Development of non-ß-Lactam covalent allosteric inhibitors targeting PBP2a in Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA), a Gram-positive bacterial pathogen, continues to pose a serious threat to the current public health system in our society. The high level of resistance to ß-lactam antibiotics in MRSA is attributed to the expression of penicillin-binding protein 2a (PBP2a), which catalyzes cell wall cross-linking. According to numerous research reports, the activity of the PBP2a protein is known to be regulated by an allosteric site distinct from the active site where cell wall cross-linking occurs. Here, we conducted a screening of 113 compounds containing a 1,3,4-oxadiazole core to design new covalent inhibitors targeting the allosteric site of PBP2a and establish their structural-activity relationship. The stereochemically selective synthesis of sulfonyl oxadiazole compounds identified in the initial screening resulted in a maximum eightfold enhancement in cell inhibition activity. The sulfonyl oxadiazole-based compounds formulated as PEG-based ointments, with low toxicity test results on human cells (CC 50 : >78µM), demonstrated potent antimicrobial effects not only in a mouse skin wound infection model but also against oxacillin-resistant clinical isolate MRSA (IC 50 ≈ 1µM), as evidenced by the results. Furthermore, additional studies utilizing LC-MS/MS and in-silico approaches clearly support the allosteric site covalent binding mechanism through the nucleophilic aromatic substitution (S N Ar) reaction, as well as its association with the closure of the major active site of PBP2a.

Carbapenem-induced ß-lactamase-isoform expression trends in Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii (CRAb) is an urgent bacterial threat to public health, with only a few treatment options and a >50% fatality rate. Although several resistance mechanisms are understood, the appearance of these mutations is generally considered stochastic. Recent reports have, however, begun to challenge this assumption. Here, we demonstrate that independent samples of Ab, exposed to different carbapenems with escalating concentrations, show concentration- and carbapenem-dependent trends in ß-lactamase-isoform expression. This result, based on the isoforms identified through label-free-quantification LC-MS/MS measurements of cell-free, gel-separated ß-lactamases, suggests that the appearance of antibiotic resistance may be somewhat non-stochastic. Specifically, several minor AmpC/ADC ß-lactamase-isoforms were found to exhibit both dose- and carbapenem-dependent expression, suggesting the possibility of non-stochastic mutations. Additionally, these also have high sequence similarity to major expressed isoforms, indicating a potential path over which resistance occurred in independent samples. Antibiotic resistance maybe somewhat antibiotic-directed by a hitherto unknown mechanism and further investigation may lead to new strategies for mitigating antibiotic resistance. Teaser: The emergence of antibiotic-resistant ß-lactamase proteins from mutations may exhibit patterns based on specific antibiotics.

Library Screening, In Vivo Confirmation, and Structural and Bioinformatic Analysis of Pentapeptide Sequences as Substrates for Protein Farnesyltransferase.

Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This process often causes proteins to associate with the membrane and participate in signal transduction pathways. The most common substrates of FTase are proteins that have C-terminal tetrapeptide CaaX box sequences where the cysteine is the site of modification. However, recent work has shown that five amino acid sequences can also be recognized, including the pentapeptides CMIIM and CSLMQ. In this work, peptide libraries were initially used to systematically vary the residues in those two parental sequences using an assay based on Matrix Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS). In addition, 192 pentapeptide sequences from the human proteome were screened using that assay to discover additional extended CaaaX-box motifs. Selected hits from that screening effort were rescreened using an in vivo yeast reporter protein assay. The X-ray crystal structure of CMIIM bound to FTase was also solved, showing that the C-terminal tripeptide of that sequence interacted with the enzyme in a similar manner as the C-terminal tripeptide of CVVM, suggesting that the tripeptide comprises a common structural element for substrate recognition in both tetrapeptide and pentapeptide sequences. Molecular dynamics simulation of CMIIM bound to FTase further shed light on the molecular interactions involved, showing that a putative catalytically competent Zn(II)-thiolate species was able to form. Bioinformatic predictions of tetrapeptide (CaaX-box) reactivity correlated well with the reactivity of pentapeptides obtained from in vivo analysis, reinforcing the importance of the C-terminal tripeptide motif. This analysis provides a structural framework for understanding the reactivity of extended CaaaX-box motifs and a method that may be useful for predicting the reactivity of additional FTase substrates bearing CaaaX-box sequences.

In Vitro and In Silico Explorations of the Protein Conformational Changes of Corynebacterium glutamicum MshA, a Model Retaining GT-B Glycosyltransferase.

MshA is a GT-B glycosyltransferase catalyzing the first step in the biosynthesis of mycothiol. While many GT-B enzymes undergo an open-to-closed transition, MshA is unique because its 97° rotation is beyond the usual range of 10-25°. Molecular dynamics (MD) simulations were carried out for MshA in both ligand bound and unbound states to investigate the effect of ligand binding on localized protein dynamics and its conformational free energy landscape. Simulations showed that both the unliganded "opened" and liganded "closed" forms of the enzyme sample a wide degree of dihedral angles and interdomain distances with relatively low overlapping populations. Calculation of the free energy surface using replica exchange MD for the apo "opened" and an artificial generated apo "closed" structure revealed overlaps in the geometries sampled, allowing calculation of a barrier of 2 kcal/mol for the open-to-closed transition in the absence of ligands. MD simulations of fully liganded MshA revealed a smaller sampling of the dihedral angles. The localized protein fluctuation changes suggest that UDP-GlcNAc binding activates the motions of loops in the 1-l-myo-inositol-1-phosphate (I1P)-binding site despite little change in the interactions with UDP-GlcNAc. Circular dichroism, intrinsic fluorescence spectroscopy, and mutagenesis studies were used to confirm the ligand-induced structural changes in MshA. The results support a proposed mechanism where UDP-GlcNAc binds with rigid interactions to the C-terminal domain of MshA and activates flexible loops in the N-terminal domain for binding and positioning of I1P. This model can be used for future structure-based drug development of inhibitors of the mycothiol biosynthetic pathway.

Functional profiling of the G protein-coupled receptor C3aR1 reveals ligand-mediated biased agonism.

G protein-coupled receptors (GPCRs) are leading druggable targets for several medicines, but many GPCRs are still untapped for their therapeutic potential due to poor understanding of specific signaling properties. The complement C3a receptor 1 (C3aR1) has been extensively studied for its physiological role in C3a-mediated anaphylaxis/inflammation, and in TLQP-21-mediated lipolysis, but direct evidence for the functional relevance of the C3a and TLQP-21 ligands and signal transduction mechanisms are still limited. In addition, C3aR1 G protein coupling specificity is still unclear, and whether endogenous ligands, or drug-like compounds, show ligand-mediated biased agonism is unknown. Here, we demonstrate that C3aR1 couples preferentially to Gi/o/z proteins and can recruit ß-arrestins to cause internalization. Furthermore, we showed that in comparison to C3a63-77, TLQP-21 exhibits a preference toward Gi/o-mediated signaling compared to ß-arrestin recruitment and internalization. We also show that the purported antagonist SB290157 is a very potent C3aR1 agonist, where antagonism of ligand-stimulated C3aR1 calcium flux is caused by potent ß-arrestin-mediated internalization. Finally, ligand-mediated signaling bias impacted cell function as demonstrated by the regulation of calcium influx, lipolysis in adipocytes, phagocytosis in microglia, and degranulation in mast cells. Overall, we characterize C3aR1 as a Gi/o/z-coupled receptor and demonstrate the functional relevance of ligand-mediated signaling bias in key cellular models. Due to C3aR1 and its endogenous ligands being implicated in inflammatory and metabolic diseases, these results are of relevance toward future C3aR1 drug discovery.

Characterization of a novel inhibitor for the New Delhi metallo-ß-lactamase-4: Implications for drug design and combating bacterial drug resistance.

The bacterial metallo-ß-lactamases (MBLs) catalyze the inactivation of ß-lactam antibiotics. Identifying novel pharmacophores remains crucial for the clinical development of additional MBL inhibitors. Previously, 1-hydroxypyridine-2(1H)-thione-6-carboxylic acid, hereafter referred to as 1,2-HPT-6-COOH, was reported as a low cytotoxic nanomolar ß-lactamase inhibitor of Verona-integron-encoded metallo-ß-lactamase 2, capable of rescuing ß-lactam antibiotic activity. In this study, we explore its exact mechanism of inhibition and the extent of its activity through structural characterization of its binding to New Delhi metallo-ß-lactamase 4 (NDM-4) and its inhibitory activity against both NDM-1 and NDM-4. Of all the structure-validated MBL inhibitors available, 1,2-HPT-6-COOH is the first discovered compound capable of forming an octahedral coordination sphere with Zn2 of the binuclear metal center. This unexpected mechanism of action provides important insight for the further optimization of 1,2-HPT-6-COOH and the identification of additional pharmacophores for MBL inhibition.

Targeted and selective knockout of the TLQP-21 neuropeptide unmasks its unique role in energy homeostasis.

OBJECTIVE: Pro-peptide precursors are processed into biologically active peptide hormones or neurotransmitters, each playing an essential role in physiology and disease. Genetic loss of function of a pro-peptide precursor results in the simultaneous ablation of all biologically-active peptides within that precursor, often leading to a composite phenotype that can be difficult to align with the loss of specific peptide components. Due to this biological constraint and technical limitations, mice carrying the selective ablation of individual peptides encoded by pro-peptide precursor genes, while leaving the other peptides unaffected, have remained largely unaddressed. METHODS: We developed and characterized a mouse model carrying the selective knockout of the TLQP-21 neuropeptide (ΔTLQP-21) encoded by the Vgf gene. To achieve this goal, we used a knowledge-based approach by mutating a codon in the Vgf sequence leading to the substitution of the C-terminal Arginine of TLQP-21, which is the pharmacophore as well as an essential cleavage site from its precursor, into Alanine (R21→A). RESULTS: We provide several independent validations of this mouse, including a novel in-gel digestion targeted mass spectrometry identification of the unnatural mutant sequence, exclusive to the mutant mouse. ΔTLQP-21 mice do not manifest gross behavioral and metabolic abnormalities and reproduce well, yet they have a unique metabolic phenotype characterized by an environmental temperature-dependent resistance to diet-induced obesity and activation of the brown adipose tissue. CONCLUSIONS: The ΔTLQP-21 mouse line can be a valuable resource to conduct mechanistic studies on the necessary role of TLQP-21 in physiology and disease, while also serving as a platform to test the specificity of novel antibodies or immunoassays directed at TLQP-21. Our approach also has far-reaching implications by informing the development of knowledge-based genetic engineering approaches to generate selective loss of function of other peptides encoded by pro-hormones genes, leaving all other peptides within the pro-protein precursor intact and unmodified.

A novel strategy to characterize the pattern of ß-lactam antibiotic-induced drug resistance in Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii (CRAb) is an urgent public health threat, according to the CDC. This pathogen has few treatment options and causes severe nosocomial infections with > 50% fatality rate. Although previous studies have examined the proteome of CRAb, there have been no focused analyses of dynamic changes to ß-lactamase expression that may occur due to drug exposure. Here, we present our initial proteomic study of variation in ß-lactamase expression that occurs in CRAb with different ß-lactam antibiotics. Briefly, drug resistance to Ab (ATCC 19606) was induced by the administration of various classes of ß-lactam antibiotics, and the cell-free supernatant was isolated, concentrated, separated by SDS-PAGE, digested with trypsin, and identified by label-free LC-MS-based quantitative proteomics. Thirteen proteins were identified and evaluated using a 1789 sequence database of Ab ß-lactamases from UniProt, the majority of which were Class C ß-lactamases (≥ 80%). Importantly, different antibiotics, even those of the same class (e.g. penicillin and amoxicillin), induced non-equivalent responses comprising various isoforms of Class C and D serine-ß-lactamases, resulting in unique resistomes. These results open the door to a new approach of analyzing and studying the problem of multi-drug resistance in bacteria that rely strongly on ß-lactamase expression.

Targeted and selective knockout of the TLQP-21 neuropeptide unmasks its unique role in energy homeostasis.

Pro-peptide precursors are processed into biologically active peptide hormones or neurotransmitters, each playing an essential role in physiology and disease. Genetic loss of function of a pro-peptide precursor results in the simultaneous ablation of all biologically-active peptides within that precursor, often leading to a composite phenotype that can be difficult to align with the loss of specific peptide components. Due to this biological constraint and technical limitations, mice carrying the selective ablation of individual peptides encoded by pro-peptide precursor genes, while leaving the other peptides unaffected, have remained largely unaddressed. Here, we developed and characterized a mouse model carrying the selective knockout of the TLQP-21 neuropeptide (ΔTLQP-21) encoded by the Vgf gene. To achieve this goal, we used a knowledge-based approach by mutating a codon in the Vgf sequence leading to the substitution of the C-terminal Arginine of TLQP-21, which is the pharmacophore as well as an essential cleavage site from its precursor, into Alanine (R 21 →A). We provide several independent validations of this mouse, including a novel in-gel digestion targeted mass spectrometry identification of the unnatural mutant sequence, exclusive to the mutant mouse. ΔTLQP-21 mice do not manifest gross behavioral and metabolic abnormalities and reproduce well, yet they have a unique metabolic phenotype characterized by a temperature-dependent resistance to diet-induced obesity and activation of the brown adipose tissue.

A novel strategy to characterize the pattern of ß-lactam antibiotic-induced drug resistance in Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii (CRAb) is an urgent public health threat, according to the CDC. This pathogen has few treatment options and causes severe nosocomial infections with > 50% fatality rate. Although previous studies have examined the proteome of CRAb, there have been no focused analyses of dynamic changes to ß-lactamase expression that may occur due to drug exposure. Here, we present our initial proteomic study of variation in ß-lactamase expression that occurs in CRAb with different ß-lactam antibiotics. Briefly, drug resistance to Ab (ATCC 19606) was induced by the administration of various classes of ß-lactam antibiotics, and the cell-free supernatant was isolated, concentrated, separated by SDS-PAGE, digested with trypsin, and identified by label-free LC-MS-based quantitative proteomics. Peptides were identified and evaluated using a 1789 sequence database of Ab ß-lactamases from UniProt. Importantly, we observed that different antibiotics, even those of the same class ( e.g. penicillin and amoxicillin), induce non-equivalent responses comprising various Class C and D serine-ß-lactamases, resulting in unique resistomes. These results open the door to a new approach of analyzing and studying the problem of multi-drug resistance in bacteria that rely strongly on ß-lactamase expression.

Kinetic Characterization and Computational Modeling of Escherichia coli Heptosyltransferase II: Exploring the Role of Protein Dynamics in Catalysis for GT-B Glycosyltransferase.

Glycosyltransferase (GT) enzymes promote the formation of glycosidic bonds between a sugar molecule and a diversity of substrates. Heptosyltransferase II (HepII) is a GT involved in the lipopolysaccharide (LPS) biosynthetic pathway that transfers the seven-carbon sugar (l-glycero-d-manno-heptose, Hep) onto a lipid-anchored glycopolymer (heptosylated Kdo2-Lipid A, Hep-Kdo2-Lipid A, or HLA). LPS plays a key role in Gram-negative bacterial sepsis, biofilm formation, and host colonization, and as such, LPS biosynthetic enzymes are targets for novel antimicrobial therapeutics. Three heptosyltransferases are involved in the inner-core LPS biosynthesis, with Escherichia coli HepII being the last to be quantitatively characterized in vivo. HepII shares modest sequence similarity with heptosyltransferase I (HepI) while maintaining a high degree of structural homology. Here, we report the first kinetic and biophysical characterization of HepII and demonstrate the properties of HepII that are shared with HepI, including sugar donor promiscuity and sugar acceptor-induced secondary structural changes, which results in significant thermal stabilization. HepII also has an increased catalytic efficiency and a significantly tighter binding affinity for both of its substrates compared to HepI. A structural model of the HepII ternary complex, refined by molecular dynamics simulations, was developed to probe the potentially important substrate-protein contacts. Ligand binding-induced changes in Trp fluorescence in HepII enabled the determination of substrate dissociation constants. Combined, these efforts meaningfully enhance our understanding of the heptosyltransferase family of enzymes and will aid in future efforts to design novel, potent, and specific inhibitors for this family of enzymes.

Discovery of first-in-class nanomolar inhibitors of heptosyltransferase I reveals a new aminoglycoside target and potential alternative mechanism of action.

A clinically relevant inhibitor for Heptosyltransferase I (HepI) has been sought after for many years because of its critical role in the biosynthesis of lipopolysaccharides on bacterial cell surfaces. While many labs have discovered or designed novel small molecule inhibitors, these compounds lacked the bioavailability and potency necessary for therapeutic use. Extensive characterization of the HepI protein has provided valuable insight into the dynamic motions necessary for catalysis that could be targeted for inhibition. Structural inspection of Kdo2-lipid A suggested aminoglycoside antibiotics as potential inhibitors for HepI. Multiple aminoglycosides have been experimentally validated to be first-in-class nanomolar inhibitors of HepI, with the best inhibitor demonstrating a Ki of 600 ± 90 nM. Detailed kinetic analyses were performed to determine the mechanism of inhibition while circular dichroism spectroscopy, intrinsic tryptophan fluorescence, docking, and molecular dynamics simulations were used to corroborate kinetic experimental findings. While aminoglycosides have long been described as potent antibiotics targeting bacterial ribosomes' protein synthesis leading to disruption of the stability of bacterial cell membranes, more recently researchers have shown that they only modestly impact protein production. Our research suggests an alternative and novel mechanism of action of aminoglycosides in the inhibition of HepI, which directly leads to modification of LPS production in vivo. This finding could change our understanding of how aminoglycoside antibiotics function, with interruption of LPS biosynthesis being an additional and important mechanism of aminoglycoside action. Further research to discern the microbiological impact of aminoglycosides on cells is warranted, as inhibition of the ribosome may not be the sole and primary mechanism of action. The inhibition of HepI by aminoglycosides may dramatically alter strategies to modify the structure of aminoglycosides to improve the efficacy in fighting bacterial infections.

Ligand-Induced Conformational and Dynamical Changes in a GT-B Glycosyltransferase: Molecular Dynamics Simulations of Heptosyltransferase I Complexes.

Understanding the dynamical motions and ligand recognition motifs of heptosyltransferase I (HepI) can be critical to discerning the behavior of other glycosyltransferase (GT) enzymes. Prior studies in our lab have demonstrated that GTs in the GT-B structural class, which are characterized by their connection of two Rossman-like domains by a linker region, have conserved structural fold and dynamical motions, despite low sequence homology, therefore making discoveries found in HepI transferable to other GT-B enzymes. Through molecular dynamics simulations and ligand binding free energy analysis of HepI in the apo and bound complexes (for all kinetically relevant combinations of the native substrates/products), we have determined the energetically favored enzymatic pathway for ligand binding and release. Our principal component, dynamic cross correlation, and network analyses of the simulations have revealed correlated motions involving residues within the N-terminal domain communicating with C-terminal domain residues via both proximal amino acid residues and also functional groups of the bound substrates. Analyses of the structural changes, energetics of substrate/product binding, and changes in pKa have elucidated a variety of inter and intradomain interactions that are critical for enzyme catalysis. These data corroborate our experimental observations of protein conformational changes observed in both presteady state kinetic and circular dichroism analyses of HepI. These simulations provided invaluable structural insights into the regions involved in HepI conformational rearrangement upon ligand binding. Understanding the specific interactions governing conformational changes is likely to enhance our efforts to develop novel dynamics disrupting inhibitors against GT-B structural enzymes in the future.

The molecular identity of the TLQP-21 peptide receptor.

The TLQP-21 neuropeptide has been implicated in functions as diverse as lipolysis, neurodegeneration and metabolism, thus suggesting an important role in several human diseases. Three binding targets have been proposed for TLQP-21: C3aR1, gC1qR and HSPA8. The aim of this review is to critically evaluate the molecular identity of the TLQP-21 receptor and the proposed multi-receptor mechanism of action. Several studies confirm a critical role for C3aR1 in TLQP-21 biological activity and a largely conserved mode of binding, receptor activation and signaling with C3a, its first-identified endogenous ligand. Conversely, data supporting a role of gC1qR and HSPA8 in TLQP-21 activity remain limited, with no signal transduction pathways being described. Overall, C3aR1 is the only receptor for which a necessary and sufficient role in TLQP-21 activity has been confirmed thus far. This conclusion calls into question the validity of a multi-receptor mechanism of action for TLQP-21 and should inform future studies.

Fragment-based screening and hit-based substructure search: Rapid discovery of 8-hydroxyquinoline-7-carboxylic acid as a low-cytotoxic, nanomolar metallo ß-lactamase inhibitor.

Metallo-ß-lactamases (MBLs) are zinc-containing carbapenemases that inactivate a broad range of ß-lactam antibiotics. There is a lack of ß-lactamase inhibitors for restoring existing ß-lactam antibiotics arsenals against common bacterial infections. Fragment-based screening of a non-specific metal chelator library demonstrates 8-hydroxyquinoline as a broad-spectrum nanomolar inhibitor against VIM-2 and NDM-1. A hit-based substructure search provided an early structure-activity relationship of 8-hydroxyquinolines and identified 8-hydroxyquinoline-7-carboxylic acid as a low-cytotoxic ß-lactamase inhibitor that can restore ß-lactam activity against VIM-2-expressing E. coli. Molecular modeling further shed structural insight into its potential mode of binding within the dinuclear zinc active site. 8-Hydroxyquinoline-7-carboxylic acid is highly stable in human plasma and human liver microsomal study, making it an ideal lead candidate for further development.

CD200 Immune-Checkpoint Peptide Elicits an Anti-glioma Response Through the DAP10 Signaling Pathway.

Numerous therapies aimed at driving an effective anti-glioma response have been employed over the last decade; nevertheless, survival outcomes for patients remain dismal. This may be due to the expression of immune-checkpoint ligands such as PD-L1 by glioblastoma (GBM) cells which interact with their respective receptors on tumor-infiltrating effector T cells curtailing the activation of anti-GBM CD8+ T cell-mediated responses. Therefore, a combinatorial regimen to abolish immunosuppression would provide a powerful therapeutic approach against GBM. We developed a peptide ligand (CD200AR-L) that binds an uncharacterized CD200 immune-checkpoint activation receptor (CD200AR). We sought to test the hypothesis that CD200AR-L/CD200AR binding signals via he DAP10&12 pathways through in vitro studies by analyzing transcription, protein, and phosphorylation, and in vivo loss of function studies using inhibitors to select signaling molecules. We report that CD200AR-L/CD200AR binding induces an initial activation of the DAP10&12 pathways followed by a decrease in activity within 30 min, followed by reactivation via a positive feedback loop. Further in vivo studies using DAP10&12KO mice revealed that DAP10, but not DAP12, is required for tumor control. When we combined CD200AR-L with an immune-stimulatory gene therapy, in an intracranial GBM model in vivo, we observed increased median survival, and long-term survivors. These studies are the first to characterize the signaling pathway used by the CD200AR, demonstrating a novel strategy for modulating immune checkpoints for immunotherapy currently being analyzed in a phase I adult trial.

Conserved Conformational Hierarchy across Functionally Divergent Glycosyltransferases of the GT-B Structural Superfamily as Determined from Microsecond Molecular Dynamics.

It has long been understood that some proteins undergo conformational transitions en route to the Michaelis Complex to allow chemistry. Examination of crystal structures of glycosyltransferase enzymes in the GT-B structural class reveals that the presence of ligand in the active site triggers an open-to-closed conformation transition, necessary for their catalytic functions. Herein, we describe microsecond molecular dynamics simulations of two distantly related glycosyltransferases that are part of the GT-B structural superfamily, HepI and GtfA. Simulations were performed using the open and closed conformations of these unbound proteins, respectively, and we sought to identify the major dynamical modes and communication networks that interconnect the open and closed structures. We provide the first reported evidence within the scope of our simulation parameters that the interconversion between open and closed conformations is a hierarchical multistep process which can be a conserved feature of enzymes of the same structural superfamily. Each of these motions involves of a collection of smaller molecular reorientations distributed across both domains, highlighting the complexities of protein dynamic involved in the interconversion process. Additionally, dynamic cross-correlation analysis was employed to explore the potential effect of distal residues on the catalytic efficiency of HepI. Multiple distal nonionizable residues of the C-terminal domain exhibit motions anticorrelated to positively charged residues in the active site in the N-terminal domain involved in substrate binding. Mutations of these residues resulted in a reduction in negatively correlated motions and an altered enzymatic efficiency that is dominated by lower Km values with kcat effectively unchanged. The findings suggest that residues with opposing conformational motions involved in the opening and closing of the bidomain HepI protein can allosterically alter the population and conformation of the "closed" state, essential to the formation of the Michaelis complex. The stabilization effects of these mutations likely equally influence the energetics of both the ground state and the transition state of the catalytic reaction, leading to the unaltered kcat. Our study provides new insights into the role of conformational dynamics in glycosyltransferase's function and new modality to modulate enzymatic efficiency.

NAD+ enhances ribitol and ribose rescue of α-dystroglycan functional glycosylation in human FKRP-mutant myotubes.

Mutations in the fukutin-related protein (FKRP) cause Walker-Warburg syndrome (WWS), a severe form of congenital muscular dystrophy. Here, we established a WWS human induced pluripotent stem cell-derived myogenic model that recapitulates hallmarks of WWS pathology. We used this model to investigate the therapeutic effect of metabolites of the pentose phosphate pathway in human WWS. We show that functional recovery of WWS myotubes is promoted not only by ribitol but also by its precursor ribose. Moreover, we found that the combination of each of these metabolites with NAD+ results in a synergistic effect, as demonstrated by rescue of α-dystroglycan glycosylation and laminin binding capacity. Mechanistically, we found that FKRP residual enzymatic capacity, characteristic of many recessive FKRP mutations, is required for rescue as supported by functional and structural mutational analyses. These findings provide the rationale for testing ribose/ribitol in combination with NAD+ to treat WWS and other diseases associated with FKRP mutations.

Healthy muscles are complex machines that require a myriad of finely tuned molecules to work properly. For instance, a protein called alpha-DG sits at the surface of healthy muscle cells, where it strengthens the tissue by latching onto other proteins in the environment. To perform its role correctly, it first needs to be coated with sugar molecules, a complex process which requires over 20 proteins, including the enzyme FKRP. Faulty forms of FKRP reduce the number of sugars added to alpha-DG, causing the muscle tissue to weaken and waste away, potentially leading to severe forms of diseases known as muscular dystrophies. Drugs that can restore alpha-DG sugar molecules could help to treat these conditions. Previous studies on mice and fish have highlighted two potential candidates, known as ribitol and NAD+, which can help to compensate for reduced FKRP activity and allow sugars to be added to alpha-DG again. Yet no model is available to test these molecules on actual human muscle cells. Here, Ortiz-Cordero et al. developed such a model in the laboratory by growing muscle cells from naïve, undifferentiated cells generated from skin given by a muscular dystrophy patient with a faulty form of FKRP. The resulting muscle fibers are in essence identical to the ones present in the individual. As such, they can help to understand the effect various drugs have on muscular dystrophies. The cells were then put in contact with either NAD+, ribitol, or a precursor of ribitol known as ribose. Ortiz-Cordero et al. found that ribitol and ribose restored the ability of FKRP to add sugars to alpha-DG, reducing muscle damage. Combining NAD+ with ribitol or ribose had an even a bigger impact, further increasing the number of sugars on alpha-DG. The human muscle cell model developed by Ortiz-Cordero et al. could help to identify new compounds that can treat muscular conditions. In particular, the findings point towards NAD+, ribose and ribitol as candidates for treating FKRP-related muscular dystrophies. Further safety studies are now needed to evaluate whether these compounds could be used in patients.