Recent work with approaches like recombinant mutants and X-ray crystallography has given much new information about the ligand-binding properties of human serum albumin (HSA). The information increases the understanding of this unique transport and depot protein and could give a structural basis for the possible construction of therapeutic agents with altered HSA-binding properties. A tabulation of high-affinity binding sites for both endogenous and exogenous compounds has been made; it could be useful for the above-mentioned purpose, but it could also be of value when trying to predict potential drug interactions at the protein-binding level. Drug displacement is not always a complication to therapy; it can be used to increase the biological effect of a drug. However, due to rebinding at other sites, the increase in the free concentration of a displaced ligand can be less than expected. Drugs and drug metabolites can also interact covalently with HSA; thiol-containing drugs often bind to the single free cysteine residue of HSA, and glucuronidated drugs react irreversibly with other residues of the protein. Reversible binding of ligands is often stereospecific, and therefore immobilized HSA can be used to separate drug isomers. Albumin-containing dialysates are useful for extracorporeal removal of endogenous toxins and in the treatment of drug overdoses. HSA has different types of hydrolytic activities, which also can be stereospecific. The esterase-like property seems especially useful in converting prodrugs to active drugs in plasma.

Practical Aspects of the Ligand-Binding and Enzymatic Properties of Human Serum Albumin

Ligand binding strategies of human serum albumin: how can the cargo be utilized?

Human serum albumin (HSA), being the most abundant carrier protein in blood and a modern day clinical tool for drug delivery, attracts high attention among biologists. Hence, its unfolding/refolding strategies and exogenous/endogenous ligand binding preference are of immense use in therapeutics and clinical biochemistry. Among its fellow proteins albumin is known to carry almost every small molecule. Thus, it is a potential contender for being a molecular cargo/or nanovehicle for clinical, biophysical and industrial purposes. Nonetheless, its structure and function are largely regulated by various chemical and physical factors to accommodate HSA to its func- tional purpose. This multifunctional protein also possesses enzymatic properties which may be used to convert prodrugs to active therapeutics. This review aims to highlight current overview on the binding strategies of protein to various ligands that may be expected to lead to significant clinical applications. Chirality 00:000-000, 2009. V C 2009 Wiley-Liss, Inc.

Review Article Ligand Binding Strategies of Human Serum Albumin: How Can the Cargo be Utilized?

Publisher Summary The role of plasma protein binding is recognized as an important factor in drug disposition and efficacy. This chapter discusses the structures of drug-binding proteins, particularly human serum albumin (HSA) and alpha-1-acid glycoprotein (AAG), the relationship between ligand structure and protein binding and the effect of plasma protein binding on drug disposition and pharmacological activity. Human serum albumin (HSA) binds a wide variety of endogenous and exogenous ligands with association constants typically in the range of 10 4 to 10 6 M –1 . As the most abundant protein in plasma, it plays an important role in the maintenance of blood pH and colloidal osmotic pressure and accounts for most of the thiol content of plasma. Numerous competition and spectroscopic studies, using various ligands, have identified two sites (I and II) most relevant for drug binding. The interaction of various nonsteroidal anti-inflammatory drugs (NSAIDs) with HSA has been studied extensively. The interaction of other drug classes with HSA has also been investigated. Covalent modification of plasma proteins can be produced by direct attachment of an electrophilic moiety of the parent molecule to nucleophilic protein residues or by metabolic activation of the parent to a more reactive species. These reactions are important as their potential to form immunogenic adducts, with the possibility of producing idiosyncratic hypersensitivity reactions. Microdialysis is a potent technique to measure the extent of binding in vivo or in vitro in plasma. high-performance liquid chromatography (HPLC) methods, utilizing protein stationary phases, offer the potential for rapid screening, especially for those molecules bound to albumin. The blood brain barrier (BBB) effectively restricts the access of polar, hydrophilic drugs into the central nervous system (CNS), thus establishing a significant drug delivery impediment.

Chapter 33. Plasma Protein Binding of Drugs

A number of therapeutic drugs with different structures and mechanisms of action have been reported to undergo metabolic activation by Phase I or Phase II drug-metabolizing enzymes. The bioactivation gives rise to reactive metabolites/intermediates, which readily confer covalent binding to various target proteins by nucleophilic substitution and/or Schiff's base mechanism. These drugs include analgesics (e.g., acetaminophen), antibacterial agents (e.g., sulfonamides and macrolide antibiotics), anticancer drugs (e.g., irinotecan), antiepileptic drugs (e.g., carbamazepine), anti-HIV agents (e.g., ritonavir), antipsychotics (e.g., clozapine), cardiovascular drugs (e.g., procainamide and hydralazine), immunosupressants (e.g., cyclosporine A), inhalational anesthetics (e.g., halothane), nonsteroidal anti-inflammatory drugs (NSAIDSs) (e.g., diclofenac), and steroids and their receptor modulators (e.g., estrogens and tamoxifen). Some herbal and dietary constituents are also bioactivated to reactive metabolites capable of binding covalently and inactivating cytochrome P450s (CYPs). A number of important target proteins of drugs have been identified by mass spectrometric techniques and proteomic approaches. The covalent binding and formation of drug-protein adducts are generally considered to be related to drug toxicity, and selective protein covalent binding by drug metabolites may lead to selective organ toxicity. However, the mechanisms involved in the protein adduct-induced toxicity are largely undefined, although it has been suggested that drug-protein adducts may cause toxicity either through impairing physiological functions of the modified proteins or through immune-mediated mechanisms. In addition, mechanism-based inhibition of CYPs may result in toxic drug-drug interactions. The clinical consequences of drug bioactivation and covalent binding to proteins are unpredictable, depending on many factors that are associated with the administered drugs and patients. Further studies using proteomic and genomic approaches with high throughput capacity are needed to identify the protein targets of reactive drug metabolites, and to elucidate the structure-activity relationships of drug's covalent binding to proteins and their clinical outcomes.

Drug Bioactivation Covalent Binding to Target Proteins and Toxicity Relevance

The structures of adducts formed from in vitro incubation of a drug (tolmetin) glucuronide (TG) and human serum albumin (HSA), and the preferred binding sites on this protein were determined by mass spectrometry. In addition, the concentration dependence of covalent modification of HSA by TG was studied at three different concentration ratios of TG to HSA. Protein adducts were enzymatically digested and peptide fragments were separated by HPLC. Tolmetin-containing peptides (indicated by absorbance at 313 nm) were analyzed by liquid secondary-ion mass spectrometry, continuous flow-fast atom bombardment mass spectrometry, and collision-induced dissociation using a four-sector tandem mass spectrometer, matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry, and in selected cases by Edman sequencing. The identified peptides contained tolmetin linked covalently via a glucuronic acid to a protein lysine group (lysine 199 and to a lesser extent lysines 195 and 525) or tolmetin directly linked to lysines (lysines 199 and 541), serines (serines 220, 232, and 480), or arginines (arginine 222). In addition, there was indirect evidence for binding of TG to lysine 541, and binding of tolmetin to arginine 521. Our results establish that the binding of these reactive metabolites to nucleophilic sites of proteins occur via two different mechanisms: one involving imine (Schiff base) formation and the other involving nucleophilic displacement of glucuronic acid. Our data suggest, however, that the former, in which the glucuronic acid moiety of the acyl glucuronide is retained within the adducts, is favored at lower (closer to physiological) metabolite concentrations.

Reactivity of tolmetin glucuronide with human serum albumin. Identification of binding sites and mechanisms of reaction by tandem mass spectrometry.

Human serum albumins modified by covalently bound tolmetin or zomepirac were synthesized as models for similar products formed in vivo from acyl glucuronides. Activated esters of both drugs were prepared with 1-ethyl-3-(3-dimethylaminopropyl)-carbodi-imide, and then allowed to react with human serum albumin. Tryptic digests of both protein products were analysed by HPLC to identify peptides containing covalently bound drugs, and binding sites on albumin were identified by high-performance tandem MS. Three binding sites were common to both products, i.e. lysine-195, -199 and -351. Three further modified residues were identified for the tolmetin-albumin product, i.e. aspartic acid 1, and lysine-524 and -536.

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Synthesis and mass-spectrometric characterization of human serum albumins modified by covalent binding of two non-steroidal anti-inflammatory drugs: tolmetin and zomepirac.

A. Ding

Papers

Reactivity of tolmetin glucuronide with human serum albumin. Identification of binding sites and mechanisms of reaction by tandem mass spectrometry.

Synthesis and mass-spectrometric characterization of human serum albumins modified by covalent binding of two non-steroidal anti-inflammatory drugs: tolmetin and zomepirac.