ABSTRACT
For the last two decades, the aromatic aldehyde 5-hydroxymethyl-furfural (5-HMF) has been the subject of several investigations for its pharmacologic potential. In 2004, the Safo group reported that 5-HMF has potent antisickling activity by targeting and ameliorating the primary pathophysiology of hypoxia-induced sickling of erythrocytes (red blood cells [RBC]). Following the encouraging outcome of the preclinical and phase I/II clinical studies of 5-HMF for the treatment of sickle cell disease (SCD), there have been multiple studies suggesting 5-HMF has several other biological or pharmacologic activities, including anti-allergic, antioxidant, anti-hypoxic, anti-ischemic, cognitive improvement, anti-tyrosinase, anti-proliferation, cytoprotective, and anti-inflammatory activities. The wide range of its effects makes 5-HMF a potential candidate for treating a variety of diseases including cognitive disorders, gout, allergic disorders, anemia, hypoxia, cancers, ischemia, hemorrhagic shock, liver fibrosis, and oxidative injury. Several of these therapeutic claims are currently under investigation and, while promising, vary in terms of the strength of their evidence. This review presents the research regarding the therapeutic potential of 5-HMF in addition to its sources, physicochemical properties, safety, absorption, distribution, metabolism, and excretion (ADME) profiles.
ABSTRACT
Sickle cell disease (SCD) is the most common genetic disorder, affecting millions of people worldwide. Aromatic aldehydes, which increase the oxygen affinity of human hemoglobin to prevent polymerization of sickle hemoglobin and inhibit red blood cell (RBC) sickling, have been the subject of keen interest for the development of effective treatment against SCD. However, the aldehyde functional group metabolic instability has severly hampered their development, except for voxelotor, which was approved in 2019 for SCD treatment. To improve the metabolic stability of aromatic aldehydes, we designed and synthesized novel molecules by incorporating Michael acceptor reactive centers into the previously clinically studied aromatic aldehyde, 5-hydroxymethylfurfural (5-HMF). Eight such derivatives, referred to as MMA compounds were synthesized and studied for their functional and biological activities. Unlike 5-HMF, which forms Schiff-base interaction with αVal1 nitrogen of hemoglobin, the MMA compounds covalently interacted with ßCys93, as evidenced by reverse-phase HPLC and disulfide exchange reaction, explaining their RBC sickling inhibitory activities, which at 2 mM and 5 mM, range from 0% to 21% and 9% to 64%, respectively. Additionally, the MMA compounds showed a second mechanism of sickling inhibition (12%-41% and 13%-62% at 2 mM and 5 mM, respectively) by directly destabilizing the sickle hemoglobin polymer. In vitro studies demonstrated sustained pharmacologic activities of the compounds compared to 5-HMF. These findings hold promise for advancing SCD therapeutics.
Subject(s)
Anemia, Sickle Cell , Antisickling Agents , Humans , Antisickling Agents/pharmacology , Antisickling Agents/therapeutic use , Hemoglobins/metabolism , Hemoglobins/therapeutic use , Anemia, Sickle Cell/drug therapy , Anemia, Sickle Cell/metabolism , Hemoglobin, Sickle/metabolism , Hemoglobin, Sickle/therapeutic use , Furans , Aldehydes/therapeutic use , Oxygen/metabolismABSTRACT
The synthetic allosteric effector of hemoglobin, TD-7 has been investigated as a potential therapeutic agent for the treatment of sickle cell disease. The pharmacologic activity of TD-7 is due to formation of a Schiff-base interaction between its aldehyde group and the two N-terminal αVal1 amines of hemoglobin, effectively inhibiting sickling of red blood cells. However, TD-7 faces a challenge in terms of poor oral bioavailability due to rapid in-vivo oxidative metabolism of its aldehyde functional group. To address this shortcoming, researches have explored the use of a L-cysteine ethyl ester group to cap the aldehyde group to form a thiazolidine aromatic aldehyde prodrug complex, resulting in the improvement of the metabolic stability of this class of compounds. This report details the synthesis of a thiazolidine prodrug of TD-7, referred to as Pro-7, along with a comprehensive investigation of Pro-7 functional and biological properties. In an in-vitro Hb modification and Hb oxygen affinity studies using normal whole blood, as well as erythrocyte sickling inhibition using sickle whole blood, Pro-7 exhibited a gradual onset but progressive increase in all activities. Additionally, in-vivo pharmacokinetic studies conducted with Sprague Dawley rats demonstrated that Pro-7 can undergo hydrolysis to release TD-7. However, the blood concentration of TD-7 did not reach the desired therapeutic level. These findings suggest that the incorporation of the L-cysteine ethyl ester group to TD-7 represents a promising strategy to enhance the metabolic stability of aromatic aldehydes that could lead to the development of a more effective drug for the treatment of sickle cell disease.
ABSTRACT
Pyridoxine 4-dehydrogenase (PdxI), a NADPH-dependent pyridoxal reductase, is one of the key players in the Escherichia coli pyridoxal 5'-phosphate (PLP) salvage pathway. This enzyme, which catalyses the reduction of pyridoxal into pyridoxine, causes pyridoxal to be converted into PLP via the formation of pyridoxine and pyridoxine phosphate. The structural and functional properties of PdxI were hitherto unknown, preventing a rational explanation of how and why this longer, detoured pathway occurs, given that, in E. coli, two pyridoxal kinases (PdxK and PdxY) exist that could convert pyridoxal directly into PLP. Here, we report a detailed characterisation of E. coli PdxI that explains this behaviour. The enzyme efficiently catalyses the reversible transformation of pyridoxal into pyridoxine, although the reduction direction is thermodynamically strongly favoured, following a compulsory-order ternary-complex mechanism. In vitro, the enzyme is also able to catalyse PLP reduction and use NADH as an electron donor, although with lower efficiency. As with all members of the aldo-keto reductase (AKR) superfamily, the enzyme has a TIM barrel fold; however, it shows some specific features, the most important of which is the presence of an Arg residue that replaces the catalytic tetrad His residue that is present in all AKRs and appears to be involved in substrate specificity. The above results, in conjunction with kinetic and static measurements of vitamins B6 in cell extracts of E. coli wild-type and knockout strains, shed light on the role of PdxI and both kinases in determining the pathway followed by pyridoxal in its conversion to PLP, which has a precise regulatory function.
Subject(s)
Pyridoxine , Vitamin B 6 , Vitamin B 6/chemistry , Pyridoxine/metabolism , Escherichia coli/metabolism , Pyridoxal Phosphate/metabolism , Pyridoxal/metabolismABSTRACT
Phenylhydroxylamine (PhNHOH) and nitrosobenzene (PhNO) interact with human tetrameric hemoglobin (Hb) to form the nitrosobenzene adduct Hb(PhNO). These interactions also frequently lead to methemoglobin formation in red blood cells. We utilize UV-vis spectroscopy and X-ray crystallography to identify the primary and secondary products that form when PhNHOH and related alkylhydroxylamines (RNHOH; R = Me, t-Bu) react with human ferric Hb. We show that with MeNHOH, the primary product is Hb[α-FeIII(H2O)][ß-FeII(MeNO)], in which nitrosomethane is bound to the ß subunit but not the α subunit. Attempts to isolate a nitrosochloramphenicol (CAMNO) adduct resulted in our isolation of a Hb[α-FeII][ß-FeII-cySOx]{CAMNO} product (cySOx = oxidized cysteine) in which CAMNO was located outside of the protein in the solvent region between the ß2 and α2 subunits of the same tetramer. We also observed that the ßcys93 residue had been oxidized. In the case of t-BuNHOH, we demonstrate that the isolated product is the ß-hemichrome Hb[α-FeIII(H2O)][ß-FeIII(His)2]{t-BuNHOH}, in which the ß heme has slipped â¼4.4 Å towards the solvent exterior to accommodate the bis-His heme coordination. When PhNHOH is used, a similar ß-hemichrome Hb[α-FeIII(H2O)][ß-FeIII(His)2-cySOx]{PhNHOH} was obtained. Our results reveal, for the first time, the X-ray structural determination of a ß-hemichrome in a human Hb derivative. Our UV-vis and X-ray crystal structural result reveal that although Hb(PhNO) and Hb(RNO) complexes may form as primary products, attempted isolation of these products by crystallization may result in the structural determination of their secondary products which may contain ß-hemichromes en route to further protein degradation.
Subject(s)
Ferric Compounds , Hemeproteins , Humans , Heme/chemistry , Hemoglobins/chemistry , Solvents , Ferrous CompoundsABSTRACT
X-ray crystallography and structure-based drug discovery have played a major role in the discovery of antisickling agents that target hemoglobin (Hb) for the treatment of sickle cell disease (SCD). Sickle cell disease, the most common inherited hematologic disorder, occurs as a result of a single point mutation of ßGlu6 in normal human adult hemoglobin (HbA) to ßVal6 in sickle hemoglobin (HbS). The disease is characterized by polymerization of HbS and sickling of red blood cells (RBCs), leading to several secondary pathophysiologies, including but not limited to vaso-occlusion, hemolytic anemia, oxidative stress, inflammation, stroke, pain crisis, and organ damage. Despite the fact that SCD was the first disease to have its molecular basis established, the development of therapies was for a very long time a challenge and took several decades to find therapeutic agents. The determination of the crystal structure of Hb by Max Perutz in the early 60s, and the pioneering X-ray crystallography research by Donald J. Abraham in the early 80s, which resulted in the first structures of Hb in complex with small molecule allosteric effectors of Hb, gave much hope that structure-based drug discovery (SBDD) could be used to accelerate development of antisickling drugs that target the primary pathophysiology of hypoxia-induced HbS polymerization to treat SCD. This article, which is dedicated to Donald J. Abraham, briefly reviews structural biology, X-ray crystallography and structure-based drug discovery from the perspective of Hb. The review also presents the impact of X-ray crystallography in SCD drug development using Hb as a target, emphasizing the major and important contributions by Don Abraham in this field.
ABSTRACT
Valsartan (VST) is a poorly soluble antihypertensive drug characterized by its limited dissolution rate and low bioavailability. This study aims to improve VST solubility and dissolution rate via developing liquisolid tablets (LSTs) containing a self-nanoemulsifying drug delivery system (SNEDDS), which is expected to enhance VST bioavailability. This aim was achieved via two designs of experiment. The first was the simplex-lattice design to optimize VST-loaded-SNEDDS using sesame oil, Tween 80, and polyethylene glycol 400. The second was the 32-3-level factorial design to optimize the liquisolid system using the SNEDDS-loaded VST and Neusilin®US2 as a carrier and fumed silica as a coating material. Different excipient ratios (X1) and varioussuper-disintegrants (X2) were also used in developing the optimized VST-LSTs. Thein vitrodissolution of VST from LSTs was compared with the marketed product (Diovan®). Non-compartmental analysis of plasma data after extravascular input with the linear trapezoidal method was used to calculate thepharmacokinetic parameters of the optimized VST-LSTs compared with the marketed tablet in male Wistar rats. The optimized SNEDDS compromised 24.9% sesame oil, 33.3% surfactant, and 41.8% cosurfactant, giving 173.9 nm size and 63.9 mg/ml loading capacity. Also, the SNEDDS-loaded VST tablet revealed good quality attributes with the release of 75% of its content in 5 min and 100% within 15 min. On the other hand, the marketed product took 1 h for the entire drug to be released.Moreover, the maximum plasma concentration (Cmax) of the optimizedVST-LSTwas6585.33 ng/ml within 1 h (Tmax), compared to 2884.67 ng/ml within 2 h of the marketed tablet.The relative bioavailability of the SNEDDS-loaded VST tablet was 213.7% compared to that of the marketed tablet, indicating that this formulation approach could be applied for increasing solubility, dissolution behavior in GIT, and bioavailability of poorly water-soluble drugs.
Subject(s)
Nanoparticles , Sesame Oil , Rats , Animals , Male , Valsartan , Biological Availability , Rats, Wistar , Emulsions , Drug Delivery Systems/methods , Excipients , Solubility , TabletsABSTRACT
In this work, a novel promising hybrid mode of uracil/thiouracil based quinoline pharmacophore i.e. 5a-f was rationalized and synthesized based on rigidification and lipophilic principles, and following the reported pharmacophoric features of camptothecin & doxorubicin. Concurrently, a non-rigid mode pharmacophore i.e. 7a-f was also designed and synthesized. The anti-proliferative activity of the compounds was assessed against three different cancer cell lines, namely A549 lung cancer, MCF-7 breast adenocarcinoma, and HepG-2 hepatic carcinoma. Further, promising candidates were evaluated against A549, and MCF-7 and for their ability to inhibit topoisomerases I &II. Compound 5f was observed to be the most active congener, displaying the highest cell inhibition of 84.4% for topoisomerase I and 92%, for topoisomerase II at a concentration of 100 µM. When its cytotoxicity was evaluated against A549 cells, 5f arrested the cell cycle at the S phase and increased the apoptosis ratio by 46.31%. DFT calculation of 5f showed higher dipole moment and greater negative energy values (-247531.510 kcal/mol) with positive & negative poles, and better stability reflection. Furthermore, molecular docking of 5f to both enzymes showed good agreement with the biological assessment. This study has given insight for further consideration of the highly promising hybrid 5f.
Subject(s)
Antineoplastic Agents , Quinolines , Molecular Structure , Structure-Activity Relationship , Thiouracil/pharmacology , Molecular Docking Simulation , Uracil/pharmacology , Density Functional Theory , Drug Screening Assays, Antitumor , Topoisomerase II Inhibitors/pharmacology , Quinolines/pharmacology , Cell Proliferation , Cell Line, Tumor , DNA Topoisomerases, Type II/metabolismABSTRACT
The aliphatic hydrophobic amino acid residues-alanine, isoleucine, leucine, proline and valine-are among the most common found in proteins. Their structural role in proteins is seemingly obvious: engage in hydrophobic interactions to stabilize secondary, and to a lesser extent, tertiary and quaternary structure. However, favorable hydrophobic interactions involving the sidechains of these residue types are generally less significant than the unfavorable set arising from interactions with polar atoms. Importantly, the constellation of interactions between residue sidechains and their environments can be recorded as three-dimensional maps that, in turn, can be clustered. The clustered average map sets compose a library of interaction profiles encoding interaction strengths, interaction types and the optimal 3D position for the interacting partners. This library is backbone angle-dependent and suggests solvent and lipid accessibility for each unique interaction profile. In this work, in addition to analysis of soluble proteins, a large set of membrane proteins that contained optimized artificial lipids were evaluated by parsing the structures into three distinct components: soluble extramembrane domain, lipid facing transmembrane domain, core transmembrane domain. The aliphatic residues were extracted from each of these sets and passed through our calculation protocol. Notable observations include: the roles of aliphatic residues in soluble proteins and in the membrane protein's soluble domains are nearly identical, although the latter are slightly more solvent accessible; by comparing maps calculated with sidechain-lipid interactions to maps ignoring those interactions, the potential extent of residue-lipid and residue-interactions can be assessed and likely exploited in structure prediction and modeling; amongst these residue types, the levels of lipid engagement show isoleucine as the most engaged, while the other residues are largely interacting with neighboring helical residues.
ABSTRACT
Protecting food from bacterial contamination is crucial for ensuring its safety and avoiding foodborne illness. Serratia marcescens is one of the food bacterial contaminants that can form biofilms and pigments that spoil the food product and could cause infections and illness to the consumer. Food preservation is essential to diminish such bacterial contaminants or at least reduce their pathogenesis; however, it should not affect food odor, taste, and consistency and must be safe. Sodium citrate is a well-known safe food additive and the current study aims to evaluate its anti-virulence and anti-biofilm activity at low concentrations against S. marcescens. The anti-virulence and antibiofilm activities of sodium citrate were evaluated phenotypically and genotypically. The results showed the significant effect of sodium citrate on decreasing the biofilm formation and other virulence factors, such as motility and the production of prodigiosin, protease, and hemolysins. This could be owed to its downregulating effect on the virulence-encoding genes. An in vivo investigation was conducted on mice and the histopathological examination of isolated tissues from the liver and kidney of mice confirmed the anti-virulence activity of sodium citrate. In addition, an in silico docking study was conducted to evaluate the sodium citrate binding ability to S. marcescens quorum sensing (QS) receptors that regulates its virulence. Sodium citrate showed a marked virtual ability to compete on QS proteins, which could explain sodium citrate's anti-virulence effect. In conclusion, sodium citrate is a safe food additive and can be used at low concentrations to prevent contamination and biofilm formation by S. marcescens and other bacteria.
ABSTRACT
The Department of Medicinal Chemistry, together with the Institute for Structural Biology, Drug Discovery and Development, at Virginia Commonwealth University (VCU) has evolved, organically with quite a bit of bootstrapping, into a unique drug discovery ecosystem in response to the environment and culture of the university and the wider research enterprise. Each faculty member that joined the department and/or institute added a layer of expertise, technology and most importantly, innovation, that fertilized numerous collaborations within the University and with outside partners. Despite moderate institutional support with respect to a typical drug discovery enterprise, the VCU drug discovery ecosystem has built and maintained an impressive array of facilities and instrumentation for drug synthesis, drug characterization, biomolecular structural analysis and biophysical analysis, and pharmacological studies. Altogether, this ecosystem has had major impacts on numerous therapeutic areas, such as neurology, psychiatry, drugs of abuse, cancer, sickle cell disease, coagulopathy, inflammation, aging disorders and others. Novel tools and strategies for drug discovery, design and development have been developed at VCU in the last five decades; e.g., fundamental rational structure-activity relationship (SAR)-based drug design, structure-based drug design, orthosteric and allosteric drug design, design of multi-functional agents towards polypharmacy outcomes, principles on designing glycosaminoglycans as drugs, and computational tools and algorithms for quantitative SAR (QSAR) and understanding the roles of water and the hydrophobic effect.
Subject(s)
Chemistry, Pharmaceutical , Computational Chemistry , Humans , Ecosystem , Universities , Virginia , Drug Discovery/methods , Quantitative Structure-Activity Relationship , Molecular BiologyABSTRACT
The highly effective phosphodiesterase type 5 inhibitor (avanafil; AVA) is routinely prescribed to treat erectile dysfunction. The drug has poor oral bioavailability and undergoes a significant first-pass metabolism. Therefore, altering AVA's solubility and choosing a different delivery method may boost its effectiveness. Nine different solid dispersion formulations utilizing polyvinylpyrrolidone (PVP) at three different ratios were prepared and characterized. The Box-Behnken design was employed to optimize AVA-buccal tablets. The pre-compression and post-compression characteristics of the tablets were assessed. The mucoadhesion strength of the optimized tablet was investigated using cow buccal mucosal tissue. In vivo performance of the optimized tablets was examined on human volunteers compared to the commercial tablets. PVP K90 at 2:1 drug to polymer ratio showed the highest solubilization capacity. The mucoadhesive polymer type and percentage and the mucopenetration enhancer percentage were significantly affect the mucoadhesion strength, tablet hardness, and the initial and cumulative AVA released from the prepared tablets. The optimized AVA-buccal tablet showed 4.96 folds increase in the mean residence time, higher plasma exposure, and an improvement in the relative bioavailability of AVA by 1076.27% compared with the commercial tablet. Therefore, a successful approach to deal with AVA first-pass metabolism and low bioavailability could be to employ buccal tablets containing a solubility-enhanced form of AVA.
Subject(s)
Chemistry, Pharmaceutical , Deoxycholic Acid , Male , Humans , Administration, Buccal , Biological Availability , Polymers , Povidone , Tablets , SolubilityABSTRACT
Aromatic aldehydes act as allosteric effectors of hemoglobin (AEH), forming Schiff-base adducts with the protein to increase its oxygen (O2) affinity; a desirable property in sickle cell disease (SCD) treatment, as the high-O2 affinity hemoglobin (Hb) does not polymerize and subsequently prevents erythrocytes sickling. This study reports the development, validation, and application of a weak cation-exchange HPLC assay - quantifying the appearance of Hb-AEH adduct - as a "universal" method, allowing for the prioritization of AEH candidates through an understanding of their Hb binding affinity and kinetics. Concentration- and time-dependent Hb binding profiles of ten AEHs were determined with HPLC, followed by the appropriate non-linear modeling to characterize their steady-state binding affinity (KDss), and binding kinetics second-order association (kon) and first-order dissociation (koff) rate constants. Vanillin-derived AEHs exhibited enhanced binding affinity to Hb, primarily due to their faster kon. Across AEH, kon and koff values are strongly correlated (r = 0.993, n = 7), suggesting that modifications of the AEH scaffold enhanced their interactions with Hb as intended, but inadvertently increased their Hb-AEH adduct dissociation. To our knowledge, the present study is the first to provide valuable insight into Hb binding kinetics of antisickling aromatic aldehydes, and the assay will be a useful platform in screening/prioritizing drug candidates for SCD treatment.
Subject(s)
Aldehydes , Hemoglobin A , Chromatography, High Pressure Liquid , Schiff Bases , OxygenABSTRACT
Inflammation is a complicated disorder that is produced as a result of consecutive processes. 5-LOX (5-lipoxygenase) is accountable for various inflammation mediators and leukotrienes synthesis, and its inhibition is the target of anti-inflammation therapeutics. Fungi have acquired enormous attentiveness because of their capability to biosynthesize novel bio-metabolites that reveal diversified bio-activities. A new tetracyclic triterpenoid, integracide L (1), along with integracides B (2) and F (3), were separated from Mentha longifolia-associated Fusarium sp. (FS No. MAR2014). Their structures were verified utilizing varied spectral analyses. The isolated metabolites (1-3), alongside the earlier reported integracides G (4), H (5), and J (6), were inspected for 5-LOX inhibition capacity. Interestingly, 1-6 possessed marked 5-LOX inhibition potentials with IC50s ranging from 1.18 to 3.97 µM compared to zileuton (IC50 1.17 µM). Additionally, molecular docking was executed to examine the interaction among these metabolites and 5-LOX, as well as to validate the in vitro findings. The docking study revealed their inhibitory activity interactions in the binding pocket. These findings highlighted the potential of integracides as lead metabolites for anti-inflammation drug discovery.
ABSTRACT
The development of an oral anti-diabetic medication characterized by enhanced hypoglycemic activity is in high demand. The goal was to study the hypoglycemic activity and pancreatic histopathology after the black-seed-based self-nanoemulsifying drug delivery system (SNEDDS) loaded with glimepiride liquisolid tablets to diabetic rats. The solubility of glimepiride in various vehicles was investigated. An optimization SNEDDS formulation was developed using a mixture of the experimental design approach. Box-Behnken design (BBD) was used to develop glimepiride liquisolid tablets utilizing Avicel PH 101 and Neusilin as a carrier mixture and FujiSil as a coating material. The quality attributes of the prepared tablets were assessed. Following the administration of the optimized tablets to diabetic rats, the pharmacodynamics and histopathological changes were investigated and compared to a commercial drug product. Results revealed that the optimized SNEDDS formulation that contains 15.43% w/w black seed oil, 40% w/w Tween 80, and 44.57% w/w Polyethylene glycol 400 showed an average droplet size of 34.64 ± 2.01 nm and a drug load of 36.67 ± 3.13 mg/mL. The optimized tablet formulation contained 0.31% Avicel in the carrier mixture, a 14.99 excipient ratio, and 8% superdisintegrant. Pre- and post-compression properties were satisfactory, and the optimized glimepiride liquisolid tablet showed a two-fold increase in dissolution. The optimized tablet demonstrated superior pharmacodynamics. The pancreatic tissues of the group treated with the optimized tablet displayed normal histological structure. The obtained data offered a commercially viable alternative for manufacturing solid dosage forms containing water-insoluble drugs, but additional clinical research is required.
ABSTRACT
Sickle cell disease (SCD) is caused by a single-point mutation, and the ensuing deoxygenation-induced polymerization of sickle hemoglobin (HbS), and reduction in bioavailability of vascular nitric oxide (NO), contribute to the pathogenesis of the disease. In a proof-of-concept study, we successfully incorporated nitrate ester groups onto two previously studied potent antisickling aromatic aldehydes, TD7 and VZHE039, to form TD7-NO and VZHE039-NO hybrids, respectively. These compounds are stable in buffer but demonstrated the expected release of NO in whole blood in vitro and in mice. The more promising VZHE039-NO retained the functional and antisickling activities of the parent VZHE039 molecule. Moreover, VZHE039-NO, unlike VZHE039, significantly attenuated RBC adhesion to laminin, suggesting this compound has potential in vivo RBC anti-adhesion properties relevant to vaso-occlusive events. Crystallographic studies show that, as with VZHE039, VZHE039-NO also binds to liganded Hb to make similar protein interactions. The knowledge gained during these investigations provides a unique opportunity to generate a superior candidate drug in SCD with enhanced benefits.
Subject(s)
Anemia, Sickle Cell , Hemoglobin, Sickle , Mice , Animals , Hemoglobin, Sickle/metabolism , Antisickling Agents/pharmacology , Antisickling Agents/therapeutic use , Nitric Oxide , Aldehydes/pharmacology , Nitrates , Laminin , Anemia, Sickle Cell/drug therapy , Anemia, Sickle Cell/metabolism , EstersABSTRACT
The pyridoxal 5'-phosphate (PLP) homeostasis protein (PLPHP) is a ubiquitous member of the COG0325 family with apparently no catalytic activity. Although the actual cellular role of this protein is unknown, it has been observed that mutations of the PLPHP encoding gene affect the activity of PLP-dependent enzymes, B6 vitamers and amino acid levels. Here we report a detailed characterization of the Escherichia coli ortholog of PLPHP (YggS) with respect to its PLP binding and transfer properties, stability, and structure. YggS binds PLP very tightly and is able to slowly transfer it to a model PLP-dependent enzyme, serine hydroxymethyltransferase. PLP binding to YggS elicits a conformational/flexibility change in the protein structure that is detectable in solution but not in crystals. We serendipitously discovered that the K36A variant of YggS, affecting the lysine residue that binds PLP at the active site, is able to bind PLP covalently. This observation led us to recognize that a number of lysine residues, located at the entrance of the active site, can replace Lys36 in its PLP binding role. These lysines form a cluster of charged residues that affect protein stability and conformation, playing an important role in PLP binding and possibly in YggS function.
Subject(s)
Escherichia coli Proteins , Escherichia coli , Escherichia coli/metabolism , Lysine/metabolism , Pyridoxal Phosphate , Proteins/chemistry , Protein Stability , Homeostasis , Phosphates/metabolism , Carrier Proteins/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolismABSTRACT
Sickle cell disease (SCD) is a genetic disorder that affects millions of individuals worldwide. Chronic anemia, hemolysis, and vasculopathy are associated with SCD, and their role has been well characterized. These symptoms stem from hemoglobin (Hb) polymerization, which is the primary event in the molecular pathogenesis of SCD and contributes to erythrocyte or red blood cell (RBC) sickling, stiffness, and vaso-occlusion. The disease is caused by a mutation at the sixth position of the ß-globin gene, coding for sickle Hb (HbS) instead of normal adult Hb (HbA), which under hypoxic conditions polymerizes into rigid fibers to distort the shapes of the RBCs. Only a few therapies are available, with the universal effectiveness of recently approved therapies still being monitored. In this review, we first focus on how sickle RBCs have altered metabolism and then highlight how this understanding reveals potential targets involved in the pathogenesis of the disease, which can be leveraged to create novel therapeutics for SCD.