ABSTRACT
Protein-polymer conjugates combine the unique properties of both proteins and synthetic polymers, making them important materials for biomedical applications. In this work, we synthesized and characterized protein-branched polymer bioconjugates that were precisely designed to retain protein functionality while preventing unwanted interactions. Using chymotrypsin as a model protein, we employed a controlled radical branching polymerization (CRBP) technique utilizing a water-soluble inibramer, sodium 2-bromoacrylate. The green-light-induced atom transfer radical polymerization (ATRP) enabled the grafting of branched polymers directly from the protein surface in the open air. The resulting bioconjugates exhibited a predetermined molecular weight, well-defined architecture, and high branching density. Conformational analysis by SEC-MALS validated the controlled grafting of branched polymers. Furthermore, enzymatic assays revealed that densely grafted polymers prevented protein inhibitor penetration, and the resulting conjugates retained up to 90% of their enzymatic activity. This study demonstrates a promising strategy for designing protein-polymer bioconjugates with tunable sieving behavior, opening avenues for applications in drug delivery and biotechnology.
Subject(s)
Chymotrypsin , Polymers , Chymotrypsin/metabolism , Polymerization , Membrane ProteinsABSTRACT
We are entering an era in which therapeutic proteins are assembled using building block-like strategies, with no standardized schema to discuss these formats. Existing nomenclatures, like AbML, sacrifice human readability for precision. Therefore, considering even a dozen such formats, in combination with hundreds of possible targets, can create confusion and increase the complexity of drug discovery. To address this challenge, we introduce Verified Taxonomy for Antibodies (VERITAS). This classification and nomenclature scheme is extensible to multispecific therapeutic formats and beyond. VERITAS names are easy to understand while drawing direct connections to the structure of a given format, with or without specific target information, making these names useful to adopt in scientific discourse and as inputs to machine learning algorithms for drug development.
Subject(s)
Antibodies, Bispecific , Biological Products , Humans , Drug Development , Drug DiscoveryABSTRACT
Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by absence of the protein dystrophin, which acts as a structural link between the basal lamina and contractile machinery to stabilize muscle membranes in response to mechanical stress. In DMD, mechanical stress leads to exaggerated membrane injury and fiber breakdown, with fast fibers being the most susceptible to damage. A major contributor to this injury is muscle contraction, controlled by the motor protein myosin. However, how muscle contraction and fast muscle fiber damage contribute to the pathophysiology of DMD has not been well characterized. We explored the role of fast skeletal muscle contraction in DMD with a potentially novel, selective, orally active inhibitor of fast skeletal muscle myosin, EDG-5506. Surprisingly, even modest decreases of contraction (<15%) were sufficient to protect skeletal muscles in dystrophic mdx mice from stress injury. Longer-term treatment also decreased muscle fibrosis in key disease-implicated tissues. Importantly, therapeutic levels of myosin inhibition with EDG-5506 did not detrimentally affect strength or coordination. Finally, in dystrophic dogs, EDG-5506 reversibly reduced circulating muscle injury biomarkers and increased habitual activity. This unexpected biology may represent an important alternative treatment strategy for Duchenne and related myopathies.
Subject(s)
Muscular Dystrophy, Animal , Muscular Dystrophy, Duchenne , Mice , Animals , Dogs , Muscular Dystrophy, Duchenne/metabolism , Mice, Inbred mdx , Muscle, Skeletal/metabolism , Dystrophin/genetics , Muscle Contraction/physiology , Disease Models, Animal , Muscular Dystrophy, Animal/genetics , Muscular Dystrophy, Animal/metabolismABSTRACT
BACKGROUND: Becker muscular dystrophy (BMD) is a genetic neuromuscular disease of growing importance caused by in-frame, partial loss-of-function mutations in the dystrophin (DMD) gene. BMD presents with reduced severity compared with Duchenne muscular dystrophy (DMD), the allelic disorder of complete dystrophin deficiency. Significant therapeutic advancements have been made in DMD, including four FDA-approved drugs. BMD, however, is understudied and underserved-there are no drugs and few clinical trials. Discordance in therapeutic efforts is due in part to lack of a BMD mouse model which would enable greater understanding of disease and de-risk potential therapeutics before first-in-human trials. Importantly, a BMD mouse model is becoming increasingly critical as emerging DMD dystrophin restoration therapies aim to convert a DMD genotype into a BMD phenotype. METHODS: We use CRISPR/Cas9 technology to generate bmx (Becker muscular dystrophy, X-linked) mice, which express an in-frame ~40 000 bp deletion of exons 45-47 in the murine Dmd gene, reproducing the most common BMD patient mutation. Here, we characterize muscle pathogenesis using molecular and histological techniques and then test skeletal muscle and cardiac function using muscle function assays and echocardiography. RESULTS: Overall, bmx mice present with significant muscle weakness and heart dysfunction versus wild-type (WT) mice, despite a substantial improvement in pathology over dystrophin-null mdx52 mice. bmx mice show impaired motor function in grip strength (-39%, P < 0.0001), wire hang (P = 0.0025), and in vivo as well as ex vivo force assays. In aged bmx, echocardiography reveals decreased heart function through reduced fractional shortening (-25%, P = 0.0036). Additionally, muscle-specific serum CK is increased >60-fold (P < 0.0001), indicating increased muscle damage. Histologically, bmx muscles display increased myofibre size variability (minimal Feret's diameter: P = 0.0017) and centrally located nuclei indicating degeneration/regeneration (P < 0.0001). bmx muscles also display dystrophic pathology; however, levels of the following parameters are moderate in comparison with mdx52: inflammatory/necrotic foci (P < 0.0001), collagen deposition (+1.4-fold, P = 0.0217), and sarcolemmal damage measured by intracellular IgM (P = 0.0878). Like BMD patients, bmx muscles show reduced dystrophin protein levels (~20-50% of WT), whereas Dmd transcript levels are unchanged. At the molecular level, bmx muscles express increased levels of inflammatory genes, inflammatory miRNAs and fibrosis genes. CONCLUSIONS: The bmx mouse recapitulates BMD disease phenotypes with histological, molecular and functional deficits. Importantly, it can inform both BMD pathology and DMD dystrophin restoration therapies. This novel model will enable further characterization of BMD disease progression, identification of biomarkers, identification of therapeutic targets and new preclinical drug studies aimed at developing therapies for BMD patients.
Subject(s)
Dystrophin , Muscular Dystrophy, Duchenne , Animals , Humans , Mice , Dystrophin/genetics , Dystrophin/metabolism , Exons/genetics , Muscle, Skeletal/pathology , Muscular Dystrophy, Duchenne/genetics , Muscular Dystrophy, Duchenne/pathology , Oligonucleotides, Antisense , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Disease Models, AnimalABSTRACT
BACKGROUND: Selective androgen receptor modulators (SARMs) increase muscle mass via the androgen receptor. This phase 2A trial investigated the effects of a SARM, GSK2881078, in conjunction with exercise, on leg strength in patients with chronic obstructive pulmonary disease (COPD) and impaired physical function. METHODS: 47 postmenopausal women and 50 men with COPD (forced expiratory volume in 1 s 30%-65% predicted; short physical performance battery score: 3-11) were enrolled into a randomised double-blind, placebo control trial. Patients were randomised 1:1 to once daily placebo or oral GSK2881078 (females: 1.0 mg; males: 2.0 mg) for 13 weeks with a concurrent home-exercise programme, involving strength training and physical activity. Primary endpoints were change from baseline in leg strength at 90 days (one-repetition maximum; absolute (kg) and relative (% change)) and multiple safety outcomes. Secondary endpoints included lean body mass, physical function and patient-reported outcomes. RESULTS: GSK2881078 increased leg strength in men. The difference in adjusted mean change from baseline and adjusted mean percentage change from baseline between treatment and placebo were: for women, 8.0 kg (90% CI -2.5 to 18.4) and 5.2% (90% CI -4.7 to 15.0), respectively; for men, 11.8 kg (90% CI -0.5 to 24.0) and 7.0% (90% CI 0.5 to 13.6), respectively. Lean body mass increased, but no changes in patient-reported outcomes were observed. Reversible reductions in high-density lipoprotein-cholesterol and transient elevations in hepatic transaminases were the main treatment-related safety findings. CONCLUSIONS: GSK2881078 was well tolerated and short-term treatment increased leg strength, when expressed as per cent predicted, in men with COPD more than physical training alone. TRIAL REGISTRATION NUMBER: NCT03359473.
Subject(s)
Pulmonary Disease, Chronic Obstructive , Receptors, Androgen , Male , Humans , Female , Receptors, Androgen/therapeutic use , Pulmonary Disease, Chronic Obstructive/drug therapy , Muscle Weakness/etiology , Exercise , Double-Blind MethodABSTRACT
One of the most straightforward and commonly used chemical modifications of proteins is to react surface amino groups (lysine residues) with activated esters. This chemistry has been used to generate protein-polymer conjugates, many of which are now approved therapeutics. Similar conjugates have also been generated by reacting activated ester atom transfer polymerization initiators with lysine residues to create biomacromolecular initiators for polymerization reactions. The reaction between activated esters and lysine amino groups is rapid and has been consistently described in almost every publication on the topic as a "random reaction". A random reaction implies that every accessible lysine amino group on a protein molecule is equally reactive, and as a result, that the reaction is indiscriminate. Nonetheless, the literature contradicts itself by also suggesting that some lysine amino groups are more reactive than others (as a function of pKa, surface accessibility, temperature, and local environment). If the latter assumption is correct, then the outcome of these reactions cannot be random at all, and we should be able to predict the outcome from the structure of the protein. Predicting the non-random outcome of a reaction between surface lysines and reactive esters could transform the speed at which active bioconjugates can be developed and engineered. Herein, we describe a robust integrated tool that predicts the activated ester reactivity of every lysine in a protein, thereby allowing us to calculate the non-random sequence of reaction as a function of reaction conditions. Specifically, we have predicted the intrinsic reactivity of each lysine in multiple proteins with a bromine-functionalised N-hydroxysuccinimide initiator molecule. We have also shown that the model applied to PEGylation. The rules-based analysis has been coupled together in a single Python program that can bypass tedious trial and error experiments usually needed in protein-polymer conjugate design and synthesis.
Subject(s)
Bromine , Lysine , Esters , Lysine/metabolism , Polymers/chemistry , Protein Processing, Post-Translational , Proteins/metabolismABSTRACT
Protease-protease interactions lie at the heart of the biological cascades that provide rapid molecular responses to living systems. Blood clotting cascades, apoptosis signaling networks, bacterial infection, and virus trafficking have all evolved to be activated and sustained by protease-protease interactions. Biomimetic strategies designed to target drugs to specific locations have generated proprotein drugs that can be activated by proteolytic cleavage to release native protein. We have previously demonstrated that the modification of enzymes with a custom-designed comb-shaped polymer nanoarmor can shield the enzyme surface and eliminate almost all protein-protein interactions. We now describe the synthesis and characterization of protease-sensitive comb-shaped nanoarmor cages using poly(ethylene glycol) methacrylate macromonomers where the PEG tines of the comb are connected to the backbone of the growing polymer chain by peptide linkers. Protease-induced cleavage of the tines of the comb releases a polymer-modified protein that can once again participate in protein-protein interactions. Atom transfer radical polymerization (ATRP) was used to copolymerize the macromonomer and carboxybetaine methacrylate from initiator-labeled chymotrypsin and trypsin enzymes, yielding proprotease conjugates that retained activity toward small peptide substrates but prevented activity against proteins. Native proteases triggered the release of the PEG side chains from the polymer backbone within 20 min, thereby increasing the activity of the conjugate toward larger protein substrates by 100%. Biomimetic cascade initiation of nanoarmored protease-sensitive protein-polymer conjugates may open the door to a new class of responsive targeted therapies.
Subject(s)
Peptide Hydrolases , Polymers , Methacrylates , Peptides , Polymerization , Polymers/chemistry , ProteinsABSTRACT
PEGylation is a well-established and clinically proven half-life extension strategy for protein delivery. Protein modification with amine-reactive poly(ethylene glycol) (PEG) generates heterogeneous and complex bioconjugate mixtures, often composed of several PEG positional isomers with varied therapeutic efficacy. Laborious and costly experiments for reaction optimization and purification are needed to generate a therapeutically useful PEG conjugate. Kinetic models which accurately predict the outcome of so-called "random" PEGylation reactions provide an opportunity to bypass extensive wet lab experimentation and streamline the bioconjugation process. In this study, we propose a protein tertiary structure-dependent reactivity model that describes the rate of protein-amine PEGylation and introduces "PEG chain coverage" as a tangible metric to assess the shielding effect of PEG chains. This structure-dependent reactivity model was implemented into three models (linear, structure-based, and machine-learned) to gain insight into how protein-specific molecular descriptors (exposed surface areas, pKa, and surface charge) impacted amine reactivity at each site. Linear and machine-learned models demonstrated over 75% prediction accuracy with butylcholinesterase. Model validation with Somavert, PEGASYS, and phenylalanine ammonia lyase showed good correlation between predicted and experimentally determined degrees of modification. Our structure-dependent reactivity model was also able to simulate PEGylation progress curves and estimate "PEGmer" distribution with accurate predictions across different proteins, PEG linker chemistry, and PEG molecular weights. Moreover, in-depth analysis of these simulated reaction curves highlighted possible PEG conformational transitions (from dumbbell to brush) on the surface of lysozyme, as a function of PEG molecular weight.
Subject(s)
Data Science , Muramidase , Amines , Muramidase/chemistry , Phenylalanine Ammonia-Lyase , Polyethylene Glycols/chemistry , Proteins/chemistryABSTRACT
Polymer-based protein engineering has enabled the synthesis of a variety of protein-polymer conjugates that are widely applicable in therapeutic, diagnostic and biotechnological industries. Accurate characterizations of physical-chemical properties, in particular, molar masses, sizes, composition and their dispersities are critical parameters that determine the functionality and conformation of protein-polymer conjugates and are important for creating reproducible manufacturing processes. Most of the current characterization techniques suffer from fundamental limitations and do not provide an accurate understanding of a sample's true nature. In this paper, we demonstrate the advantage of asymmetrical flow field-flow fractionation (AF4) coupled with multiple detectors for the characterization of a library of complex, zwitterionic and neutral protein-polymer conjugates. This method allows for determination of intrinsic physical properties of protein-polymer chimeras from a single, rapid measurement.
ABSTRACT
Government-sanctioned use of nerve agents (NA) has escalated dramatically in recent years. Oxime reactivators of organophosphate (OP)-inhibited acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) serve as antidotes toward poisoning by OPNAs. The oximes used as therapeutics are quaternary compounds that cannot penetrate the blood-brain barrier (BBB). There remains an urgent need for the development of next generation OPNA therapeutics. We have developed two high-throughput screening (HTS) assays using a fluorogenic NA surrogate, O-ethyl methylphosphonyl O-4-methyl-3-cyano-coumarin (EMP-MeCyC). EMP-MeCyC detoxification and EMP-BChE reactivation screening campaigns of ~155,000 small molecules resulted in the identification of 33 nucleophile candidates, including non-quaternary oximes. Four of the oximes were reactivators of both Sarin- and VX-inhibited BChE and directly detoxified Sarin. One oxime also detoxified VX. The novel reactivators included a non-quaternary pyridine amidoxime, benzamidoxime, benzaldoxime and a piperidyl-ketoxime. The VX-inhibited BChE reactivation reaction rates by these novel molecules were similar to those observed with known bis-quaternary reactivators and faster than mono-quaternary pyridinium oximes. Notably, we discovered the first ketoxime reactivator of OP-ChEs and detoxifier of OPNAs. Preliminary toxicological studies demonstrated that the newly discovered non-quaternary oximes were relatively non-toxic in mice. The discovery of unique non-quaternary oximes opens the door to the design of novel therapeutics and decontamination agents following OPNA exposure.
Subject(s)
Blood-Brain Barrier/drug effects , Butyrylcholinesterase/chemistry , Butyrylcholinesterase/metabolism , Cholinesterase Inhibitors/pharmacology , Nerve Agents/toxicity , Oximes/pharmacology , Animals , Enzyme Activation , Humans , Male , Mice , Mice, Inbred ICRABSTRACT
Even the most advanced protein-polymer conjugate therapeutics do not eliminate antibody-protein and receptor-protein recognition. Next-generation bioconjugate drugs will need to replace stochastic selection with rational design to select desirable levels of protein-protein interaction while retaining function. The "Holy Grail" for rational design would be to generate functional enzymes that are fully catalytic with small molecule substrates while eliminating interaction between the protein surface and larger molecules. Using chymotrypsin, an important enzyme that is used to treat pancreatic insufficiency, we have designed a series of molecular chimeras with varied grafting densities and shapes. Guided by molecular dynamic simulations and next-generation molecular chimera characterization with asymmetric flow field-flow fractionation chromatography, we grew linear, branched, and comb-shaped architectures from the surface of the protein by atom-transfer radical polymerization. Comb-shaped polymers, grafted from the surface of chymotrypsin, completely prevented enzyme inhibition with protein inhibitors without sacrificing the ability of the enzyme to catalyze the hydrolysis of a peptide substrate. Asymmetric flow field-flow fractionation coupled with multiangle laser light scattering including dynamic light scattering showed that nanoarmor designed with comb-shaped polymers was particularly compact and spherical. The polymer structure significantly increased protein stability and reduced protein-protein interactions. Atomistic molecular dynamic simulations predicted that a dense nanoarmor with long-armed comb-shaped polymer would act as an almost perfect molecular sieve to filter large ligands from substrates. Surprisingly, a conjugate that was composed of 99% polymer was needed before the elimination of protein-protein interactions.
Subject(s)
Polymerization , Polymers/chemistry , Proteins/chemistry , Fractionation, Field Flow , Ligands , Light , Molecular Dynamics Simulation , Protein Binding , Scattering, RadiationABSTRACT
INTRODUCTION: One of the hallmarks of injured skeletal muscle is the appearance of elevated skeletal muscle proteins in circulation. Human skeletal muscle generally consists of a mosaic of slow (type I) and fast (type IIa, IIx/d) fibers, defined by their myosin isoform expression. Recently, measurement of circulating fiber-type specific isoforms of troponin I has been used as a biomarker to suggest that muscle injury in healthy volunteers (HV) results in the appearance of muscle proteins from fast but not slow fibers. We sought to understand if this is also the case in severe myopathy patients with Becker and Duchenne muscular dystrophy (BMD, DMD). METHODS: An enzyme-linked immunosorbent assay (ELISA) that selectively measures fast and slow skeletal troponin I (TNNI2 and TNNI1) was used to measure a cross-section of patient plasma samples from HV (N = 50), BMD (N = 49), and DMD (N = 132) patients. Creatine kinase (CK) activity was also measured from the same samples for comparison. RESULTS: TNNI2 was elevated in BMD and DMD and correlated with the injury biomarker, CK. In contrast, TNNI1 levels were indistinguishable from levels in HV. There was an inverse relationship between CK and TNNI2 levels and age, but no relationship for TNNI1. DISCUSSION: We define a surprising discrepancy between TNNI1 and TNNI2 in patient plasma that may have implications for the interpretation of elevated muscle protein levels in dystrophinopathies.
Subject(s)
Muscular Dystrophy, Duchenne/blood , Muscular Dystrophy, Duchenne/diagnosis , Troponin I/blood , Adolescent , Adult , Biomarkers/blood , Child , Cross-Sectional Studies , Female , Humans , Male , Middle Aged , Retrospective Studies , Young AdultABSTRACT
Biotherapeutics have achieved global economic success due to their high specificity towards their drug targets, providing exceptional safety and efficiency. The ongoing shift away from small molecule drugs towards biotherapeutics heightens the need to further improve the pharmacokinetics of these biological drugs. Three pervasive obstacles that limit the therapeutic capacity of biotherapeutics are proteolytic degradation, circulating half-life, and the development of anti-drug antibodies. These challenges can culminate in limited efficiency and consequently warrant the need for higher drug doses and more frequent administration. We have explored the coupling of biotherapeutics to long-lived and biocompatible red blood cells (RBCs) to address limited pharmacokinetics. Butyrylcholinesterase (BChE), for example, provides prophylactic protection against organophosphate nerve agents (OPNAs), yet the short circulation life of the drug requires extraordinary doses. Herein, we report the rapid and tunable chemical engineering of BChE to RBC membranes to create a cell-based delivery system that retains the enzyme activity and enhances stability. In a three-step process that first pre-modifies BChE with a cell-reactive polymer chain, primes the cells for engineering, and then grafts the conjugates to the cells, we attached over 2 million BChE molecules to the surface of each RBC without diminishing the bioscavenging capacity of the enzyme. Critically, this membrane-engineering approach was cell-tolerated with minimal hemolysis observed. These results provide strong evidence for the ability of engineered RBCs to serve as an enhanced biotherapeutic delivery vehicle. STATEMENT OF SIGNIFICANCE: Organophosphate nerve agents (OPNAs) are one of the most lethal forms of chemical warfare. After exposure to OPNAs, a patient is given life-saving therapeutics, such as atropine and oxime. However, these drugs are limited, and the patient can still suffer from irreparable injuries. Given the toxicity of OPNAs, access to a prophylactic is vital. We have created an enhanced delivery system for prophylactic butyrylcholinesterase (BChE) by engineering this biotherapeutic to the red blood cell (RBC) surface. In three simple steps that first pre-modifies BChE with a cell-reactive polymer, primes the cells for engineering, and then grafts the conjugates to the cells, we attached over 2 million BChE molecules to a single RBC while retaining the enzyme's activity and enhancing its stability.
Subject(s)
Organophosphates , Pharmaceutical Preparations , Butyrylcholinesterase , Erythrocytes , Humans , OximesABSTRACT
Each year approximately 1.3 billion tons of food is either wasted or lost. One of the most wasted foods in the world is bread. The ability to reuse wasted food in another area of need, such as water scarcity, would provide a tremendous sustainable outcome. To address water scarcity, many areas of the world are now implementing desalination. One desalination technology that could benefit from food waste reuse is capacitive deionization (CDI). CDI has emerged as a powerful desalination technology that essentially only requires a pair of electrodes and a low-voltage power supply. Developing freestanding carbon electrodes from food waste could lower the overall cost of CDI systems and the environmental and economic impact from food waste. We created freestanding CDI electrodes from bread. The electrodes possessed a hierarchical pore structure that enabled both high salt adsorption capacity and one of the highest reported values for hydraulic permeability to date in a flow-through CDI system. We also developed a sustainable technique for electrode fabrication that does not require the use of common laboratory equipment and could be deployed in decentralized locations and developing countries with low-financial resources.
ABSTRACT
Organophosphorus nerve agents (OPNAs), used in chemical warfare, irreversibly inhibit essential cholinesterases (ChEs) in the cholinergic neurotransmission system. Several potent nucleophilic oximes have been approved for the treatment of acute poisoning by OPNAs, but they are rapidly cleared from blood circulation. Butyrylcholinesterase (BChE) stoichiometrically binds nerve agents, but because the molecular weight of a nerve agent is about 500-fold less than the enzyme, the bioscavenger has had limited utility. We synthesized BChE-polymer-oxime conjugates using atom transfer radical polymerization (ATRP) and azide-alkyne "click" chemistry. The activity of the BChE-polymer-oxime conjugates was dependent on the degree of oxime loading within the copolymer side chains. The covalent modification of oxime-containing copolymers prolonged the activity of BChE in the presence of the VX- and cyclosarin-fluorogenic analogues EMP-MeCyC and CMP-MeCyC, respectively. After complete inactivation by VX and cyclosarin fluorogenic analogues, the conjugates demonstrated efficient self-reactivation of up to 80% within 3-6 h. Repeated inhibition and high-level self-reactivation assays revealed that the BChE-polymer-oxime conjugates were excellent reactivators of OPNA-inhibited BChE. Recurring self-reactivation of BChE-polymer-oxime conjugates following repeated BChE inhibition by fluorogenic OPNAs (Flu-OPNAs) opens the door to developing the next generation of nerve agent "catalytic" bioscavengers.
Subject(s)
Butyrylcholinesterase , Nerve Agents , Cholinesterase Inhibitors , Organophosphorus Compounds , Oximes , PolymersABSTRACT
Red blood cells (RBC) have great potential as drug delivery systems, capable of producing unprecedented changes in pharmacokinetics, pharmacodynamics, and immunogenicity. Despite this great potential and nearly 50 years of research, it is only recently that RBC-mediated drug delivery has begun to move out of the academic lab and into industrial drug development. RBC loading with drugs can be performed in several ways-either via encapsulation within the RBC or surface coupling, and either ex vivo or in vivo-depending on the intended application. In this review, we briefly summarize currently used technologies for RBC loading/coupling with an eye on how pharmacokinetics is impacted. Additionally, we provide a detailed description of key ADME (absorption, distribution, metabolism, elimination) changes that would be expected for RBC-associated drugs and address unique features of RBC pharmacokinetics. As thorough understanding of pharmacokinetics is critical in successful translation to the clinic, we expect that this review will provide a jumping off point for further investigations into this area.
ABSTRACT
OBJECTIVES: To report the effect of a selective androgen receptor modulators (SARMs) on the urethral continence mechanisms in a rat model of stress urinary incontinence (SUI) induced by bilateral ovariectomy (OVX). MATERIALS AND METHODS: Female Sprague-Dawley rats with bilateral OVX were used. Rats were divided into five groups; sham operated, vehicle-treated OVX, low-dose SARM-treated OVX (GSK2849466A: 0.005 mg/kg/day, per os [p.o.]), high-dose SARM-treated OVX (GSK2849466A: 0.03 mg/kg/day, p.o.) and dihydrotestosterone (DHT)-treated OVX (1 mg/kg/day, subcutaneous) groups. After 4 weeks of SARM treatments or 3 weeks of DHT treatment (6 weeks after OVX), rats were subjected to evaluation of the sneeze-induced continence reflex using microtransducer-tipped catheter methods, sneeze-induced leak-point pressure, and continuous cystometry measurements, followed by histological analyses of urethral tissues. RESULTS: (i) OVX significantly impaired urethral continence function after 6 weeks to induce SUI during sneezing. (ii) Low-dose SARM treatment restored urethral baseline pressure (UBP) without affecting the amplitude of urethral response during sneezing (A-URS), partially reversing OVX-induced SUI during sneezing. (iii) High-dose SARM treatment reversed decreases in both UBP and A-URS, more effectively preventing SUI during sneezing. (iv) DHT treatment only restored A-URS without affecting UBP, partially preventing OVX-induced SUI during sneezing. (v) The high-dose SARM treatment induced hypertrophy of the striated and smooth muscle around the urethra. (vi) SARM treatment did not affect bladder function in sham or OVX rats. CONCLUSION: Treatment with SARMs could be a more effective modality for the treatment of SUI than DHT, without affecting bladder function, by enhancing smooth- and striated muscle-mediated urethral function under stress conditions such as sneezing.
Subject(s)
Androgen Receptor Antagonists/pharmacology , Ovariectomy , Urinary Bladder/drug effects , Urinary Incontinence, Stress , Androgen Receptor Antagonists/administration & dosage , Animals , Female , Rats , Rats, Sprague-Dawley , Sneezing/physiologyABSTRACT
Organophosphate nerve agents rapidly inhibit cholinesterases thereby destroying the ability to sustain life. Strong nucleophiles, such as oximes, have been used as therapeutic reactivators of cholinesterase-organophosphate complexes, but suffer from short half-lives and limited efficacy across the broad spectrum of organophosphate nerve agents. Cholinesterases have been used as long-lived therapeutic bioscavengers for unreacted organophosphates with limited success because they react with organophosphate nerve agents with one-to-one stoichiometries. The chemical power of nucleophilic reactivators is coupled to long-lived bioscavengers by designing and synthesizing cholinesterase-polymer-oxime conjugates using atom transfer radical polymerization and azide-alkyne "click" chemistry. Detailed kinetic studies show that butyrylcholinesterase-polymer-oxime activity is dependent on the electrostatic properties of the polymers and the amount of oxime within the conjugate. The covalent coupling of oxime-containing polymers to the surface of butyrylcholinesterase slows the rate of inactivation of paraoxon, a model nerve agent. Furthermore, when the enzyme is covalently inhibited by paraoxon, the covalently attached oxime induced inter- and intramolecular reactivation. Intramolecular reactivation will open the door to the generation of a new class of nerve agent scavengers that couple the speed and selectivity of biology to the ruggedness and simplicity of synthetic chemicals.
ABSTRACT
The field of protein-polymer conjugates has suffered from a lack of predictive tools and design guidelines to synthesize highly active and stable conjugates. In order to develop this type of information, structure-function-dynamics relationships must be understood. These relationships depend strongly on protein-polymer interactions and how these influence protein dynamics and conformations. Probing nanoscale interactions is experimentally difficult, but computational tools, such as molecular dynamics simulations, can easily obtain atomic resolution. Atomistic molecular dynamics simulations were used to study α-chymotrypsin (CT) densely conjugated with either zwitterionic, positively charged, or negatively charged polymers. Charged polymers interacted with the protein surface to varying degrees and in different regions of the polymer, depending on their flexibilities. Specific interactions of the negatively charged polymer with CT caused structural deformations in CT's substrate binding pocket and active site while no deformations were observed for zwitterionic and positively charged polymers. Attachment of polymers displaced water molecules from CT's surface into the polymer phase and polymer hydration correlated with the Hofmeister series.
Subject(s)
Chymotrypsin/chemistry , Polymers/chemistry , Animals , Cattle , Molecular Dynamics SimulationABSTRACT
The global and economic success of immunoglobulin-based therapeutics in treating a wide range of diseases has heightened the need to further enhance their efficacy and lifetime while diminishing deleterious side effects. The three most ubiquitous challenges of therapeutic immunoglobulin delivery are their relatively short lifetimes in vivo, the immunologic consequences of soluble antibody-antigen complexes, and the emergence of anti-drug antibodies. We describe the rapid, cell-tolerated chemical engineering of the erythrocyte membrane in order to display any antibody, our model system being the display of anti-Tumor Necrosis Factor (anti-TNFα), on the surface of long-lived red blood cells (RBCs) while masking the antibody's Fc region. We developed four synthetic approaches to generate RBC-Staphylococcal protein A (RBC-SpA) complexes: amino group targeting through N-hydrosuccinidyl ester-functionalized homobifunctional poly(ethylene glycol) (NHS-PEG-NHS), direct thiol group targeting using heterobifunctional NHS-PEG-maleimide (NHS-PEG-MAL), converted thiol targeting using heterobifunctional NHS-PEG-MAL, and click chemistry using heterobifunctional NHS-PEG-azido (NHS-PEG-N3) and NHS-PEG-alkyne (NHS-PEG-alk). The RBC-PEG-SpA complexes were formed within minutes, followed by the attachment of over 105 antibodies per RBC to the accessible RBC-bound SpA via Fc-Protein A coupling. The RBC-PEG-SpA-antibody arrays were shown to be stable for more than 60 days in PBS and for more than 42 days in serum containing buffer. RBC-PEG-SpA-antibody complexes were shown to remove TNFα from physiological buffer and had similar mechanical properties to unmodified RBCs. Out of the four approaches, the converted thiol method provided the most controlled chemistry and construct stability. We are now ideally positioned to determine the long-term in vivo efficacy of chemically membrane-engineered RBCs to remove antigens, like TNFα, from serum. STATEMENT OF SIGNIFICANCE: The global and economic success of immunoglobulin-based therapeutics in treating a wide range of diseases has heightened the need to further enhance their efficacy and lifetime while diminishing deleterious side effects. The three most ubiquitous challenges of therapeutic immunoglobulin delivery are their relatively short lifetimes in vivo, the immunologic consequences of soluble antibody-antigen complexes, and the emergence of anti-drug antibodies. We describe the rapid, cell-tolerated chemical engineering of the erythrocyte membrane to display any antibody, our model system being the display of anti-Tumor Necrosis Factor (anti-TNFα), on the surface of long-lived red blood cells (RBCs) while masking the antibody's Fc region. Conversion of RBCs into therapeutic delivery vehicles, we argue, would enhance the circulation life of immunoglobulin-based therapeutics while simultaneously evading deleterious immune response.