA activity that can be exploited in the rational design of inhibitors. Thus, our primary goal here is to apply solution NMR techniques to probe the residue specific structural and dynamic coupling active in RTA as a prerequisite to understand the functional implications of an allosteric network. In this report we present de novo sequence specific amide and sidechain methyl chemical shift assignments of the 267 residue RTA in the free state and in complex with an 11-residue peptide (P11) representing the identical C-terminal sequence of the ribosomal P-stalk proteins. These assignments will facilitate future studies detailing the propagation of binding induced conformational changes in RTA complexed with inhibitors, antibodies, and biologically relevant targets.

The catalytic mechanism of the RNA methyltransferase METTL3.

The complex of methyltransferase-like proteins 3 and 14 (METTL3-14) is the major enzyme that deposits N6-methyladenosine (m6A) modifications on messenger RNA (mRNA) in humans. METTL3-14 plays key roles in various biological processes through its methyltransferase (MTase) activity. However, little is known about its substrate recognition and methyl transfer mechanism from its cofactor and methyl donor S-adenosylmethionine (SAM). Here, we study the MTase mechanism of METTL3-14 by a combined experimental and multiscale simulation approach using bisubstrate analogues (BAs), conjugates of a SAM-like moiety connected to the N6-atom of adenosine. Molecular dynamics simulations based on crystal structures of METTL3-14 with BAs suggest that the Y406 side chain of METTL3 is involved in the recruitment of adenosine and release of m6A. A crystal structure with a BA representing the transition state of methyl transfer shows a direct involvement of the METTL3 side chains E481 and K513 in adenosine binding which is supported by mutational analysis. Quantum mechanics/molecular mechanics (QM/MM) free energy calculations indicate that methyl transfer occurs without prior deprotonation of adenosine-N6. Furthermore, the QM/MM calculations provide further support for the role of electrostatic contributions of E481 and K513 to catalysis. The multidisciplinary approach used here sheds light on the (co)substrate binding mechanism, catalytic step, and (co)product release, and suggests that the latter step is rate-limiting for METTL3. The atomistic information on the substrate binding and methyl transfer reaction of METTL3 can be useful for understanding the mechanisms of other RNA MTases and for the design of transition state analogues as their inhibitors.

The catalytic mechanism of the RNA methyltransferase METTL3

The complex of methyltransferase-like proteins 3 and 14 (METTL3-14) is the major enzyme that deposits N6-methyladenosine (m6A) modifications on messenger RNA (mRNA) in humans. METTL3-14 plays key roles in various biological processes through its methyltransferase (MTase) activity. However, little is known about its substrate recognition and methyl transfer mechanism from its cofactor and methyl donor S-adenosylmethionine (SAM). Here, we study the MTase mechanism of METTL3-14 by a combined experimental and multiscale simulation approach using bisubstrate analogues (BAs), conjugates of a SAM-like moiety connected to the N6-atom of adenosine. Molecular dynamics simulations based on crystal structures of METTL3-14 with BAs suggest that the Y406 side chain of METTL3 is involved in the recruitment of adenosine and release of m6A. A crystal structure with a BA representing the transition state of methyl transfer shows a direct involvement of the METTL3 side chains E481 and K513 in adenosine binding which is supported by mutational analysis. Quantum mechanics/molecular mechanics (QM/MM) free energy calculations indicate that methyl transfer occurs without prior deprotonation of adenosine-N6. Furthermore, the QM/MM calculations provide further support for the role of electrostatic contributions of E481 and K513 to catalysis. The multidisciplinary approach used here sheds light on the (co)substrate binding mechanism, catalytic step, and (co)product release, and suggests that the latter step is rate-limiting for METTL3. The atomistic information on the substrate binding and methyl transfer reaction of METTL3 can be useful for understanding the mechanisms of other RNA MTases and for the design of transition state analogues as their inhibitors.

Snapshots of the Reaction Coordinate of a Thermophilic 2'-Deoxyribonucleoside/ribonucleoside Transferase.

Nucleosides are ubiquitous to life and are required for the synthesis of DNA, RNA, and other molecules crucial for cell survival. Despite the notoriously difficult organic synthesis of nucleosides, 2'-deoxynucleoside analogues can interfere with natural DNA replication and repair and are successfully employed as anticancer, antiviral, and antimicrobial compounds. Nucleoside 2'-deoxyribosyltransferase (dNDT) enzymes catalyze transglycosylation via a covalent 2'-deoxyribosylated enzyme intermediate with retention of configuration, having applications in the biocatalytic synthesis of 2'-deoxynucleoside analogues in a single step. Here, we characterize the structure and function of a thermophilic dNDT, the protein from Chroococcidiopsis thermalis (CtNDT). We combined enzyme kinetics with structural and biophysical studies to dissect mechanistic features in the reaction coordinate, leading to product formation. Bell-shaped pH-rate profiles demonstrate activity in a broad pH range of 5.5-9.5, with two very distinct pKa values. A pronounced viscosity effect on the turnover rate indicates a diffusional step, likely product (nucleobase1) release, to be rate-limiting. Temperature studies revealed an extremely curved profile, suggesting a large negative activation heat capacity. We trapped a 2'-fluoro-2'-deoxyarabinosyl-enzyme intermediate by mass spectrometry and determined high-resolution structures of the protein in its unliganded, substrate-bound, ribosylated, 2'-difluoro-2'-deoxyribosylated, and in complex with probable transition-state analogues. We reveal key features underlying (2'-deoxy)ribonucleoside selection, as CtNDT can also use ribonucleosides as substrates, albeit with a lower efficiency. Ribonucleosides are the building blocks of RNA and other key intracellular metabolites participating in energy and metabolism, expanding the scope of use of CtNDT in biocatalysis.

Polymorphic positions 349 and 725 of the autoimmunity-protective allotype 10 of ER aminopeptidase 1 are key in determining its unique enzymatic properties.

ER aminopeptidase 1 (ERAP1) is a polymorphic intracellular aminopeptidase with key roles in antigen presentation and adaptive immune responses. ERAP1 allotype 10 is highly protective toward developing some forms of autoimmunity and displays unusual functional properties, including very low activity versus some substrates. To understand the molecular mechanisms that underlie the biology of allotype 10, we studied its enzymatic and biophysical properties focusing on its unique polymorphisms V349M and Q725R. Compared to ancestral allotype 1, allotype 10 is much less effective in trimming small substrates but presents allosteric kinetics that ameliorate activity differences at high substrate concentrations. Furthermore, it is inhibited by a transition-state analogue via a non-competitive mechanism and is much less responsive to an allosteric small-molecule modulator. It also presents opposite enthalpy, entropy, and heat capacity of activation compared to allotype 1, and its catalytic rate is highly dependent on viscosity. Polymorphisms V349M and Q725R significantly contribute to the lower enzymatic activity of allotype 10 for small substrates, especially at high substrate concentrations, influence the cooperation between the regulatory and active sites, and regulate viscosity dependence, likely by limiting product release. Overall, our results suggest that allotype 10 is not just an inactive variant of ERAP1 but rather carries distinct enzymatic properties that largely stem from changes at positions 349 and 725. These changes affect kinetic and thermodynamic parameters that likely control rate-limiting steps in the catalytic cycle, resulting in an enzyme optimized for sparing small substrates and contributing to the homeostasis of antigenic epitopes in the ER.

Mutual induced fit transition structure stabilization of corannulene's bowl-to-bowl inversion in a perylene bisimide cyclophane.

Corannulene is known to undergo a fast bowl-to-bowl inversion at r.t. via a planar transition structure (TS). Herein we present the catalysis of this process within a perylene bisimide (PBI) cyclophane composed of chirally twisted, non-planar chromophores, linked by para-xylylene spacers. Variable temperature NMR studies reveal that the bowl-to-bowl inversion is significantly accelerated within the cyclophane template despite the structural non-complementarity between the binding site of the host and the TS of the guest. The observed acceleration corresponds to a decrease in the bowl-to-bowl inversion barrier of 11.6 kJ mol-1 compared to the uncatalyzed process. Comparative binding studies for corannulene (20 π-electrons) and other planar polycyclic aromatic hydrocarbons (PAHs) with 14 to 24 π-electrons were applied to rationalize this barrier reduction. They revealed high binding constants that reach, in tetrachloromethane as a solvent, the picomolar range for the largest guest coronene. Computational models corroborate these experimental results and suggest that both TS stabilization and ground state destabilization contribute to the observed catalytic effect. Hereby, we find a "mutual induced fit" between host and guest in the TS complex, such that mutual geometric adaptation of the energetically favored planar TS and curved π-systems of the host results in an unprecedented non-planar TS of corannulene. Concomitant partial planarization of the PBI units optimizes noncovalent TS stabilization by π-π stacking interactions. This observation of a "mutual induced fit" in the TS of a host-guest complex was further validated experimentally by single crystal X-ray analysis of a host-guest complex with coronene as a qualitative transition state analogue.

Structural analysis and molecular substrate recognition properties of Arabidopsis thaliana ornithine transcarbamylase, the molecular target of phaseolotoxin produced by Pseudomonas syringae.

Halo blight is a plant disease that leads to a significant decrease in the yield of common bean crops and kiwi fruits. The infection is caused by Pseudomonas syringae pathovars that produce phaseolotoxin, an antimetabolite which targets arginine metabolism, particularly by inhibition of ornithine transcarbamylase (OTC). OTC is responsible for production of citrulline from ornithine and carbamoyl phosphate. Here we present the first crystal structures of the plant OTC from Arabidopsis thaliana (AtOTC). Structural analysis of AtOTC complexed with ornithine and carbamoyl phosphate reveals that OTC undergoes a significant structural transition when ornithine enters the active site, from the opened to the closed state. In this study we discuss the mode of OTC inhibition by phaseolotoxin, which seems to be able to act only on the fully opened active site. Once the toxin is proteolytically cleaved, it mimics the reaction transition state analogue to fit inside the fully closed active site of OTC. Additionally, we indicate the differences around the gate loop region which rationally explain the resistance of some bacterial OTCs to phaseolotoxin.

Rationally Designed Novel Phenyloxazoline Synthase Inhibitors: Chemical Synthesis and Biological Evaluation to Accelerate the Discovery of New Antimycobacterial Antibiotics.

The uncontrolled spread of drug-resistant tuberculosis (DR-TB) clinical cases necessitates the urgent discovery of newer chemotypes with novel mechanisms of action. Here, we report the chemical synthesis of rationally designed novel transition-state analogues (TSAs) by targeting the cyclization (Cy) domain of phenyloxazoline synthase (MbtB), a key enzyme of the conditionally essential siderophore biosynthesis pathway. Following bio-assay-guided evaluation of TSA analogues preferentially in iron-deprived and iron-rich media to understand target preferentiality against a panel of pathogenic and non-pathogenic mycobacteria strains, we identified a hit, i.e., TSA-5. Molecular docking, dynamics, and MMPBSA calculations enabled us to comprehend TSA-5's stable binding at the active site pocket of MbtB_Cy and the results imply that the MbtB_Cy binding pocket has a strong affinity for electron-withdrawing functional groups and contributes to stable polar interactions between enzyme and ligand. Furthermore, enhanced intracellular killing efficacy (8 μg/mL) of TSA-5 against Mycobacterium aurum in infected macrophages is noted in comparison to moderate in vitro antimycobacterial efficacy (64 μg/mL) against M. aurum. TSA-5 also demonstrates whole-cell efflux pump inhibitory activity against Mycobacterium smegmatis. Identification of TSA-5 by focusing on the modular MbtB_Cy domain paves the way for accelerating novel anti-TB antibiotic discoveries.

Combined inhibition of MTAP and MAT2a mimics synthetic lethality in tumor models via PRMT5 inhibition

Homozygous 5'-methylthioadenosine phosphorylase (MTAP) deletions occur in approximately 15% of human cancers. Co-deletion of MTAP and methionine adenosyltransferase 2 alpha (MAT2a) induces a synthetic lethal phenotype involving protein arginine methyltransferase 5 (PRMT5) inhibition. MAT2a inhibitors are now in clinical trials for genotypic MTAP-/- cancers, however the MTAP-/- genotype represents fewer than 2% of human colorectal cancers (CRCs), limiting the utility of MAT2a inhibitors in these and other MTAP+/+ cancers. Methylthio-DADMe-immucillin-A (MTDIA) is a picomolar transition state analog inhibitor of MTAP that renders cells enzymatically MTAP-deficient to induce the MTAP-/- phenotype. Here, we demonstrate that MTDIA and MAT2a inhibitor AG-270 combination therapy mimics synthetic lethality in MTAP+/+ CRC cell lines with similar effects in mouse xenografts and without adverse histology on normal tissues. Combination treatment is synergistic with a 104-fold increase in drug potency for inhibition of CRC cell growth in culture. Combined MTDIA and AG-270 decreases S-adenosyl-L-methionine and increases 5'-methylthioadenosine in cells. The increased intracellular methylthioadenosine:S-adenosyl-L-methionine ratio inhibits PRMT5 activity, leading to cellular arrest and apoptotic cell death by causing MDM4 alternative splicing and p53 activation. Combination MTDIA and AG-270 treatment differs from direct inhibition of PRMT5 by GSK3326595 by avoiding toxicity caused by cell death in the normal gut epithelium induced by the PRMT5 inhibitor. The combination of MTAP and MAT2a inhibitors expands this synthetic lethal approach to include MTAP+/+ cancers, especially the remaining 98% of CRCs without the MTAP-/- genotype.

Conformation state-specific monobodies regulate the functions of flexible proteins through conformation trapping.

Synthetic binding proteins have emerged as modulators of protein functions through protein-protein interactions (PPIs). Because PPIs are influenced by the structural dynamics of targeted proteins, investigating whether the synthetic-binders-based strategy is applicable for proteins with large conformational changes is important. This study demonstrates the applicability of monobodies (fibronectin type-III domain-based synthetic binding proteins) in regulating the functions of proteins that undergo tens-of-angstroms-scale conformational changes, using an example of the A55C/C77S/V169C triple mutant (Adktm ; a phosphoryl transfer-catalyzing enzyme with a conformational change between OPEN/CLOSED forms). Phage display successfully developed monobodies that recognize the OPEN form (substrate-unbound form), but not the CLOSED form of Adktm . Two OPEN form-specific clones (OP-2 and OP-4) inhibited Adktm kinase activity. Epitope mapping with a yeast-surface display/flow cytometry indicated that OP-2 binds to the substrate-entry side of Adktm , whereas OP-4 binding occurs at another site. Small angle X-ray scattering coupled with size-exclusion chromatography (SEC-SAXS) indicated that OP-4 binds to the hinge side opposite to the substrate-binding site of Adktm , retaining the whole OPEN-form structure of Adktm . Titration of the OP-4-Adktm complex with Ap5 A, a transition-state analog of Adktm , showed that the conformational shift to the CLOSED form was suppressed although Adktm retained the OPEN-form (i.e., substrate-binding ready form). These results show that OP-4 captures and stabilizes the OPEN-form state, thereby affecting the hinge motion. These experimental results indicate that monobody-based modulators can regulate the functions of proteins that show tens-of-angstroms-scale conformational changes, by trapping specific conformational states generated during large conformational change process that is essential for function exertion.

Synthesis of a novel fluorinated phosphonyl C-glycoside, (3-deoxy-3-fluoro-β-d-glucopyranosyl)methylphosphonate, a potential inhibitor of β-phosphoglucomutase

β-phosphoglucomutase (βPGM) catalyzes the conversion of β-glucose 1-phosphate (βG1P) to glucose-6-phosphate (G6P), a universal source of cellular energy, in a two-step process. Transition state analogue (TSA) complexes formed from substrate analogues and a metal fluoride (MgF3− and AlF4−) enable analysis of each of these enzymatic steps independently. Novel substrate analogues incorporating fluorine offer opportunities to interrogate the enzyme mechanism using 19F NMR spectroscopy. Herein, the synthesis of a novel fluorinated phosphonyl C-glycoside (3-deoxy-3-fluoro-β-d-glucopyranosyl)methylphosphonate (1), in 12 steps (0.85 % overall yield) is disclosed. A four-stage synthetic strategy was employed, involving: 1) fluorine addition to the monosaccharide, 2) selective anomeric deprotection, 3) phosphonylation of the anomeric centre, and 4) global deprotection. Analysis of βPGM and 1 will be reported in due course.

Crystal structures of the DExH-box RNA helicase DHX9.

DHX9 is a DExH-box RNA helicase with versatile functions in transcription, translation, RNA processing and regulation of DNA replication. DHX9 has recently emerged as a promising target for oncology, but to date no mammalian structures have been published. Here, crystal structures of human, dog and cat DHX9 bound to ADP are reported. The three mammalian DHX9 structures share identical structural folds. Additionally, the overall architecture and the individual domain structures of DHX9 are highly conserved with those of MLE, the Drosophila orthologue of DHX9 previously solved in complex with RNA and a transition-state analogue of ATP. Due to differences in the bound substrates and global domain orientations, the localized loop conformations and occupancy of dsRNA-binding domain 2 (dsRBD2) differ between the mammalian DHX9 and MLE structures. The combined effects of the structural changes considerably alter the RNA-binding channel, providing an opportunity to compare active and inactive states of the helicase. Finally, the mammalian DHX9 structures provide a potential tool for structure-based drug-design efforts.

Phosphate Binding in PNP Alters Transition-State Analogue Affinity and Subunit Cooperativity.

Purine nucleoside phosphorylases (PNPs) catalyze the phosphorolysis of 6-oxypurine nucleosides with an HPO42- dianion nucleophile. Nucleosides and phosphate occupy distinct pockets in the PNP active site. Evaluation of the HPO42- site by mutagenesis, cooperative binding studies, and thermodynamic and structural analysis demonstrate that alterations in the HPO42- binding site can render PNP inactive and significantly impact subunit cooperativity and binding to transition-state analogue inhibitors. Cooperative interactions between the cationic transition-state analogue and the anionic HPO42- nucleophile demonstrate the importance of reforming the transition-state ensemble for optimal inhibition with transition-state analogues. Altered phosphate binding in the catalytic site mutants helps to explain one of the known lethal PNP deficiency syndromes in humans.