Design Of Novel Prodrugs Research Articles

Toward Orally Absorbed Prodrugs of the Antibiotic Aztreonam. Design of Novel Prodrugs of Sulfate Containing Drugs. Part 2.

Aztreonam, first discovered in 1980, is an FDA approved, intravenous, monocyclic beta-lactam antibiotic. Aztreonam is active against Gram-negative bacteria and is still used today. The oral bioavailability of aztreonam in humans is less than 1%. Herein we describe the design and synthesis of potential oral prodrugs of aztreonam.

Computational modeling and in-vitro/in-silico correlation of phospholipid-based prodrugs for targeted drug delivery in inflammatory bowel disease.

Targeting drugs to the inflamed intestinal tissue(s) represents a major advancement in the treatment of inflammatory bowel disease (IBD). In this work we present a powerful in-silico modeling approach to guide the molecular design of novel prodrugs targeting the enzyme PLA2, which is overexpressed in the inflamed tissues of IBD patients. The prodrug consists of the drug moiety bound to the sn-2 position of phospholipid (PL) through a carbonic linker, aiming to allow PLA2 to release the free drug. The linker length dictates the affinity of the PL-drug conjugate to PLA2, and the optimal linker will enable maximal PLA2-mediated activation. Thermodynamic integration and Weighted Histogram Analysis Method (WHAM)/Umbrella Sampling method were used to compute the changes in PLA2 transition state binding free energy of the prodrug molecule (∆∆Gtr) associated with decreasing/increasing linker length. The simulations revealed that 6-carbons linker is the optimal one, whereas shorter or longer linkers resulted in decreased PLA2-mediated activation. These in-silico results were shown to be in excellent correlation with experimental in-vitro data. Overall, this modern computational approach enables optimization of the molecular design of novel prodrugs, which may allow targeting the free drug specifically to the diseased intestinal tissue of IBD patients.

Crystal structure of the fungal nitroreductase Frm2 from Saccharomyces cerevisiae.

Nitroreductases are flavoenzymes that catalyze nitrocompounds and are widely utilized in industrial applications due to their detoxification potential and activation of biomedicinal prodrugs. Type I nitroreductases are classified into subgroups depending on the use of NADPH or NADH as the electron donor. Here, we report the crystal structure of the fungal nitroreductase Frm2 from Saccharomyces cerevisiae, one of the uncharacterized subgroups of proteins, to reveal its minimal architecture previously observed in bacterial nitroreductases such as CinD and YdjA. The structure lacks protruding helical motifs that form part of the cofactor and substrate binding site, resulting in an open and wide active site geometry. Arg82 is uniquely conserved in proximity to the substrate binding site in Frm2 homologues and plays a crucial role in the activity of the active site. Frm2 primarily utilizes NADH to reduce 4-NQO. Because missing helical elements are involved in the direct binding to the NAD(P)H in group A or group B in Type I family, Frm2 and its homologues may represent a distinctive subgroup with an altered binding mode for the reducing compound. This result provides a structural basis for the rational design of novel prodrugs with the ability to reduce nitrogen-containing hazardous molecules.

Prodrugs Design Based on Inter‐ and Intramolecular Chemical Processes

This review provides the reader a concise overview of the majority of prodrug approaches with the emphasis on the modern approaches to prodrug design. The chemical approach catalyzed by metabolic enzymes which is considered as widely used among all other approaches to minimize the undesirable drug physicochemical properties is discussed. Part of this review will shed light on the use of molecular orbital methods such as DFT, semiempirical and ab initio for the design of novel prodrugs. This novel prodrug approach implies prodrug design based on enzyme models that were utilized for mimicking enzyme catalysis. The computational approach exploited for the prodrug design involves molecular orbital and molecular mechanics (DFT, ab initio, and MM2) calculations and correlations between experimental and calculated values of intramolecular processes that were experimentally studied to assign the factors determining the reaction rates in certain processes for better understanding on how enzymes might exert their extraordinary catalysis.

Crystal Structure of a Minimal Nitroreductase, ydjA, from Escherichia coli K12 with and without FMN Cofactor

Nitroreductases (NTR) are enzymes that reduce hazardous nitroaromatic compounds and are of special interest due to their potential use in bioremediation and their activation of prodrugs in directed anticancer therapies. We elucidated the crystal structures of ydjA from Escherichia coli (Ec_ydjA), one of the smallest NTRs, in its flavin mononucleotide (FMN)-bound and cofactor-free forms. The α + β mixed monomeric Ec_ydjA forms a homodimeric structure through the interactions of the long central helices and the extended regions at both termini. Two FMN molecules are bound at the dimeric interface. The absence of the 30 internal amino acids in Ec_ydjA, which forms two helices and restricts the cofactor and substrate binding in other NTR family members, creates a wider and more flexible active site. Unlike the bent FMN ring structures present in most NTR complexes currently known, the flavin system in the Ec_ydjA structure maintains a flat ring conformation, which is sandwiched between a Trp and a His residue from each monomer. The analysis of our Ec_ydjA structure explains its specificity for larger substrates and provides structural information for the rational design of novel prodrugs with the ability to reduce nitrogen-containing hazardous molecules.

Effects of steric bulk and stereochemistry on the rates of diketopiperazine formation from N-aminoacyl-2,2-dimethylthiazolidine-4-carboxamides (Dmt dipeptide amides)—a model for a new prodrug linker system

A peptide-like self-immolative molecular clip is required for release of active drugs from prodrugs by endopeptidases. Upon cleavage from the carrier, this clip must collapse and release the drug rapidly. A series of aminoacyl-5,5-dimethylthiaproline (Aaa-Dmt) N-(2-(4-nitrophenyl)ethyl)amides were designed. Boc- l-aminoacyl fluorides were coupled with R-DmtOH to give Boc- l-Aaa- R-DmtOH, which were converted to the Boc- l-Aaa- R-Dmt N-(2-(4-nitrophenyl)ethyl)amides. The l, S diastereomeric series was prepared by the reaction of Boc-Aaa PFP esters with S-DmtOH. The l-Aaa-Dmt N-(2-(4-nitrophenyl)ethyl)amides were allowed to cyclise to diketopiperazines (DKPs) in aqueous buffers, expelling 2-(4-nitrophenyl)ethylamine as a model for amine-containing drugs. Reaction rates were dependant on pH. In the l, R diastereomeric series, increasing steric bulk of the Aaa side-chain (Gly, Ala, Phe, Val) led to decrease in the reaction rate. However, in the l, S series, the greatest rate of reaction was observed for the most bulky amino-acid (Val), with t ½=15 min at pH 8.0. The effects of steric bulk and stereochemistry are rationalised through conformational analysis (NMR and X-ray crystallography) of the starting dipeptide amides, the product diketopiperazines and key analogues. Since the dipeptides are (almost) exclusively in the cis-amide conformation, trans–cis interconversion is not relevant. The data suggest that steric interactions in the reacting conformations of the dipeptide amides, as they form the tetrahedral intermediates, are the controlling factors. Thus, l-Aaa- S-Dmt amides are shown to be excellent candidates for incorporation into the design of novel prodrugs.

Aerobic nitroreduction by flavoproteins: enzyme structure, mechanisms and role in cancer chemotherapy.

NQO1 (DT-diaphorase) and its truncated isoenzyme, the metalloenzyme NQO2, can reduce quinone substrates by two-electron transfer. While NQO1 is a known detoxification enzyme, the function of NQO2 is less well understood. Both rat NQO1 and human NQO2 reductively bioactivate the dinitroarene CB 1954 to a cytotoxic product that behaves as a difunctional DNA-crosslinking species with potent anti-tumour activity, although human NQO1 is much less effective. A FMN-dependent nitroreductase from E. coli B also reduces quinones and reductively bioactivates CB 1954. However, this enzyme reduces CB 1954 to the 2- and 4-hydroxylamines in equivalent yield, whereas NQO1 and NQO2 generate only the 4-isomer. The reduction profile is a key factor in the development of anti-tumour prodrugs, where distinct delivery strategies are being evaluated: prodrug therapy, antibody-, macromolecule and gene-directed enzyme prodrug therapy (ADEPT, MDEPT or GDEPT). The flavoprotein enzymes are explored in terms of structure and bioreduction mechanism, particularly for use in the design of novel prodrugs with potential application as chemotherapeutic agents.

The structure of Escherichia coli nitroreductase complexed with nicotinic acid: three crystal forms at 1.7 Å, 1.8 Å and 2.4 Å resolution

Escherichia coli nitroreductase is a flavoprotein that reduces a variety of quinone and nitroaromatic substrates. Its ability to convert relatively non-toxic prodrugs such as CB1954 (5-{aziridin-1-yl}-2,4-dinitrobenzamide) into highly cytotoxic derivatives has led to interest in its potential for cancer gene therapy. We have determined the structure of the enzyme bound to a substrate analogue, nicotinic acid, from three crystal forms at resolutions of 1.7 Å, 1.8 Å and 2.4 Å, representing ten non-crystallographically related monomers. The enzyme is dimeric, and has a large hydrophobic core; each half of the molecule consists of a five-stranded β-sheet surrounded by α-helices. Helices E and F protrude from the core region of each monomer. There is an extensive dimer interface, and the 15 C-terminal residues extend around the opposing monomer, contributing the fifth β-strand. The active sites lie on opposite sides of the molecule, in solvent-exposed clefts at the dimer interface. The FMN forms hydrogen bonds to one monomer and hydrophobic contacts to both; its si face is buried. The nicotinic acid stacks between the re face of the FMN and Phe124 in helix F, with only one hydrogen bond to the protein. If the nicotinamide ring of the coenzyme NAD(P)H were in the same position as that of the nicotinic acid ligand, its C4 atom would be optimally positioned for direct hydride transfer to flavin N5. Comparison of the structure with unliganded flavin reductase and NTR suggests reduced mobility of helices E and F upon ligand binding. Analysis of the structure explains the broad substrate specificity of the enzyme, and provides the basis for rational design of novel prodrugs and for site-directed mutagenesis for improved enzyme activity.

Extended Substrate Acceptance of Herpes Simplex Virus Type 1 Thymidine Kinase: a New Chance for Gene and Antiviral Therapy

Herpes simplex virus type 1 thymidine kinase (HSV1-TK) has become increasingly important as a target in medicinal chemistry because of its links to therapy of viral infection, gene therapy of cancer and allogeneic transplantation. These applications are based on the differences in binding properties between the human and the viral enzyme. Several problems have been encountered in the clinic, e.g. the increase of resistance for antiviral drugs and the immunosuppressive effects of the dosages needed for tumor regression. Thus intensive efforts have been directed towards understanding substrate diversity to overcome the clinical limitations. In this context, kinetic and thermodynamic studies revealed that substrates bind in compulsory order and that the binding event is enthalpy driven. The structural evaluation of aciclovir resistant HSV strains shows that loss of electrostatic interactions, change in steric accessibility and modification of the 3D conformation of HSV1-TK are responsible for the encountered resistance. Further crystallography studies revealed the role of water in substrate binding, the advantage of a fixed ribose ring and that substrate acceptance of HSV1-TK is extended to all five nucleobases. The reviewed results give new rationale for the design of novel prodrugs and engineered HSV1-TK for antiviral and gene therapy.

The use of esters as prodrugs for oral delivery of beta-lactam antibiotics.

It is apparent that the sequence of events that has been followed in the approach to the discovery and development of a new beta-lactam prodrug has been similar in many of the case histories we have studied and indeed similar to the approach we have followed. Initially, we select a suitable series of prodrug moieties, which either comprises totally novel structures or is deduced from the data bases available (bearing in mind reports of potential toxicity) or both. The successful preparation of these prodrugs and the studies undertaken to ensure they are of known purity and stability is not easy and, as would be expected, is the initial go/no-go decision. Usually, the next stage has involved the assessment of whether or not bioavailability of the parent molecule is increased after administration of the prodrug ester by gavage to laboratory animal species. The selection of which species to use has very often been made according to which has the most information available in those particular laboratories and in the literature. It is this process that can be dishearteningly misleading as was demonstrated in Table IV and Fig. 1. Increasing the range of animal species does not lead to a better ability to predict bioavailability in humans. Hydrolysis studies are important to ensure that any novel prodrug will hydrolyze in human tissues, and also in the clarification of why a particular prodrug is not performing as expected in animals. After selection, it is essential to determine where and how rapidly hydrolysis takes place in the animal species to be used for safety evaluation prior to the first bioavailability studies in humans. The assessment of absolute oral bioavailability has not always been undertaken. This would seem critical for studies in not only the selected animal species but also in humans. In the absence of these data it is difficult to judge whether oral uptake can be increased further by modifying the ester moieties and at the development stage to determine whether or not modifications in formulation could increase bioavailability. When the prodrug is being developed for an injectable beta-lactam already available for humans, there would be no problem, but it would be an important consideration during the development of an entirely novel beta-lactam antibiotic for which no parenteral data are available in humans. Animal data are not totally predictive. The development of prodrugs is not easy, as a consequence of species differences in the properties of the prodrug superimposed on those of the parent compound during the evaluation. However, technical advances have enabled us to assay concentrations more precisely, determine basic physicochemical properties more efficiently, understand absorption processes by the use of in vitro systems, and analyze data far more comprehensively by the use of ever-evolving computer software. The prodrug approach to increasing the oral bioavailability of beta-lactam antibiotics has provided clinically valuable agents and continues. Despite the inherent difficulties, knowledge gained over the years, of the relationships between physicochemical and biological properties of the parent compound and the intact prodrug ester, has contributed to the design of novel prodrugs and a number of novel auxiliaries have been developed.

Design of novel prodrugs for the enhancement of the transdermal penetration of indomethacin

A series of esters of indomethacin containing tertiary amine functional groups and designed for transdermal delivery were synthesized. The rates of chemical hydrolysis of all the esters in pH 7.4 phosphate buffer were determined. For N,N-diethylaminopropyl N-(4-chlorobenzoyl)-5-methoxy-2-methyl-3-indole acetate (ester 4), the rates of chemical hydrolysis in pH 5.5–11.25 buffers were investigated in detail. Ester 4 was chemically stable and had a higher n-octanol/water partition coefficient and a greater solubility in water at pH 7.4 than indomethacin. Furthermore, the rate of transdermal penetration of ester 4 through cadaver skin in Franz cells was found to be more than 10-times faster than that of indomethacin itself. Ester 4 was found to possess surface-active properties (CMC = 0.5 mg/ml). Assuming that micelles cannot penetrate biologic membranes, a corrected permeability coefficient was calculated for ester 4 using only the monomer concentration. This value, 3.6 × 10 −2 cm/h, was 100-times greater than that of indomethacin. These results suggest that prodrugs with structures similar to that of ester 4 may be useful for enhancing transdermal penetration of other carboxylic acid-containing anti-inflammatory agents.