Random screening provided no suitable lead structures in a search for novel inhibitors of the bacterial enzyme DNA gyrase. Therefore, an alternative approach had to be developed. Relying on the detailed 3D structural information of the targeted ATP binding site, our approach combines as key techniques (1) an in silico screening for potential low molecular weight inhibitors, (2) a biased high throughput DNA gyrase screen, (3) validation of the screening hits by biophysical methods, and (4) a 3D guided optimization process. When the in silico screening was performed, the initial data set containing 350 000 compounds could be reduced to 3000 molecules. Testing these 3000 selected compounds in the DNA gyrase assay provided 150 hits clustered in 14 classes. Seven classes could be validated as true, novel DNA gyrase inhibitors that act by binding to the ATP binding site located on subunit B: phenols, 2-amino-triazines, 4-amino-pyrimidines, 2-amino-pyrimidines, pyrrolopyrimidines, indazoles, and 2-hydroxymethyl-indoles. The 3D guided optimization provided highly potent DNA gyrase inhibitors, e. g., the 3,4-disubstituted indazole 23 being a 10 times more potent DNA gyrase inhibitor than novobiocin (3).

Sphingosine-1-phosphate (S1P) is a widespread lysophospholipid which displays a wealth of biological effects. Extracellular S1P conveys its activity through five specific G-protein coupled receptors numbered S1P(1) through S1P(5). Agonists of the S1P(1) receptor block the egress of T-lymphocytes from thymus and lymphoid organs and hold promise for the oral treatment of autoimmune disorders. Here, we report on the discovery and detailed structure-activity relationships of a novel class of S1P(1) receptor agonists based on the 2-imino-thiazolidin-4-one scaffold. Compound 8bo (ACT-128800) emerged from this series and is a potent, selective, and orally active S1P(1) receptor agonist selected for clinical development. In the rat, maximal reduction of circulating lymphocytes was reached at a dose of 3 mg/kg. The duration of lymphocyte sequestration was dose dependent. At a dose of 100 mg/kg, the effect on lymphocyte counts was fully reversible within less than 36 h. Pharmacokinetic investigation of 8bo in beagle dogs suggests that the compound is suitable for once daily dosing in humans.

The malaria parasite Plasmodium falciparum degrades host cell hemoglobin inside an acidic food vacuole during the blood stage of the infectious cycle. A number of aspartic proteinases called plasmepsins (PMs) have been identified to play important roles in this degradation process and therefore generated significant interest as new antimalarial targets. Several x-ray structures of PMII have been described previously, but thus far, structure-guided drug design has been hampered by the fact that only inhibitors comprising a statine moiety or derivatives thereof have been published. Our drug discovery efforts to find innovative, cheap, and easily synthesized inhibitors against aspartic proteinases yielded some highly potent non-peptidic achiral inhibitors. A highly resolved (1.6 Å) x-ray structure of PMII is presented, featuring a potent achiral inhibitor in an unprecedented orientation, contacting the catalytic aspartates indirectly via the "catalytic" water. Major side chain rearrangements in the active site occur, which open up a new pocket and allow a new binding mode of the inhibitor. Moreover, a second inhibitor molecule could be located unambiguously in the active site of PMII. These newly obtained structural insights will further guide our attempts to improve compound properties eventually leading to the identification of molecules suitable as antimalarial drugs.Malaria is a major public health issue in many areas of the world, with Plasmodium falciparum being the causative agent of the most severe and deadliest form of this disease. Each year, 500 million new infections resulting in up to 2 million deaths and enormous economic damage (1) are attributed to this parasite. Drug resistance in P. falciparum has been aggravating the problem in many parts of the world during the last two decades, and new antimalarial agents addressing new targets are desperately needed.The protozoan parasite resides in erythrocytes of infected individuals during the asexual part of its life cycle. Recent studies indicated that hemoglobin degradation in a parasitic acidic organelle represents a major metabolic pathway and is crucial for survival of the parasite. Multiple proteinases appear to be actively involved in hemoglobin degradation (2-5). In particular, three members of a family of P. falciparum aspartic proteinases (PMI, 1 PMII, and PMIV) have been localized in the food vacuole (4, 5) and shown to be able to degrade hemoglobin in vitro. Another sequence-related proteinase with a new catalytic apparatus called PMIII or histo-aspartic proteinase (6) is also involved in hemoglobin catabolism in vitro. A number of research groups have reported attempts to find potent inhibitors of plasmepsins (7-12). Many of the identified molecules are peptidomimetic in nature, a compound class often associated with relatively low bioavailability and, importantly for use in developing countries, unfeasible due to significant cost of goods. We have discovered and subsequently optimized a new class of potent PMII inhibitors that could potential...

Two aspartic proteinases, plasmepsins 1 and 11, are present in the digestive vacuole of the human malarial parasite Plasmodium falciparum and are believed to be essential for parasite degradation of haemoglobin. Here we report the expression and kinetic characterisation of functional recombinant plasmepsin 1. In order to generate active plasmepsin I from its precursor, an autocatalytic cleavage site was introduced into the propart of the zymogen by mutation of LysllOP to Val (P indicates a propart residue). Appropriate refolding of the mutated zymogen then permitted pH-dependent autocatalytic processing of the zymogen to the active mature proteinase. A purification scheme was devised that removed aggregated and misfolded protein to yield pure, fully processable, proplasmepsin 1. Kinetic constants for two synthetic peptide substrates and four inhibitors were determined for both recombinant plasmepsin I and recombinant plasmepsin 11. Plasmepsin I had 5-10-fold lower kc.,,/K,n values than plasmepsin I1 for the peptide substrates, while the aspartic proteinase inhibitors, selected for their ability to inhibit P falciparum growth, were found to have up to 80-fold lower inhibition constants for plasmepsin I compared to plasmepsin 11. The most active plasmepsin I inhibitors were antagonistic to the antimalarial action of chloroquine on cultured parasites. Northern blot analysis of RNA, isolated from specific stages of the erythrocytic cycle of I? falciparum, showed that the proplasmepsin I gene is expressed in the ring stages whereas the proplasmepsin I1 gene is not transcribed until the later trophozoite stage of parasite growth. The differences in kinetic properties and temporal expression of the two plasmepsins suggest they are not functionally redundant but play distinct roles in the parasite.

There is an urgent need for new antibacterial drugs that are effective against infections caused by multidrug-resistant pathogens. Novel nonfluoroquinolone inhibitors of bacterial type II topoisomerases (DNA gyrase and topoisomerase IV) have the potential to become such drugs because they display potent antibacterial activity and exhibit no target-mediated cross-resistance with fluoroquinolones. Bacterial topoisomerase inhibitors that are built on a tetrahydropyran ring linked to a bicyclic aromatic moiety through a syn-diol linker show potent anti-Gram-positive activity, covering isolates with clinically relevant resistance phenotypes. For instance, analog 49c was found to be a dual DNA gyrase-topoisomerase IV inhibitor, with broad antibacterial activity and low propensity for spontaneous resistance development, but suffered from high hERG K(+) channel block. On the other hand, analog 49e displayed lower hERG K(+) channel block while retaining potent in vitro antibacterial activity and acceptable frequency for resistance development. Furthermore, analog 49e showed moderate clearance in rat and promising in vivo efficacy against Staphylococcus aureus in a murine infection model.