Antimicrobial Preservatives for Protein and Peptide Formulations: An Overview.

Biological drugs intended for multi-dose application require the presence of antimicrobial preservatives to avoid microbial growth. As the presence of certain preservatives has been reported to increase protein and peptide particle formation, it is essential to choose a preservative compatible with the active pharmaceutical ingredient in addition to its preservation function. Thus, this review describes the current status of the use of antimicrobial preservatives in biologic formulations considering (i) appropriate preservatives for protein and peptide formulations, (ii) their physico-chemical properties, (iii) their in-/compatibilities with other excipients or packaging material, and (iv) their interactions with the biological compound. Further, (v) we present an overview of licensed protein and peptide formulations.

Imatinib treatment causes substantial transcriptional changes in adult Schistosoma mansoni in vitro exhibiting pleiotropic effects.

BACKGROUND: Schistosome parasites cause schistosomiasis, one of the most important infectious diseases worldwide. For decades Praziquantel (PZQ) is the only drug widely used for controlling schistosomiasis. The absence of a vaccine and fear of PZQ resistance have motivated the search for alternatives. Studies on protein kinases (PKs) demonstrated their importance for diverse physiological processes in schistosomes. Among others two Abl tyrosine kinases, SmAbl1 and SmAbl2, were identified in Schistosoma mansoni and shown to be transcribed in the gonads and the gastrodermis. SmAbl1 activity was blocked by Imatinib, a known Abl-TK inhibitor used in human cancer therapy (Gleevec/Glivec). Imatinib exhibited dramatic effects on the morphology and physiology of adult schistosomes in vitro causing the death of the parasites. METHODOLOGY/PRINCIPAL FINDINGS: Here we show modeling data supporting the targeting of SmAbl1/2 by Imatinib. A biochemical assay confirmed that SmAbl2 activity is also inhibited by Imatinib. Microarray analyses and qRT-PCR experiments were done to unravel transcriptional processes influenced by Imatinib in adult schistosomes in vitro demonstrating a wide influence on worm physiology. Surface-, muscle-, gut and gonad-associated processes were affected as evidenced by the differential transcription of e.g. the gynecophoral canal protein gene GCP, paramyosin, titin, hemoglobinase, and cathepsins. Furthermore, transcript levels of VAL-7 and egg formation-associated genes such as tyrosinase 1, p14, and fs800-like were affected as well as those of signaling genes including a ribosomal protein S6 kinase and a glutamate receptor. Finally, a comparative in silico analysis of the obtained microarray data sets and previous data analyzing the effect of a TGFßR1 inhibitor on transcription provided first evidence for an association of TGFß and Abl kinase signaling. Among others GCP and egg formation-associated genes were identified as common targets. CONCLUSIONS/SIGNIFICANCE: The data affirm broad negative effects of Imatinib on worm physiology substantiating the role of PKs as interesting targets.

Identification of semicarbazones, thiosemicarbazones and triazine nitriles as inhibitors of Leishmania mexicana cysteine protease CPB.

Cysteine proteases of the papain superfamily are present in nearly all eukaryotes. They play pivotal roles in the biology of parasites and inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas' disease and leishmaniasis. Homology modeling of the mature Leishmania mexicana cysteine protease CPB2.8 suggested that it differs significantly from bovine cathepsin B and thus could be a good drug target. High throughput screening of a compound library against this enzyme and bovine cathepsin B in a counter assay identified four novel inhibitors, containing the warhead-types semicarbazone, thiosemicarbazone and triazine nitrile, that can be used as leads for antiparasite drug design. Covalent docking experiments confirmed the SARs of these lead compounds in an effort to understand the structural elements required for specific inhibition of CPB2.8. This study has provided starting points for the design of selective and highly potent inhibitors of L. mexicana cysteine protease CPB that may also have useful efficacy against other important cysteine proteases.

Docking-based virtual screening of covalently binding ligands: an orthogonal lead discovery approach.

In pharmaceutical industry, lead discovery strategies and screening collections have been predominantly tailored to discover compounds that modulate target proteins through noncovalent interactions. Conversely, covalent linkage formation is an important mechanism for a quantity of successful drugs in the market, which are discovered in most cases by hindsight instead of systematical design. In this article, the implementation of a docking-based virtual screening workflow for the retrieval of covalent binders is presented considering human cathepsin K as a test case. By use of the docking conditions that led to the best enrichment of known actives, 44 candidate compounds with unknown activity on cathepsin K were finally selected for experimental evaluation. The most potent inhibitor, 4-(N-phenylanilino)-6-pyrrolidin-1-yl-1,3,5-triazine-2-carbonitrile (CP243522), showed a K(i) of 21 nM and was confirmed to have a covalent reversible mechanism of inhibition. The presented approach will have great potential in cases where covalent inhibition is the desired drug discovery strategy.

High-throughput screening with the Eimeria tenella CDC2-related kinase2/cyclin complex EtCRK2/EtCYC3a.

The poultry disease coccidiosis, caused by infection with Eimeria spp. apicomplexan parasites, is responsible for enormous economic losses to the global poultry industry. The rapid increase of resistance to therapeutic agents, as well as the expense of vaccination with live attenuated vaccines, requires the development of new effective treatments for coccidiosis. Because of their key regulatory function in the eukaryotic cell cycle, cyclin-dependent kinases (CDKs) are prominent drug targets. The Eimeria tenella CDC2-related kinase 2 (EtCRK2) is a validated drug target that can be activated in vitro by the CDK activator XlRINGO (Xenopus laevis rapid inducer of G2/M progression in oocytes). Bioinformatics analyses revealed four putative E. tenella cyclins (EtCYCs) that are closely related to cyclins found in the human apicomplexan parasite Plasmodium falciparum. EtCYC3a was cloned, expressed in Escherichia coli and purified in a complex with EtCRK2. Using the non-radioactive time-resolved fluorescence energy transfer (TR-FRET) assay, we demonstrated the ability of EtCYC3a to activate EtCRK2 as shown previously for XlRINGO. The EtCRK2/EtCYC3a complex was used for a combined in vitro and in silico high-throughput screening approach, which resulted in three lead structures, a naphthoquinone, an 8-hydroxyquinoline and a 2-pyrimidinyl-aminopiperidine-propane-2-ol. This constitutes a promising starting point for the subsequent lead optimization phase and the development of novel anticoccidial drugs.

Drug discovery and the use of computational approaches for infectious diseases.

For centuries infectious diseases were the scourge of humanity, overcome only by the discovery of vaccination and penicillin. With an armamentarium of effective antibiotics, vaccines and drugs at hand, infectious diseases for many years were considered to be negligible. With the onset of the AIDS pandemic, the return of tuberculosis and influenza (e.g., swine influenza) this notion has changed in recent years. Drug discovery for infectious diseases, therefore, is again gaining increasing interest. This article discusses the drug-discovery process in this area and introduces major computational approaches used to identify suitable drug targets and to discover and optimize chemical lead compounds towards drug candidates using examples from antiparasitic drug discovery.

Inhibition of Eimeria tenella CDK-related kinase 2: From target identification to lead compounds.

Apicomplexan parasites encompass several human- and animal-pathogenic protozoans such as Plasmodium falciparum, Toxoplasma gondii, and Eimeria tenella. E. tenella causes coccidiosis, a disease that afflicts chickens, leading to tremendous economic losses to the global poultry industry. The considerable increase in drug resistance makes it necessary to develop new therapeutic strategies against this parasite. Cyclin-dependent kinases (CDKs) are key molecules in cell-cycle regulation and are therefore prominent target proteins in parasitic diseases. Bioinformatics analysis revealed four potential CDK-like proteins, of which one-E. tenella CDK-related kinase 2 (EtCRK2)-has already been characterized by gene cloning and expression.1 By using the CDK-specific inhibitor flavopiridol in EtCRK2 enzyme assays and schizont maturation assays (SMA), we could chemically validate CDK-like proteins as potential drug targets. An X-ray crystal structure of human CDK2 (HsCDK2) served as a template to build protein models of EtCRK2 by comparative homology modeling. Structural differences in the ATP binding site between EtCRK2 and HsCDK2, as well as chicken CDK3, were addressed for the optimization of selective ATP-competitive inhibitors. Virtual screening and "wet-bench" high-throughput screening campaigns on large compound libraries resulted in an initial set of hit compounds. These compounds were further analyzed and characterized, leading to a set of four promising lead compounds for development as EtCRK2 inhibitors.

Molecular visualization in the rational drug design process.

The visualization of molecular scenarios on an atomic level can help to interpret experimental and theoretical findings. This is demonstrated in this review article with the specific field of drug design. State-of-the-art visualization techniques are described and applied to the different stages of the rational design process. Numerous examples from the literature, in which visualization was used as a major tool in the data analysis and interpretation, are provided to show that images are not only useful for drawing the attention of the reader to a specific paper in a scientific journal.

A comparative chemogenomics strategy to predict potential drug targets in the metazoan pathogen, Schistosoma mansoni.

Schistosomiasis is a prevalent and chronic helmintic disease in tropical regions. Treatment and control relies on chemotherapy with just one drug, praziquantel and this reliance is of concern should clinically relevant drug resistance emerge and spread. Therefore, to identify potential target proteins for new avenues of drug discovery we have taken a comparative chemogenomics approach utilizing the putative proteome of Schistosoma mansoni compared to the proteomes of two model organisms, the nematode, Caenorhabditis elegans and the fruitfly, Drosophila melanogaster. Using the genome comparison software Genlight, two separate in silico workflows were implemented to derive a set of parasite proteins for which gene disruption of the orthologs in both the model organisms yielded deleterious phenotypes (e.g., lethal, impairment of motility), i.e., are essential genes/proteins. Of the 67 and 68 sequences generated for each workflow, 63 were identical in both sets, leading to a final set of 72 parasite proteins. All but one of these were expressed in the relevant developmental stages of the parasite infecting humans. Subsequent in depth manual curation of the combined workflow output revealed 57 candidate proteins. Scrutiny of these for 'druggable' protein homologs in the literature identified 35 S. mansoni sequences, 18 of which were homologous to proteins with 3D structures including co-crystallized ligands that will allow further structure-based drug design studies. The comparative chemogenomics strategy presented generates a tractable set of S. mansoni proteins for experimental validation as drug targets against this insidious human pathogen.

Eimeria tenella: identification of secretory and surface proteins from expressed sequence tags.

To identify new vaccine candidates, Eimeria tenella expressed sequence tags (ESTs) from public databases were analysed for secretory molecules with an especially developed automated in silico strategy termed DNAsignalP. A total of 12,187 ESTs were clustered into 2881 contigs followed by a blastx search, which resulted in a significant number of E. tenella contigs with homologies to entries in public databases. Amino acid sequences of appropriate homologous proteins were analysed for the occurrence of an N-terminal signal sequence using the algorithm signalP. The resulting list of 84 entries comprised 51 contigs whose deduced proteins showed homologies to proteins of apicomplexan parasites. Based on function or localisation, we selected candidate proteins classified as (i) secreted proteins of Apicomplexa parasites, (ii) secreted enzymes, and (iii) transport and signalling proteins. To verify our strategy experimentally, we used a functional complementation system in yeast. For five selected candidate proteins we found that these were indeed secreted. Our approach thus represents an efficient method to identify secretory and surface proteins out of EST databases.

The second largest subunit of Trypanosoma brucei's multifunctional RNA polymerase I has a unique N-terminal extension domain.

In the protist parasite Trypanosoma brucei, RNA polymerase (pol) I transcribes the large ribosomal RNA gene unit and, in addition, variant surface glycoprotein gene expression sites and procyclin gene transcription units. The multifunctional role of RNA pol I in this organism is unique among eukaryotes, but only its largest subunit TbRPA1 has been characterized thus far. We have recently established the procyclic cell line RPIC which exclusively expresses RNA pol I tagged with the protein C epitope at the TbRPA1 C-terminus. In the present study, we prepared RPIC cell extracts and immunopurified RNA pol I using anti-protein C affinity matrix under high stringency conditions. We were able to identify five specific polypeptides on a silver-stained polyacrylamide-SDS gel with apparent molecular weights of 200, 180, 55, 29, and 22 kDa. Interestingly, the second largest subunit, TbRPA2, is 42-58 kDa larger than counterparts of other organisms. We have cloned and sequenced the complete TbRPA2 cDNA and found an open reading frame for a polypeptide of 179.5 kDa. The deduced amino acid sequence of TbRPA2 contains a unique N-terminal domain of approximately 250 amino acids. By raising a polyclonal antibody against a N-terminal peptide sequence of TbRPA2, we could specifically detect this polypeptide in immunoblots showing that it co-purifies with epitope-tagged TbRPA1. Moreover, we identified the homologous gene sequence LmRPA2 in Leishmania major and found that it encodes a homologous extension domain. Therefore, the N-terminal extra domain in trypanosomatid RPA2 polypeptides may serve a parasite-specific function.