Joscha Kotthaus

The Fourth Molybdenum Containing Enzyme mARC: Cloning and Involvement in the Activation of N-Hydroxylated Prodrugs

The recently discovered mammalian molybdoprotein mARC1 is capable of reducing N-hydroxylated compounds. Upon reconstitution with cytochrome b(5) and b(5) reductase, benzamidoxime, pentamidine, and diminazene amidoximes, N-hydroxymelagatran, guanoxabenz, and N-hydroxydebrisoquine are efficiently reduced. These substances are amidoxime/N-hydroxyguanidine prodrugs, leading to improved bioavailability compared to the active amidines/guanidines. Thus, the recombinant enzyme allows prediction about in vivo reduction of N-hydroxylated prodrugs. Furthermore, the prodrug principle is not dependent on cytochrome P450 enzymes.

Reduction of N ω -hydroxy-L-arginine by the mitochondrial amidoxime reducing component (mARC)

NOSs (nitric oxide synthases) catalyse the oxidation of L-arginine to L-citrulline and nitric oxide via the intermediate NOHA (N(ω)-hydroxy-L-arginine). This intermediate is rapidly converted further, but to a small extent can also be liberated from the active site of NOSs and act as a transportable precursor of nitric oxide or potent physiological inhibitor of arginases. Thus its formation is of enormous importance for the nitric-oxide-generating system. It has also been shown that NOHA is reduced by microsomes and mitochondria to L-arginine. In the present study, we show for the first time that both human isoforms of the newly identified mARC (mitochondrial amidoxime reducing component) enhance the rate of reduction of NOHA, in the presence of NADH cytochrome b₅ reductase and cytochrome b₅, by more than 500-fold. Consequently, these results provide the first hints that mARC might be involved in mitochondrial NOHA reduction and could be of physiological significance in affecting endogenous nitric oxide levels. Possibly, this reduction represents another regulative mechanism in the complex regulation of nitric oxide biosynthesis, considering a mitochondrial NOS has been identified. Moreover, this reduction is not restricted to NOHA since the analogous arginase inhibitor NHAM (N(ω)-hydroxy-N(δ)-methyl-L-arginine) is also reduced by this system.

Development of Novel Potent Orally Bioavailable Oseltamivir Derivatives Active against Resistant Influenza A

With the emergence of oseltamivir-resistant influenza viruses and in view of a highly pathogenic flu pandemic, it is important to develop new anti-influenza agents. Here, the development of neuraminidase (NA) inhibitors that were designed to overcome resistance mechanisms along with unfavorable pharmacokinetic (PK) properties is described. Several 5-guanidino- and 5-amidino-based oseltamivir derivatives were synthesized and profiled for their anti-influenza activity and in vitro and in vivo PK properties. Amidine 6 and guanidine 7 were comparably effective against a panel of different A/H1N1 and A/H3N2 strains and also inhibited mutant A/H1N1 neuraminidase. Among different prodrug strategies pursued, a simple amidoxime ethyl ester (9) exhibited a superior PK profile with an oral bioavailability of 31% (rats), which is comparable to oseltamivir (36%). Thus, bioisosteric replacement of the 5-guanidine with an acetamidine-in the form of its N-hydroxy prodrug-successfully tackled the two key limitations of currently used NA inhibitors, as exemplified with oseltamivir.

Highly Potent and Selective Substrate Analogue Factor Xa Inhibitors Containing D‐Homophenylalanine Analogues as P3 Residue: Part 2

A series of highly potent substrate‐analogue factor Xa inhibitors containing D‐homophenylalanine analogues as the P3 residue has been identified by systematic optimization of a previously described inhibitor structure. An initial lead, benzylsulfonyl‐d‐hPhe‐Gly‐4‐amidinobenzylamide (3), inhibits fXa with an inhibition constant of 6.0 nM. Most modifications of the P2 amino acid and P4 benzylsulfonyl group did not improve the affinity and selectivity of the compounds as fXa inhibitors. In contrast, further variation at the P3 position led to inhibitors with significantly enhanced potency and selectivity. Inhibitor 27, benzylsulfonyl‐D‐homo‐2‐pyridylalanyl(N‐oxide)‐Gly‐4‐amidinobenzylamide, inhibits fXa with a Ki value of 0.32 nM. The inhibitor has strong anticoagulant activity in plasma and doubles the activated partial thromboplastin time and prothrombin time at concentrations of 280 nM and 170 nM, respectively. Compound 27 inhibits the prothrombinase complex with an IC50 value of 5 nM and is approximately 50 times more potent than the reference inhibitor DX‐9065a in this assay.

Incorporation of neutral C-terminal residues in 3-amidinophenylalanine-derived matriptase inhibitors

A novel series of matriptase inhibitors based on previously identified tribasic 3-amidinophenylalanine derivatives was prepared. The C-terminal basic group was replaced by neutral residues to reduce the hydrophilicity of the inhibitors. The most potent compound 22 inhibits matriptase with a K(i) value of 0.43 nM, but lacks selectivity towards factor Xa. By combination with neutral N-terminal sulfonyl residues several potent thrombin inhibitors were identified, which had reduced matriptase affinity.

Analysis of highly potent amidine containing inhibitors of serine proteases and their N-hydroxylated prodrugs (amidoximes)

The development of serine protease inhibitors often results in the discovery of new lead compounds containing strong basic amidine functions that usually suffer from poor absorption from the intestine. In order to improve oral bioavailability of these drugs, prodrug principles such as the conversion of amidines into amidoximes may be applied. In this work, two HPLC-based separation methods of serine protease inhibitors (amidines) and their N-hydroxylated prodrugs have been developed and characterised. This was performed by evaluating 11 distinct amidine-amidoxime pairs with different physicochemical parameters (clogP: −3 to 5.1). The HPLC methods developed allowed excellent separation of the compound pairs examined. Also, the possible selection of different separation techniques (i.e. adsorption- and ion-pair-chromatography) permits universal application. Moreover, both techniques are compatible with mass spectrometry and are superior to the previously described methods. In summary, both HPLC methods are suitable for the separation of most amidoxime-prodrugs currently in clinical or preclinical development.

New prodrugs of the antiprotozoal drug pentamidine.

Pentamidine is an effective antimicrobial agent that is approved for the treatment of African trypanosomiasis but suffers from poor oral bioavailability and central nervous system (CNS) penetration. This work deals with the development and systematic characterisation of new prodrugs of pentamidine. For this reason, numerous prodrugs that use different prodrug principles were synthesised and examined in vitro and in vivo. Another objective of the study was the determination of permeability of the different pentamidine prodrugs. While some of the prodrug principles applied in this study are known, such as the conversion of the amidine functions into amidoximes or the O‐alkylation of amidoximes with a carboxymethyl residue, others were developed more recently and are described here for the first time. These newly developed methods aim to increase the affinity of the prodrug for the transporters and mediate an active uptake via carrier systems by conjugation of amidoximes with compounds that improve the overall solubility of the prodrug. The different principles chosen resulted in several pentamidine prodrugs with various advantages. The objective of this investigation was the systematic characterisation and evaluation of eight pentamidine prodrugs in order to identify the most appropriate strategy to improve the properties of the parent drug. For this reason, all prodrugs were examined with respect to their solubility, stability, enzymatic activation, distribution, CNS delivery, and oral bioavailability. The results of this work have allowed reliable conclusions to be drawn regarding the best prodrug principle for the antiprotozoal drug pentamidine.

The Peptidylglycine α-Amidating Monooxygenase (PAM): A Novel Prodrug Strategy for Amidoximes and N-Hydroxyguanidines?

In recent decades the concept of targeted prodrug design has attracted much interest in the field of pharmaceutical and medicinal chemistry. The use of prodrugs allows unfavorable physicochemical, pharmacokinetic and/or pharmacodynamic properties of drug candidates to be overcome. Amidine and guanidine moieties are valuable pharmacophoric groups since they can mimic arginine residues in biological structures. They often contribute to high affinities of small molecules to their target proteins by strong ionic and Hbond interactions. In many cases, their replacement with other functionalities leads to concomitant significant loss of affinity. However, under physiological conditions these strongly basic structures exist in their cationic form and are therefore usually unable to pass through mucosal membranes. Without specific transporter mechanisms, amidines and guanidines are poorly absorbed in the gastrointestinal tract after oral application. In order to mask their basic properties and thereby improve passive diffusion, several prodrug strategies have been pursued in the past few years. To date, most of these prodrug principles were applied particularly to the amidine moiety of drugs belonging to a wellstudied class of antithrombotics. Among these principles, the amidoxime function has been a promising concept since the N-hydroxylation leads to a significant decrease in basicity and an increase in lipophilicity. Furthermore, the extensive in vivo reduction of such amidoximes, that is the required bioactivation, has been demonstrated for benzamidoxime as a model compound, the antiprotozoic prodrug pentamidine diamidoxime, the GPIIb/IIIa antagonist sibrafiban, the thrombin inhibitor ximelagatran, and several more N-hydroxylated prodrugs. 5] Notably, this bioactivation proceeds in a CYP450-independent manner; we recently identified a molybdenum-containing enzyme (mARC) that is largely responsible for this N-reduction. In contrast to amidoximes, N-hydroxyguanidines have not yet found application as prodrugs for guanidines. Their efficient N-reduction, however, has already been demonstrated for guanoxabenz and N-hydroxydebrisoquine. In this context, a major issue in the applicability of N-hydroxyguanidines as guanidine prodrugs may be their chemical instability in terms of hydrolysis and oxidation, requiring further stabilizing modifications. Previous prodrug strategies for amidoximes (and N-hydroxyguanidines) are now targeted to optimize metabolic and physicochemical stability, thereby further enhancing oral bioavailACHTUNGTRENNUNGability and their general applicability. By additional Oand/or N-substitution, presystemic reduction is expected to be prevented, which otherwise leads to decreased oral absorption. Furthermore, tendencies for chemical degradations, particularly regarding N-hydroxyguanidines, may be circumvented. Moreover, a specific prodrug group may allow for cellor tissuetargeting, thereby minimizing side effects and maximizing the ACHTUNGTRENNUNGdesired therapeutic effects. An example of an O-alkylated prodrug (such as 4, Figure 1) is the O-methylamidoxime DB844, an antiparasitic agent with good oral bioavailability and in vivo efficacy. The required

MS3 fragmentation patterns of monomethylarginine species and the quantification of all methylarginine species in yeast using MRM3

Protein arginine methylation is one of the epigenetic modifications to proteins that is studied in yeast and is known to be involved in a number of human diseases. All eukaryotes produce Nη-monomethylarginine (ηMMA), asymmetric Nη1, Nη1-dimethylarginine (aDMA), and most produce symmetric Nη1, Nη2-dimethylarginine (sDMA) on proteins, but only yeast produce Nδ-monomethylarginine (δMMA). It has proven difficult to differentiate among all of these methylarginines using mass spectrometry. Accordingly, we demonstrated that the two forms of MMA have indistinguishable primary product ion spectra. However, the secondary product ion spectra of δMMA and ηMMA exhibited distinct patterns of ions. Using incorporation of deuterated methyl-groups in yeast, we determined which secondary product ions were methylated and their structures. Utilizing distinct secondary product ions, a triple quadrupole multiple reaction monitoring cubed (MRM(3)) assay was developed to measure δMMA, ηMMA, sDMA and aDMA derived from hydrolyzed protein. As a proof-of-concept, δMMA and ηMMA were measured using the MRM(3) method in wild type and mutant strains of Saccharomyces cerevisiae and compared to the total MMA measured using an existing assay. The MRM(3) assay represents the only method to directly quantify δMMA and the only method to simultaneously quantify all yeast methylarginines.

Zanamivir Amidoxime- and N-Hydroxyguanidine-Based Prodrug Approaches to Tackle Poor Oral Bioavailability.

The neuraminidase (NA) inhibitor zanamivir (1) is potently active against a broad panel of influenza A and B strains, including mutant viruses, but suffers from pharmacokinetic (PK) shortcomings. Here, distinct prodrug approaches are described that aimed at overcoming zanamivirs lack of oral bioavailability. Lowering the high basicity of the 4-guanidino group in zanamivir and of a bioisosteric 4-acetamidine analog (5) by N-hydroxylation was deemed to be a plausible tactic. The carboxylic acid and glycerol side chain were also masked with different ester groups. The bioisosteric amidine 5 turned out to be potently active against a panel of H1N1 (IC50 = 2-10 nM) and H3N2 (IC50 = 5-10 nM) influenza A viruses (NA inhibition assay). In vitro PK studies showed that all prodrugs were highly soluble, exhibited low protein binding, and were bioactivated by N-reduction to the respective guanidines and amidines. The most promising prodrug candidates, amidoxime ester 7 and N-hydroxyguanidine ester 8, were subjected to in vivo bioavailability studies. Unfortunately, both prodrugs were not orally bioavailable to a convincing degree (F ≤ 3.7%, rats). This finding questions the general feasibility of improving the oral bioavailability of 1 by lipophilicity-increasing prodrug strategies, and suggests that intrinsic structural features represent key hurdles.