Dennis Schade

Stepwise Clearance of Repressive Roadblocks Drives Cardiac Induction in Human ESCs

Cardiac induction requires stepwise integration of BMP and WNT pathway activity. Human embryonic stem cells (hESCs) are developmentally and clinically relevant for studying the poorly understood molecular mechanisms downstream of these cascades. We show that BMP and WNT signaling drive cardiac specification by removing sequential roadblocks that otherwise redirect hESC differentiation toward competing fates, rather than activating a cardiac program per se. First, BMP and WNT signals pattern mesendoderm through cooperative repression of SOX2, a potent mesoderm antagonist. BMP signaling promotes miRNA-877 maturation to induce SOX2 mRNA degradation, while WNT-driven EOMES induction transcriptionally represses SOX2. Following mesoderm formation, cardiac differentiation requires inhibition of WNT activity. We found that WNT inhibition serves to restrict expression of anti-cardiac regulators MSX1 and CDX2/1. Conversely, their simultaneous disruption partially abrogates the requirement for WNT inactivation. These results suggest that human cardiac induction depends on multi-stage repression of alternate lineages, with implications for deriving expandable cardiac stem cells.

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.

Modulating the NO generating system from a medicinal chemistry perspective: Current trends and therapeutic options in cardiovascular disease

An impaired nitric oxide (NO) bioavailability is well-recognized in the pathology of endothelial dysfunction or atherosclerosis, respectively, and characterized by a reduced NO biosynthesis, an accelerated inactivation and/or a decreased sensitivity to NO. Therefore, attempts to increase endogenous NO concentrations or to improve responses to NO stimulation have attracted great pharmaceutical interest for the treatment of several cardiovascular diseases. The biological system of the NO/cGMP cascade is very complex and highly regulated through diverse upstream and downstream molecular and cellular elements and feedback mechanisms. This review summarizes the current options to modulate NO bioavailability for the treatment of cardiovascular disease, with a special focus on targets upstream of cGMP. We also point at the many shortcomings that are associated with the established therapy with nitrates, thereby raising general questions regarding this pharmacological approach. In fact, it is highly desirable to more selectively affect the respective pathologically altered processes in endothelial dysfunction, which ensures a safer and more effective therapy. Approaches to modulate those enzymes that predominate in the regulation of endogenous NO levels represent promising means to achieve this goal: nitric oxide synthases, arginases and dimethylarginine dimethylaminohydrolase. The herein presented novel developments essentially imply a paradigm shift from purely symptomatic to more causative therapeutics which opens up opportunities to not only treat ischemic heart disease but many more cardiovascular diseases.

Structure–activity relationship of novel and known inhibitors of human dimethylarginine dimethylaminohydrolase-1: Alkenyl-amidines as new leads

Recent studies demonstrated that inhibition of dimethylarginine dimethylaminohydrolase (DDAH) activity could be a new strategy to indirectly affect nitric oxide (NO) formation by elevating N(omega)-methylated L-arginine (NMMA, ADMA) levels. This approach is an alternate strategy for the treatment of diseases associated with increased NO-concentrations. To date, three classes of potent inhibitors are known: (1) pentafluorophenyl sulfonates (IC(50)=16-58 microM, PaDDAH), which are also inhibitors for the arginine deiminase; (2) the most potent inhibitors are based on indolylthiobarbituric acid (IC(50)=2-17 microM, PaDDAH), which were identified by virtual modelling; and (3) L-arginine analogs, whose best representative is N(omega)-(2-methoxyethyl)-L-arginine (IC(50)=22 microM, rat DDAH). Based on these known structures, we aimed to develop inhibitors for the human DDAH-1 with improved potency and better relative selectivity for DDAH-1 over NOS. Particularly, the binding pocket of the guanidine-moiety was investigated by screening differently substituted guanidines, amidines and isothioureas in order to collect information on possible binding modes in the active site. All substances were tested in a plate-reader format and HPLC assay and several potent inhibitors were identified with K(i)-values varying from 2 to 36 microM, with N(5)-(1-iminobut-3-enyl)-L-ornithine (L-VNIO) being the most potent inhibitor of the human DDAH-1 so far described. Besides these potent inhibitors alternate substrates for hDDAH-1 were identified as well.

Small molecules targeting in vivo tissue regeneration.

The field of regenerative medicine has boomed in recent years thanks to milestone discoveries in stem cell biology and tissue engineering, which has been driving paradigm shifts in the pharmacotherapy of degenerative and ischemic diseases. Small molecule-mediated replenishment of lost and/or dysfunctional tissue in vivo, however, is still in its infancy due to a limited understanding of mechanisms that control such endogenous processes of tissue homeostasis or regeneration. Here, we discuss current progress using small molecules targeting in vivo aspects of regeneration, including adult stem cells, stem cell niches, and mechanisms of homing, mobilization, and engraftment as well as somatic cell proliferation. Many of these compounds derived from both knowledge-based design and screening campaigns, illustrating the feasibility of translating in vitro discovery to in vivo regeneration. These early examples of drug-mediated in vivo regeneration provide a glimpse of the future directions of in vivo regenerative medicine approaches.

Synthetic Approaches to Nδ-Methylated l-Arginine, Nω-Hydroxy-l-arginine, l-Citrulline, and Nδ-Cyano-l-ornithine

Nomega-Methylated arginines such as asymmetric dimethyl-L-arginine (ADMA) and monomethyl-l-arginine (NMMA) are known as potent physiological inhibitors of nitric oxide synthases (NOSs). To explore a possible physiological and pharmaceutical relevance of N(delta)-methylated analogues, a synthetic scheme had to be developed that would not lead to N(delta)-methyl-L-arginine only but also to its presumed metabolites of NOS catalysis. Two basic synthetic approaches have been pursued to obtain N(delta)-methylated derivatives of L-ornithine, L-citrulline, L-arginine, and N(omega)-hydroxy-L-arginine. A first attempt utilized conventionally protected L-ornithine, i.e., the tert-butyl ester and Boc-amine, and led to three end compounds in excellent yields. Simultaneous protection of the alpha-amino acid moiety by formation of boroxazolidinones, particularly by employing 9-borabicyclo[3.3.1]nonane (9-BBN-H), proved to be a convenient option to perform side chain modifications and led to all of the desired end compounds. Additionally, enantiomeric excess (ee, %) of crucial synthetic intermediates and end compounds was determined by chiral HPLC.

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

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.

Designing modulators of dimethylarginine dimethylaminohydrolase (DDAH): a focus on selectivity over arginase.

DDAH inhibition presents a novel promising pharmaceutical strategy to lower NO formation. To date, several potent DDAH inhibitors have been published, most of them representing analogues of l-arginine. While inhibitory effects on NOSs have already been considered, selectivity over arginase has been neglected so far. In our view, the latter selectivity is more important since an additional inhibition of arginase decreases the desired effects on NO levels. Thus, we particularly focus on selectivity over arginase. We present a comprehensive selectivity profile of known DDAH inhibitors by covering their inhibitory potency on arginase. Among the studied compounds, Nω-(2-methoxyethyl)-l-arginine (2a, L-257) that is already selective over NOSs also only modestly affected arginase activity and is thus far the most suitable DDAH inhibitor for pharmacological studies.