A novel selective small-molecule PI3K inhibitor is effective against human multiple myeloma in vitro and in vivo.

Developing effective therapies against multiple myeloma (MM) is an unresolved challenge. Phosphatidylinositol-3-kinase (PI3K) activation may be associated with tumor progression and drug resistance, and inhibiting PI3K can induce apoptosis in MM cells. Thus, targeting of PI3K is predicted to increase the susceptibility of MM to anticancer therapy. The lead compound of a novel class of PI3K inhibitors, BAY80-6946 (IC50=0.5 nM against PI3K-α), was highly efficacious in four different MM cell lines, where it induced significant antitumoral effects in a dose-dependent manner. The compound inhibited cell cycle progression and increased apoptosis (P<0.001 compared with controls). Moreover, it abrogated the stimulation conferred by insulin-like growth-factor-1, a mechanism relevant for MM progression. These cellular effects were paralleled by decreased Akt phosphorylation, the main downstream target of PI3K. Likewise, profound antitumoral activity was observed ex vivo, as BAY80-6946 significantly inhibited proliferation of freshly isolated myeloma cells from three patients (P<0.001 compared with vehicle). In addition, BAY80-6946 showed convincing in vivo activity against the human AMO-1 and MOLP-8 myeloma cell lines in a preclinical murine xenograft model, where treatment with 6 mg/kg every other day for 2 weeks reduced the cell numbers by 87.0% and 69.3%, respectively (P<0.001 compared with vehicle), without overt toxicity in treated animals.

Characterization of novel MPS1 inhibitors with preclinical anticancer activity.

Monopolar spindle 1 (MPS1), a mitotic kinase that is overexpressed in several human cancers, contributes to the alignment of chromosomes to the metaphase plate as well as to the execution of the spindle assembly checkpoint (SAC). Here, we report the identification and functional characterization of three novel inhibitors of MPS1 of two independent structural classes, N-(4-{2-[(2-cyanophenyl)amino][1,2,4]triazolo[1,5-a]pyridin-6-yl}phenyl)-2-phenylacetamide (Mps-BAY1) (a triazolopyridine), N-cyclopropyl-4-{8-[(2-methylpropyl)amino]-6-(quinolin-5-yl)imidazo[1,2-a]pyrazin-3-yl}benzamide (Mps-BAY2a) and N-cyclopropyl-4-{8-(isobutylamino)imidazo[1,2-a]pyrazin-3-yl}benzamide (Mps-BAY2b) (two imidazopyrazines). By selectively inactivating MPS1, these small inhibitors can arrest the proliferation of cancer cells, causing their polyploidization and/or their demise. Cancer cells treated with Mps-BAY1 or Mps-BAY2a manifested multiple signs of mitotic perturbation including inefficient chromosomal congression during metaphase, unscheduled SAC inactivation and severe anaphase defects. Videomicroscopic cell fate profiling of histone 2B-green fluorescent protein-expressing cells revealed the capacity of MPS1 inhibitors to subvert the correct timing of mitosis as they induce a premature anaphase entry in the context of misaligned metaphase plates. Hence, in the presence of MPS1 inhibitors, cells either divided in a bipolar (but often asymmetric) manner or entered one or more rounds of abortive mitoses, generating gross aneuploidy and polyploidy, respectively. In both cases, cells ultimately succumbed to the mitotic catastrophe-induced activation of the mitochondrial pathway of apoptosis. Of note, low doses of MPS1 inhibitors and paclitaxel (a microtubular poison) synergized at increasing the frequency of chromosome misalignments and missegregations in the context of SAC inactivation. This resulted in massive polyploidization followed by the activation of mitotic catastrophe. A synergistic interaction between paclitaxel and MPS1 inhibitors could also be demonstrated in vivo, as the combination of these agents efficiently reduced the growth of tumor xenografts and exerted superior antineoplastic effects compared with either compound employed alone. Altogether, these results suggest that MPS1 inhibitors may exert robust anticancer activity, either as standalone therapeutic interventions or combined with microtubule-targeting chemicals.

PDE5 inhibitors beyond erectile dysfunction.

The phosphodiesterase type-5 (PDE5) inhibitors sildenafil, vardenafil and tadalafil are widely used first-line therapy for erectile dysfunction (ED). Since the advent of sildenafil in 1998, more than 40 million men worldwide have been successfully treated with these compounds. The safety and high tolerability of PDE5 inhibitors make them an attractive tool to investigate further physiological functions of PDE5, for example the modulation of intracellular cyclic GMP (cGMP) pools. As cGMP is a key component of intracellular signaling this may provide novel therapeutic opportunities beyond ED even for indications in which chronic administration is necessary. The approval of sildenafil for the treatment of pulmonary hypertension in 2005 was a notable success in this area of research. A number of other potential new indications are currently in various phases of preclinical research and development. In recent years, extensive but very heterogeneous information has been published in this field. The aim of this review is to summarize existing preclinical and clinical knowledge and critically discuss the evidence to support potential future indications for PDE5 inhibitors.

Novel bacterial acetyl coenzyme A carboxylase inhibitors with antibiotic efficacy in vivo.

The pseudopeptide pyrrolidinedione antibiotics, such as moiramide B, have recently been discovered to target the multisubunit acetyl coenzyme A (acetyl-CoA) carboxylases of bacteria. In this paper, we describe synthetic variations of each moiety of the modularly composed pyrrolidinediones, providing insight into structure-activity relationships of biochemical target activity, in vitro potency, and in vivo efficacy. The novel derivatives showed highly improved activities against gram-positive bacteria compared to those of previously reported variants. The compounds exhibited a MIC(90) value of 0.1 microg/ml against a broad spectrum of Staphylococcus aureus clinical isolates. No cross-resistance to antibiotics currently used in clinical practice was observed. Resistance mutations induced by pyrrolidinediones are exclusively located in the carboxyltransferase subunits of the bacterial acetyl-CoA carboxylase, indicating the identical mechanisms of action of all derivatives tested. Improvement of the physicochemical profile was achieved by salt formation, leading to aqueous solubilities of up to 5 g/liter. For the first time, the in vitro activity of this compound class was compared with its in vivo efficacy, demonstrating a path from compounds weakly active in vivo to agents with significant efficacy. In a murine model of S. aureus sepsis, the 100% effective dose of the best compound reported was 25 mg/kg of body weight, only fourfold higher than that of the comparator molecule linezolid. The obvious improvements achieved by chemical derivatization reflect the potential of this novel antibiotic compound class for future therapy.

Development of a non-radioactive, 384-well format assay to detect inhibitors of the mitogen-activated protein kinase kinase 4.

Mitogen-activated protein kinase (MAPK) kinases (MKKs, also called MAPK/extracellular signal-regulated kinase [ERK] kinase [MEK]) are constituents of numerous signal transduction pathways involved in growth, differentiation, and stress response. One of its members, MKK4, directly phosphorylates and activates the c-Jun terminal kinases (also called stress-activated protein kinase [SAPK]) in response to stress and pro-inflammatory cytokines. Recent evidence suggest that control of MKK4 activity may provide a novel approach for the treatment of cancer or as anti-inflammatory therapy. To screen for novel low-molecular-weight inhibitors of MKK4, we established a quantitative, non-radioactive in vitro kinase assay. Human MKK4 was expressed as fusion protein with glutathione S-transferase (GST) in Escherichia coli. Co-expression of a constitutive active fragment of the MAPK/ERK kinase kinase-1 yielded active GST-MKK4 using GST-SAPK alpha-kinase-negative (KN) mutant as substrate. We determined the kinetic constants for ATP and GST-SAPK alpha-KN. The apparent Km value for GST-SAPKalpha-KN was 3.7 microM, while the apparent Km value for ATP was 0.17 microM. Staurosporine inhibited GST-MKK4 with an IC50 of 70 nM. The kinase assay was adapted to a 384-well non-radioactive format. After the kinase reaction the phosphorylated product was captured onto a streptavidin-coated microtiter plate, and phosphorylation was detected with a europium-labeled anti-phosphotyrosine antibody, which allowed time-resolved fluorescence measurement.

High-throughput assay for inorganic pyrophosphatases using the cytosolic enzymes of Saccharomyces cerevisiae and human as an example.

This paper describes the development of a new, malachite green based, enzymatic assay for the identification of specific inhibitors of inorganic pyrophosphatase (iPPase) from Saccharomyces cerevisiae for antifungal drug discovery. The human iPPase was used as counterscreen. The coding regions of both enzymes were amplified, cloned into a vector providing a His-tag at the C-terminus, expressed in Escherichia coli, and purified by metal chelate affinity chromatography. Since the complete human sequence had not been published previously, the human iPPase was cloned on the basis of expressed sequence tag data. The human sequence was confirmed and showed about 55% amino acid identity with the yeast enzyme and 95% identity with an already published bovine enzyme. Both recombinant iPPases were characterized with regard to their biochemical properties, showing that the His-tag did not influence the specific activity, pH optimum, inhibitor profile, or dimerization. The enzyme activity was determined by quantifying released phosphate by complex formation with malachite green. The resulting complex was quantified spectrophotometrically. The assay was adapted to a microtiter plate format. Thus, it is possible to screen a large compound pool for iPPase inhibitors in a short period of time.

A developmentally regulated aconitase related to iron-regulatory protein-1 is localized in the cytoplasm and in the mitochondrion of Trypanosoma brucei.

Mitochondrial energy metabolism and Krebs cycle activities are developmentally regulated in the life cycle of the protozoan parasite Trypanosoma brucei. Here we report cloning of a T. brucei aconitase gene that is closely related to mammalian iron-regulatory protein 1 (IRP-1) and plant aconitases. Kinetic analysis of purified recombinant TbACO expressed in Escherichia coli resulted in a K(m) (isocitrate) of 3 +/- 0.4 mM, similar to aconitases of other organisms. This was unexpected since an arginine conserved in the aconitase protein family and crucial for substrate positioning in the catalytic center and for activity of pig mitochondrial aconitase (Zheng, L., Kennedy, M. C., Beinert, H., and Zalkin, H. (1992) J. Biol. Chem. 267, 7895-7903) is substituted by leucine in the TbACO sequence. Expression of the 98-kDa TbACO was shown to be lowest in the slender bloodstream stage of the parasite, 8-fold elevated in the stumpy stage, and increased a further 4-fold in the procyclic stage. The differential expression of TbACO protein contrasted with only minor changes in TbACO mRNA, indicating translational or post-translational mechanisms of regulation. Whereas animal cells express two distinct compartmentalized aconitases, mitochondrial aconitase and cytoplasmic aconitase/IRP-1, TbACO accounts for total aconitase activity in trypanosomes. By cell fractionation and immunofluorescence microscopy, we show that native as well as a transfected epitope-tagged TbACO localizes in both the mitochondrion (30%) and in the cytoplasm (70%). Together with phylogenetic reconstructions of the aconitase family, this suggests that animal IRPs have evolved from a multicompartmentalized ancestral aconitase. The possible functions of a cytoplasmic aconitase in trypanosomes are discussed.

High throughput assay to detect compounds that enhance the proton permeability of Candida albicans membranes.

We describe a 96-well microtiter plate format assay to detect changes in proton permeability in membranes of the pathogenic yeast, Candida albicans. Candida albicans cells were incubated with the lipophilic ester of 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), a pH-sensitive fluorescein derivative. Inside the cells, BCECF was released and trapped in the vacuole. Compounds that destroyed membrane integrity increased the pH value of the vacuole due to proton leakage into the cytoplasm. This was paralleled by an increase in BCECF fluorescence intensity, which could be quantified. The test assay was validated with amphotericin B, as well as with other membrane-active compounds known to increase membrane permeability. Possible applications and limitations of this assay in the field of antifungal drug discovery are discussed.

Computer-aided target selection-prioritizing targets for antifungal drug discovery.

The entire DNA sequence of the Saccharomyces cerevisiae genome was completed in 1996 and represents the first entirely decoded eukaryotic genome. Because major human pathogenic fungi such as Candida albicans are closely related to S. cerevisiae on a molecular level, the question arises as to how this new information can be used to identify and prioritize those genes that are most suitable as targets for antimycotic drug discovery. To tackle this challenge, a software tool called CATS (computer-aided target selection) was developed. The authors describe how it allows an automated and periodically updated assessment of all S. cerevisiae genes to be carried out with regard to their suitability as antifungal targets.

A Target-Specific Cellular Assay for Screening of Topoisomerase I Inhibitors.

We have developed a cellular, target-specific high-throughput assay for the detection of topoisomerase I inhibitors. Topoisomerase I is a nonessential enzyme involved in controlling DNA topology. Topoisomerase I is the target of anticancer drugs such as camptothecin as well as a candidate target for new antifungal drugs. A wild-type Saccharomyces cerevisiae strain and its isogenic topoisomerase I deletion mutant (DeltatopI) were labeled with S65T and wild-type green fluorescent protein (GFP), respectively. We showed that the growth of such a pair of S. cerevisiae strains labeled with this GFP combination can be independently quantified after both strains were mixed. When growth of the mixture of wild-type and DeltatopI strain was monitored in the presence of compounds, only growth of the wild-type strain was inhibited by the topoisomerase I-specific drug camptothecin. In contrast, amphotericin B, a broad-spectrum antifungal drug, inhibited growth of both strains. The two strains were used to screen compound libraries. While 0.9% of all compounds inhibited growth of both strains, only 0.06% inhibited the wild-type but not the DeltatopI strain. Thus, by using a DeltatopI strain as internal control in the same assay mixture, the number of candidate topoisomerase I inhibitors to be retested could be reduced by more than 90%. Further applications of this type of S. cerevisiae-based cellular high-throughput assays will be discussed.

Molecular mode of action of the antifungal beta-amino acid BAY 10-8888.

BAY 10-8888 is a cyclic beta-amino acid that is related to cispentacin and that has antifungal activity. Candida albicans cells accumulated BAY 10-8888 intracellularly to a concentration about 200 that in the medium when grown in media with a variety of nitrogen sources. In complex growth medium, BAY 10-8888 transport activity was markedly reduced and was paralleled by a decrease in its antifungal activity. Uptake of BAY 10-8888 was mediated by an H+-coupled amino acid transporter with specificity for branched-chain amino acids (isoleucine, leucine, and valine) and showed a KT (Michaelis constant of the transport reaction) of 0.95 mM and a Vmax of 18.9 nmol x min-1 x 10(7) cells-1. Similar to the transport of natural amino acids in Saccharomyces cerevisiae, the transport of BAY 10-8888 into the cell was unidirectional. Efflux occurred by diffusion and was not carrier mediated. Inside the cell BAY 10-8888 inhibited specifically isoleucyl-tRNA synthetase, resulting in inhibition of protein synthesis and cell growth. Intracellular isoleucine reversed BAY 10-8888-induced growth inhibition. BAY 10-8888 was not incorporated into proteins. BAY 10-8888 inhibited isoleucyl-tRNA synthetase with the same concentration dependency as protein biosynthesis in intact cells assuming 200-fold accumulation.

Multiple functions of Pmt1p-mediated protein O-mannosylation in the fungal pathogen Candida albicans.

Protein mannosylation by Pmt proteins initiates O-glycosylation in fungi. We have identified the PMT1 gene and analyzed the function of Pmt1p in the fungal human pathogen Candida albicans. Mutants defective in PMT1 alleles lacked Pmt in vitro enzymatic activity, showed reduced growth rates, and tended to form cellular aggregates. In addition, multiple specific deficiencies not known in Saccharomyces cerevisiae (including defective hyphal morphogenesis; supersensitivity to the antifungal agents hygromycin B, G418, clotrimazole, and calcofluor white; and reduced adherence to Caco-2 epithelial cells) were observed in pmt1 mutants. PMT1 deficiency also led to faster electrophoretic mobility of the Als1p cell wall protein and to elevated extracellular activities of chitinase. Homozygous pmt1 mutants were avirulent in a mouse model of systemic infection, while heterozygous PMT1/pmt1 strains showed reduced virulence. The results indicate that protein O-mannosylation by Pmt proteins occurs in different fungal species, where PMT1 deficiency can lead to defects in multiple cellular functions.

Decreased accumulation or increased isoleucyl-tRNA synthetase activity confers resistance to the cyclic beta-amino acid BAY 10-8888 in Candida albicans and Candida tropicalis.

BAY 10-8888, a cyclic beta-amino acid, exerts its antifungal activity by inhibition of isoleucyl-tRNA synthetase activity after accumulation to a millimolar concentration inside the cell. We have selected and characterized BAY 10-8888-resistant Candida albicans mutants. Reduced BAY 10-8888 accumulation as well as increased isoleucyl-tRNA synthetase activity was observed in these mutants. Some of the mutants were cross-resistant to cispentacin, a structurally related beta-amino acid, while sensitivities to 5-fluorocytosine and fluconazole remained unchanged in all mutants. All except two in vitro-resistant mutants were pathogenic in a murine candidiasis model, and BAY 10-8888 failed to cure the infection. Furthermore, we have characterized BAY 10-8888 transport and isoleucyl-tRNA synthetase activity in several Candida tropicalis strains which showed MICs higher than those of other Candida strains. An analysis of the C. tropicalis strains revealed that intracellular concentrations of BAY 10-8888 were in the millimolar range, comparable to those for C. albicans. However, these isolates expressed isoleucyl-tRNA synthetase activities about fourfold higher than those for C. albicans. To test the possibility of resistance modeling, we determined the correlations between the intracellular concentration of BAY 10-8888, the specific activity of isoleucyl-tRNA synthetase, the number of free, i.e., noninhibited, isoleucyl-tRNA synthetase molecules/cell, and growth, assuming a linear relation. We found significant correlations between growth and the intracellular concentration of BAY 10-8888 and between growth and the number of free isoleucyl-tRNA synthetase molecules/cell, but not between growth and the specific activity of isoleucyl-tRNA synthetase.

A dual labelling method for measuring uptake of low molecular weight compounds into the pathogenic yeast Candida albicans.

In contrast to other eukaryotic cells the pathogenic yeast Candida albicans is resistant to many structurally unrelated metabolic inhibitors. Reduced permeability due to the cell wall and/or altered plasma membrane composition is thought to be at least partly responsible for this phenomenon. To study the uptake of low molecular weight compounds into C. albicans we developed a dual labelling method. Intact cells, metabolically inactivated cells, spheroplasts or membrane fragments of C. albicans were incubated with various [14C]-labelled compound in the presence of [3H]-labelled water. After separation of cells and supernatant isotope ratios [3H]/[14C] were determined. Quotients of the isotope ratios from cells and supernatant, called enrichment coefficients, were calculated under all four conditions. The enrichment coefficients indicated whether a compound can enter C. albicans cells, is trapped within the cell wall, is enriched in the lipophilic membrane compartment, is actively accumulated or actively exported by multidrug resistance carriers. We used six structurally unrelated compounds to test our method. We found no evidence for a general impermeability of C. albicans.

Virulence and hyphal formation of Candida albicans require the Ste20p-like protein kinase CaCla4p.

BACKGROUND: The pathogenic fungus Candida albicans is capable of a morphological transition from a unicellular budding yeast to a filamentous form. Extensive filamentous growth leads to the formation of mycelia displaying hyphae with branches and lateral buds. Hyphae have been observed to adhere to and invade host tissues more readily than the yeast form, suggesting that filamentous growth may contribute to the virulence of this major human pathogen. A molecular and genetic understanding of the potential role of morphological switching in the pathogenicity of C. albicans would be of significant benefit in view of the increasing incidence of candidiasis. RESULTS: The CaCLA4 gene of C. albicans was cloned by functional complementation of the growth defect of cells of the budding yeast Saccharomyces cerevisiae deleted for the STE20 gene and the CLA4 gene. CaCLA4 encodes a member of the Ste20p family of serine/threonine protein kinases and is characterized by a pleckstrin homology domain and a Cdc42p-binding domain in its amino-terminal non-catalytic region. Deletion of both alleles of CaCLA4 in C. albicans caused defects in hyphal formation in vitro, in both synthetic liquid and solid media, and in vivo in a mouse model for systemic candidiasis. The gene deletions reduced colonization of the kidneys in infected mice and suppressed C. albicans virulence in the mouse model. CONCLUSIONS: Our results demonstrate that the function of the CaCla4p protein kinase is essential for virulence and morphological switching of C. albicans in a mouse model. Thus, hyphal formation of C. albicans mediated by CaCla4p may contribute to the pathogenicity of this dimorphic fungus, suggesting that regulators of morphological switching may be useful targets for antifungal drugs.

Signal transduction through homologs of the Ste20p and Ste7p protein kinases can trigger hyphal formation in the pathogenic fungus Candida albicans.

The CST20 gene of Candida albicans was cloned by functional complementation of a deletion of the STE20 gene in Saccharomyces cerevisiae. CST20 encodes a homolog of the Ste20p/p65PAK family of protein kinases. Colonies of C. albicans cells deleted for CST20 revealed defects in the lateral formation of mycelia on synthetic solid "Spider" media. However, hyphal development was not impaired in some other media. A similar phenotype was caused by deletion of HST7, encoding a functional homolog of the S. cerevisiae Ste7p protein kinase. Overexpression of HST7 partially complemented the deletion of CST20. Cells deleted for CST20 were less virulent in a mouse model for systemic candidiasis. Our results suggest that more than one signaling pathway can trigger hyphal development in C. albicans, one of which has a protein kinase cascade that is analogous to the mating response pathway in S. cerevisiae and might have become adapted to the control of mycelial formation in asexual C. albicans.

Genomic organization of an invariant surface glycoprotein gene family of Trypanosoma brucei.

The genomic organization of a gene family for the invariant surface glycoprotein, ISG75 (invariant surface glycoprotein with a molecular mass of 75 kDa), from Trypanosoma brucei is described. In T. brucei strain 427 ISG75 genes are present in tandem arrays at two loci, A and B, containing 5 and 2 copies, respectively. At the 3'-end of locus A, a single gene was identified that encodes a structural isoform of ISG75. This isoform contains a unique amino-terminal domain, whereas the rest of the protein is nearly identical to the polypeptides encoded by the other genes. This isoform is transcribed into a stable mRNA, but the expression of the derived polypeptide was below the detection limit. The ISG75 gene clusters are present on chromosomal bands 9' and 10, supporting the hypothesis of Gottesdiener et al. [25] that these bands contain allelic chromosomes. The total number of ISG75 genes is strain dependent, but at least one copy of the unique isoform is present in every variant tested.

Structure and function of GPI-anchored surface proteins of Trypanosoma brucei.

While proteins modified at their COOH-terminal end by a glycosylphosphatidylinositol (GPI) membrane anchor have been found as minor components in many eukaryotic cells, they dominate surface constituents of several parasitic protozoa. In this article, GPI-anchored proteins of Trypanosoma brucei are discussed.

Structure and function of GPI-anchored surface proteins of Trypanosoma brucei

While proteins modified at their COOH-terminal end by a glycosylphosphatidylinositol (GPI) membrane anchor have been found as minor components in many eukaryotic cells, they dominate surface constituents of several parasitic protozoa. In this article, GPI-anchored proteins of Trypanosoma brucei are discussed

Invariant surface proteins in bloodstream forms of Trypanosoma brucei.

Antigenic variation of the glycoprotein forming the coat of African trypanosomes has been a dominant field of investigation for many years. The extravagant potential of these parasites to change their surface coat has destroyed hopes for a vaccine based on the variant surface glycoprotein. Recently, there has been a rising interest in the characterization of surface proteins that are not subject to antigenic variation. In this review, Peter Overath, Maliha Chaudhri, Dietmar Steverding and Karl Ziegelbauer summarize the present evidence for the occurrence, cellular localization and function of invariant surface proteins in Trypanosoma brucei.