Myricetin allosterically inhibits Dengue NS2B-NS3 protease as studied by NMR and MD simulations

Dengue NS2B-NS3 protease existing in equilibrium between the active and inactive forms is essential for virus replication, thus representing a key drug target. Here Myricetin, a plant flavonoid, was characterized to non-competitively inhibit Dengue protease. Further NMR study identified the protease residues perturbed by binding to Myricetin, which were utilized to construct the Myricetin-protease complexes. Strikingly, in the active form Myricetin binds a new allosteric site (AS2) far away from the active site pocket and allosteric site (AS1) for binding Curcumin, while in the inactive form it binds both AS1 and AS2. To decipher the mechanism for the allosteric inhibition by Myricetin, we conducted molecular dynamics (MD) simulations on different forms of Dengue NS2B-NS3 protease. Unexpectedly, the binding of Myricetin to AS2 is sufficient to disrupt the active conformation by displacing the characteristic NS2B C-terminal b- hairpin from the active site pocket. By contrast, the binding of Myricetin to AS1 and AS2 results in locking the inactive conformation. Therefore Myricetin represents the first small molecule which allosterically inhibits Dengue protease by both disrupting the active conformation and locking the inactive conformation. The results enforce the notion that a global allosteric network exists in Dengue NS2B-NS3 protease, which is susceptible to allosteric inhibition by small molecules such as Myricetin and Curcumin. As Myricetin has been extensively used as a food additive, it might be directly utilized to fight the Dengue infections and as a promising starting for further design of potent allosteric inhibitors.

Structural and Functional Studies of 3-Deoxy-D-arabino- Heptulosonate 7-Phosphate Synthase from Prevotella nigrescens

<p>Multifunctional enzymes, bearing two or more catalytic activities, provide exceptional contributions to the efficient and coherent function of metabolic pathways. Two main benefits of multifunctional enzymes have been clearly described. Firstly, linked catalytic modules can enhance the overall catalytic rate for consecutive reactions of a metabolic pathway due to substrate channelling. Secondly, the fusion of two protein domains can impart allosteric control, such that the catalytic function of one of the protein domains is altered by a ligand binding to the second, covalently linked domain. This study examines a bifunctional enzyme comprising a 3-deoxy-D-arabino heptulosonate 7-phosphate synthase (DAH7PS) domain covalently fused to a C-terminal chorismate mutase (CM) domain from Prevotella nigrescens (PniDAH7PS). DAH7PS catalyses the first reaction of the shikimate pathway leading to the biosynthesis of aromatic amino acids, whereas CM functions at a pathway branch point, leading to the biosynthesis of tyrosine and phenylalanine. Through the investigation of PniDAH7PS, a special functional interdependence between the two non-consecutive catalytic functionalities and the derived allosteric regulation was unravelled. Chapter 2 generally characterises the biochemical and structural features of PniDAH7PS. The two catalytic activities exhibit substantial hetero-interdependency and the separation of the two distinct catalytic domains results in a dramatic loss of both the DAH7PS and CM enzymatic activities. The structural investigation into this protein revealed a unique dimeric assembly and implicates a hetero-interaction between the DAH7PS and CM domains, providing a structural basis for the functional interdependence. Moreover, allosteric inhibition of DAH7PS by prephenate, the product of the CM-catalysed reaction, was observed. This allostery is accompanied by a striking conformational change, as observed by SAXS, implying that a manipulation of the hetero-domain interaction is the mechanism underpinning the allosteric inhibition. Chapter 3 looks into the mechanism underpinning the DAH7PS and CM functional interdependence. Rearrangements of the conformation of PniDAH7PS following the addition of substrate combinations were observed. This indicates that a dynamic interaction between the DAH7PS and CM domains is important for catalysis. Furthermore, perturbation of these conformational variations by either a truncation mutation in the CM domain or the presence of a high concentration of NaCl interrupted the both the DAH7PS and CM catalytic activities, implying that a dynamic hetero-domain interaction is essential for the delivering the normal DAH7PS and CM functions. This work also reveals a dual role for the DAH7PS domain, exerting catalysis and allosteric activation on the CM activity simultaneously. Chapter 4 investigates the mechanism of the allosteric inhibition of PniDAH7PS by prephenate. The structural effect of prephenate on PniDAH7PS, with the addition of substrate combinations, was inspected, and the results unravelled the same conformation of PniDAH7PS under different conditions, exhibiting high compactness and rigidity. This finding indicates that the probable inhibitory effect of prephenate on PniDAH7PS is realised by freezing the enzyme’s structure in order to deprive PniDAH7PS of the dynamic-dependent catalytic activity. Chapter 5 describes the development of a method for producing segmentally isotopically labelled PniDAH7PS using Expressed Protein Ligation (EPL). This chapter also details attempts to couple this method with small angle neutron scattering (SANS) and nuclear magnetic resonance spectroscopy (NMR) to gain more structural information regarding the catalytic and allosteric properties of PniDAH7PS.</p>

Structural and Functional Studies of 3-Deoxy-D-arabino- Heptulosonate 7-Phosphate Synthase from Prevotella nigrescens

<p>Multifunctional enzymes, bearing two or more catalytic activities, provide exceptional contributions to the efficient and coherent function of metabolic pathways. Two main benefits of multifunctional enzymes have been clearly described. Firstly, linked catalytic modules can enhance the overall catalytic rate for consecutive reactions of a metabolic pathway due to substrate channelling. Secondly, the fusion of two protein domains can impart allosteric control, such that the catalytic function of one of the protein domains is altered by a ligand binding to the second, covalently linked domain. This study examines a bifunctional enzyme comprising a 3-deoxy-D-arabino heptulosonate 7-phosphate synthase (DAH7PS) domain covalently fused to a C-terminal chorismate mutase (CM) domain from Prevotella nigrescens (PniDAH7PS). DAH7PS catalyses the first reaction of the shikimate pathway leading to the biosynthesis of aromatic amino acids, whereas CM functions at a pathway branch point, leading to the biosynthesis of tyrosine and phenylalanine. Through the investigation of PniDAH7PS, a special functional interdependence between the two non-consecutive catalytic functionalities and the derived allosteric regulation was unravelled. Chapter 2 generally characterises the biochemical and structural features of PniDAH7PS. The two catalytic activities exhibit substantial hetero-interdependency and the separation of the two distinct catalytic domains results in a dramatic loss of both the DAH7PS and CM enzymatic activities. The structural investigation into this protein revealed a unique dimeric assembly and implicates a hetero-interaction between the DAH7PS and CM domains, providing a structural basis for the functional interdependence. Moreover, allosteric inhibition of DAH7PS by prephenate, the product of the CM-catalysed reaction, was observed. This allostery is accompanied by a striking conformational change, as observed by SAXS, implying that a manipulation of the hetero-domain interaction is the mechanism underpinning the allosteric inhibition. Chapter 3 looks into the mechanism underpinning the DAH7PS and CM functional interdependence. Rearrangements of the conformation of PniDAH7PS following the addition of substrate combinations were observed. This indicates that a dynamic interaction between the DAH7PS and CM domains is important for catalysis. Furthermore, perturbation of these conformational variations by either a truncation mutation in the CM domain or the presence of a high concentration of NaCl interrupted the both the DAH7PS and CM catalytic activities, implying that a dynamic hetero-domain interaction is essential for the delivering the normal DAH7PS and CM functions. This work also reveals a dual role for the DAH7PS domain, exerting catalysis and allosteric activation on the CM activity simultaneously. Chapter 4 investigates the mechanism of the allosteric inhibition of PniDAH7PS by prephenate. The structural effect of prephenate on PniDAH7PS, with the addition of substrate combinations, was inspected, and the results unravelled the same conformation of PniDAH7PS under different conditions, exhibiting high compactness and rigidity. This finding indicates that the probable inhibitory effect of prephenate on PniDAH7PS is realised by freezing the enzyme’s structure in order to deprive PniDAH7PS of the dynamic-dependent catalytic activity. Chapter 5 describes the development of a method for producing segmentally isotopically labelled PniDAH7PS using Expressed Protein Ligation (EPL). This chapter also details attempts to couple this method with small angle neutron scattering (SANS) and nuclear magnetic resonance spectroscopy (NMR) to gain more structural information regarding the catalytic and allosteric properties of PniDAH7PS.</p>

Allosteric Inhibition of MALT1 Is Efficacious in a Model of Chronic Graft-Versus-Host Disease and Modulates Immunometabolic Activation

Introduction Graft-versus-host disease (GVHD) is a severe complication of allogeneic hematopoietic cell transplantation. In its chronic form, GVHD causes prolonged post-transplant morbidity and significant mortality. There is a paucity of approved therapies and the current standard of care results in incomplete responses that leave significant unmet need for novel agents. Mucosa-associated lymphoid tissue lymphoma translocation protein-1 (MALT1) is a bi-functional protein that acts as a signal-propagation scaffold and protease for selective innate and adaptive immune cell receptor signaling. The established link between MALT1 and receptor-mediated activation of multiple immune cell types involved in cGVHD argues for it as a therapeutic target for cGVHD. Furthermore, we have identified MALT1 signaling to be important in linking receptor-initiated signaling to changes in cellular metabolism necessary for immune cell effector function and propose that immunometabolic profiling of immune cells from cGVHD patients may provide novel biomarkers of both disease pathophysiology and associated MALT1 activity. Methods, Results, Conclusion To assess MALT1 as a novel drug target in cGVHD, we first characterized the effectiveness of allosteric inhibition on protease, scaffolding, and metabolic activity in primary human T cells. MALT1 inhibition of T-cell receptor (TCR) activated CD4 + T cells reduced protease activity by &gt;90% and scaffolding activity by ∼50%, downstream of TCR activation. Consistent with disruption of the TCR signaling cascade, MALT1 inhibition reduced T H1 and T H17 cytokines, and the T FH cytokine IL-21 in a concentration dependent manner. B-cell receptor (BCR) driven proliferation of human B cells and C-type-lectin receptor and immune complex driven cytokine production from human macrophages were also inhibited. We next tested MALT1 inhibition in a sclerodermatous rodent model of cGVHD (scGVHD). Allogeneic bone marrow and splenocytes from LP/J donor mice were transferred to irradiated C57BL/6 recipient mice. Starting on day 21 after transfer, animals were dosed orally once daily with a MALT1 inhibitor, or twice daily with the JAK1/2 inhibitor ruxolitinib as a comparator. All animals were monitored daily for weight change, survival and GVHD score. On a modified scGVHD scoring scale, animals treated with a MALT1 inhibitor had significantly more progression-free survival throughout the study than vehicle or ruxolitinib-treated animals. No differential effect was observed on body weight and overall survival. MALT1 inhibitor treatment, but not ruxolitinib, reduced frequencies of splenic T FH cells and germinal center B cells compared with vehicle-treated mice. Importantly, MALT1 inhibition had no effect on the frequency or numbers of splenic regulatory T cells. Additionally, a delayed-type hypersensitivity (DTH) response was abrogated with MALT1 inhibition, showing that T-cell-driven responses in the skin, an important target tissue in cGVHD, can be abrogated with MALT1 blockade. Given recent studies implicating MALT1 activity as a link between immunoreceptor signaling and activation-dependent metabolic activity in T cells, we performed non-targeted metabolomics on plasma and spleens from scGVHD animals. Clustering of splenic metabolite intensities across treatment groups revealed a subset of metabolites responsive to MALT1 inhibition. This motivated us to explore the metabolic activity of cells from GVHD patients. By developing a method to infer metabolic pathway activity from transcriptional profiles of whole blood, we were able to utilize publicly available RNA-seq data. Using a data set containing 8 healthy controls and longitudinal samples from 25 GVHD patients, this approach segregated GVHD patients into four subsets, with the two largest subsets showing oppositional activity in most major metabolic pathways. Collectively, these data reveal a breadth of MALT1 activity across diverse cell types implicated in the initiation and progression of cGVHD and support development of small-molecule inhibitors of MALT1. These data also define novel heterogeneity among GHVD patients based on immunometabolic activity and motivate development of metabolic biomarkers of MALT1 activity for clinical use. Figure 1 Figure 1. Disclosures Dispirito: Rheos Medicines: Current Employment. Badur: Rheos Medicines: Current Employment. Biswas: Rheos Medicines: Current Employment. Camacho: Rheos Medicines: Current Employment. Chalishazar: Rheos Medicines: Current Employment. DeChristopher: Rheos Medicines: Current Employment. Sellers: Rheos Medicines: Current Employment. Steadman: Rheos Medicines: Current Employment. Soh: Rheos Medicines: Current Employment. Monroe: Rheos Medicines: Consultancy. Long: Rheos Medicines: Current Employment.