Antti M. Haapalainen

A Multidimensional Strategy to Detect Polypharmacological Targets in the Absence of Structural and Sequence Homology

Conventional drug design embraces the “one gene, one drug, one disease” philosophy. Polypharmacology, which focuses on multi-target drugs, has emerged as a new paradigm in drug discovery. The rational design of drugs that act via polypharmacological mechanisms can produce compounds that exhibit increased therapeutic potency and against which resistance is less likely to develop. Additionally, identifying multiple protein targets is also critical for side-effect prediction. One third of potential therapeutic compounds fail in clinical trials or are later removed from the market due to unacceptable side effects often caused by off-target binding. In the current work, we introduce a multidimensional strategy for the identification of secondary targets of known small-molecule inhibitors in the absence of global structural and sequence homology with the primary target protein. To demonstrate the utility of the strategy, we identify several targets of 4,5-dihydroxy-3-(1-naphthyldiazenyl)-2,7-naphthalenedisulfonic acid, a known micromolar inhibitor of Trypanosoma brucei RNA editing ligase 1. As it is capable of identifying potential secondary targets, the strategy described here may play a useful role in future efforts to reduce drug side effects and/or to increase polypharmacology.

Mitochondrial fatty acid synthesis--an adopted set of enzymes making a pathway of major importance for the cellular metabolism.

The highly conserved fatty acid de novo synthesis pathway in mitochondria proceeds in an acyl carrier protein-dependent manner through a discrete set of enzymes. Saccharomyces cerevisiae has served as a model for studies of mitochondrial fatty acid synthesis type II (FAS II) and as a template for identification of mammalian components. Inactivation of mitochondrial FAS II in yeast results in respiratory deficiency and loss of cytochromes. The pathway produces the octanoyl-ACP substrate for lipoic acid synthesis, but several pieces of evidence indicate that it is capable of the generation of longer fatty acids. A number of structures of mitochondrial FAS II enzymes have been published in the past few years, allowing for a comparison with their prokaryotic counterparts, several of which have been described as promising targets for antibiotics. Recently, novel links between mitochondrial FAS and RNA processing in yeast and vertebrates have been reported. In S. cerevisiae, deficiency in mitochondrial FAS results in failure of maturation of mitochondrial RNAse P, while, in mammals, mitochondrial 3-hydroxyacyl thioester dehydratase and the RPP14 subunit of RNase P are encoded by the same bicistronic transcript. The first publications linking mitochondrial FAS II to disease states in mammals are emerging.

Yeast Peroxisomal Multifunctional Enzyme: (3R)-Hydroxyacyl-CoA Dehydrogenase Domains A and B Are Required for Optimal Growth on Oleic Acid

The yeast peroxisomal (3R)-hydroxyacyl-CoA dehydrogenase/2-enoyl-CoA hydratase 2 (multifunctional enzyme type 2; MFE-2) has two N-terminal domains belonging to the short chain alcohol dehydrogenase/reductase superfamily. To investigate the physiological roles of these domains, here called A and B, Saccharomyces cerevisiae fox-2 cells (devoid of Sc MFE-2) were taken as a model system. Gly16 and Gly329 of the S. cerevisiae A and B domains, corresponding to Gly16, which is mutated in the human MFE-2 deficiency, were mutated to serine and cloned into the yeast expression plasmid pYE352. In oleic acid medium, fox-2 cells transformed with pYE352:: ScMFE-2(aΔ) and pYE352::ScMFE-2(bΔ) grew slower than cells transformed with pYE352::ScMFE-2, whereas cells transformed with pYE352::ScMFE-2(aΔbΔ) failed to grow. Candida tropicalis MFE-2 with a deleted hydratase 2 domain (Ct MFE- 2(h2Δ)) and mutational variants of the A and B domains (Ct MFE-2(h2ΔaΔ), Ct MFE- 2(h2ΔbΔ), andCt MFE- 2(h2ΔaΔbΔ)) were overexpressed and characterized. All proteins were dimers with similar secondary structure elements. Both wild type domains were enzymatically active, with the B domain showing the highest activity with short chain and the A domain with medium and long chain (3R)-hydroxyacyl-CoA substrates. The data show that the dehydrogenase domains of yeast MFE-2 have different substrate specificities required to allow the yeast to propagate optimally on fatty acids as the carbon source.

Structural Enzymological Studies of 2-Enoyl Thioester Reductase of the Human Mitochondrial Fas II Pathway: New Insights Into its Substrate Recognition Properties.

Structural and kinetic properties of the human 2-enoyl thioester reductase [mitochondrial enoyl-coenzyme A reductase (MECR)/ETR1] of the mitochondrial fatty acid synthesis (FAS) II pathway have been determined. The crystal structure of this dimeric enzyme (at 2.4 A resolution) suggests that the binding site for the recognition helix of the acyl carrier protein is in a groove between the two adjacent monomers. This groove is connected via the pantetheine binding cleft to the active site. The modeled mode of NADPH binding, using molecular dynamics calculations, suggests that Tyr94 and Trp311 are critical for catalysis, which is supported by enzyme kinetic data. A deep, water-filled pocket, shaped by hydrophobic and polar residues and extending away from the catalytic site, was recognized. This pocket can accommodate a fatty acyl tail of up to 16 carbons. Mutagenesis of the residues near the end of this pocket confirms the importance of this region for the binding of substrate molecules with long fatty acyl tails. Furthermore, the kinetic analysis of the wild-type MECR/ETR1 shows a bimodal distribution of catalytic efficiencies, in agreement with the notion that two major products are generated by the mitochondrial FAS II pathway.

Binary Structure of the Two-Domain (3R)-Hydroxyacyl-CoA Dehydrogenase from Rat Peroxisomal Multifunctional Enzyme Type 2 at 2.38 Å Resolution

The crystal structure of (3R)-hydroxyacyl-CoA dehydrogenase of rat peroxisomal multifunctional enzyme type 2 (MFE-2) was solved at 2.38 A resolution. The catalytic entity reveals an alpha/beta short chain alcohol dehydrogenase/reductase (SDR) fold and the conformation of the bound nicotinamide adenine dinucleotide (NAD(+)) found in other SDR enzymes. Of great interest is the separate COOH-terminal domain, which is not seen in other SDR structures. This domain completes the active site cavity of the neighboring monomer and extends dimeric interactions. Peroxisomal diseases that arise because of point mutations in the dehydrogenase-coding region of the MFE-2 gene can be mapped to changes in amino acids involved in NAD(+) binding and protein dimerization.

The characterization and evolutionary relationships of a trypanosomal thiolase.

Thiolases are enzymes that remove an acetyl-coenzyme A group from acyl-CoA in the catabolic β-oxidation of fatty acids, or catalyse the reverse condensation reaction for anabolic processes such as the biosynthesis of sterols and ketone bodies. In humans, six homologous isoforms of thiolase have been described, differing from each other in sequence, oligomeric state, substrate specificity and subcellular localization. A bioinformatics analysis of parasite genomes, being (i) different species of African trypanosomes, (ii) Trypanosoma cruzi and (iii) Leishmania spp., using the six human sequences as queries, showed that the distribution of thiolases in human and each of the studied Trypanosomatidae is completely different. Only one of these isoforms, called SCP2-thiolase, was found in each of the Trypanosomatidae, whereas the TFE-thiolase was also found in T. cruzi and Leishmania spp., and the AB-thiolase only in T. cruzi. Each of the trypanosomatid thiolases clusters with its orthologues from other organisms in a phylogenetic analysis and shares with them the isoform-specific sequence fingerprints. The single T. brucei SCP2-thiolase has been expressed in Escherichia coli and characterized. It shows activity in both the degradative and synthetic directions. Transcripts of this thiolase were detected in both bloodstream- and procyclic-form trypanosomes, but the protein was found only in the procyclic form. The encoded protein has both a predicted N-terminal mitochondrial signal peptide and a C-terminal candidate type 1 peroxisomal-targeting signal for sorting it into glycosomes. However experimentally, only a mitochondrial localization was found for both procyclic trypanosomes grown with glucose and cells cultured with amino acids as an energy source. When the thiolase expression in procyclic cells was knocked down by RNA interference, no important change in growth rate occurred, irrespective of whether the cells were grown with or without glucose, indicating that the metabolic pathway(s) involving this enzyme is/are not essential for the parasite under either of these growth conditions.

Response of SCP-2L Domain of Human MFE-2 to Ligand Removal: Binding Site Closure and Burial of Peroxisomal Targeting Signal

In the study of the structure and function relationship of human MFE-2, we have investigated the dynamics of human MFE-2SCP-2L (hSCP-2L) and its response to ligand removal. A comparison was made with homologous rabbit SCP-2. Breathing and a closing motion are found, identifiable with an adjustment in size and a closing off of the binding pocket. Crucial residues for structural integrity have been identified. Particularly mobile areas of the protein are loop 1 that is connecting helices A and C in space, and helix D, next to the entrance of the pocket. In hSCP-2L, the binding pocket gets occupied by Phe93, which is making a tight hydrophobic contact with Trp36. In addition, it is found that the C-terminal peroxisomal targeting signal (PTS1) that is solvent exposed in the complexed structure becomes buried when no ligand is present. Moreover, an anti-correlation exists between burial of PTS1 and the size of the binding pocket. The results are in accordance with plant nsLTPs, where a similar accommodation of binding pocket size was found after ligand binding/removal. Furthermore, the calculations support the suggestion of a ligand-assisted targeting mechanism.

CXCR3 Polymorphism and Expression Associate with Spontaneous Preterm Birth

Spontaneous preterm birth (SPTB) is a major factor associating with deaths and with lowered quality of life in humans. Environmental and genetic factors influence the susceptibility. Previously, by analyzing families with recurrent SPTB in linkage analysis, we identified a linkage peak close to the gene encoding CXCR3. Present objectives were to investigate the association of CXCR3 with SPTB in Finnish mothers (n = 443) and infants (n = 747), to analyze CXCR3 expression levels in human placenta and levels of its ligands in umbilical cord blood, and to verify the influence of Cxcr3 on SPTB-associating cytokines in mice. We detected an association between an intronic CXCR3 polymorphism, rs2280964, and SPTB in infants from families with recurrent preterm births (p = 0.009 versus term controls, odds ratio 0.52, 95% confidence interval 0.32–0.86). The minor allele was protective and undertransmitted to SPTB infants (p = 0.007). In the placenta and fetal membranes, the rs2280964 major allele homozygotes had higher expression levels than minor allele homozygotes; decidual trophoblasts showed strong CXCR3 immunoreactivity. Expression was higher in SPTB placentas compared with those from elective deliveries. Concentration of a CXCR3 ligand, CXCL9, was increased in cord blood from SPTB, and the protective rs2280964 allele was associated with low CXCL9. In CXCR3-deficient mice (Mus musculus), SPTB-associating cytokines were not acutely increased in amniotic fluid after preterm birth–inducing dose of maternal LPS. Our results indicate that CXCR3 contributes to SPTB. Activation of CXCR3 signaling may disturb the maternal–fetal tolerance, and this may promote labor.

Crystallization and preliminary crystallographic data of 2-enoyl-CoA hydratase 2 domain of Candida tropicalis peroxisomal multifunctional enzyme type 2

In yeast, the second and the third reaction of the fatty-acid beta-oxidation spiral are catalysed by peroxisomal multifunctional enzyme type 2 (Mfe2p/Fox2p). This protein has two (3R)-hydroxyacyl-CoA dehydrogenase domains and a C-terminal 2-enoyl-CoA hydratase 2 domain. Here, the purification, crystallization and X-ray diffraction analysis of the hydratase 2 domain [CtMfe2p(dh(a+b)Delta)] from Candida tropicalis Mfe2p is reported. CtMfe2p(dh(a+b)Delta) was overexpressed as an enzymatically active recombinant protein and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to space group C2, with unit-cell parameters a = 178.57, b = 60.46, c = 130.85 A, beta = 94.48 degrees. Selenomethionine-labelled protein was used for a multi-wavelength anomalous dispersion (MAD) experiment. A three-wavelength data set suitable for MAD phasing was collected to 2.25 A resolution using synchrotron radiation.

Expression of CPPED1 in human trophoblasts is associated with timing of term birth

Understanding of timing of human parturition is incomplete. Therefore, we carried out proteomic analyses of full‐term placentas from uncomplicated pregnancies to identify protein signatures associated with the onset of spontaneous delivery. We found quantitative associations of 10 proteins with spontaneous term birth, evident either in the basal or in the chorionic plates or in both. Additional 18 proteins were associated according to the location within placenta indicating local variations in protein amounts. Calcineurin‐like phosphoesterase domain‐containing 1 (CPPED1), a phosphatase previously suggested dephosphorylating AKT1/PKB, was one of the identified proteins. qRT‐PCR revealed the mRNA level of CPPED1 was higher in elective caesarean deliveries than in spontaneous births, while immunohistochemistry showed CPPED1 in cytotrophoblasts, syncytiotrophoblasts and extravillous trophoblasts. Noteworthy, phosphorylation status of AKT1 did not differ between placentas from elective caesarean and spontaneous deliveries. Additionally, analyses of samples from infants indicated that single‐nucleotide polymorphisms rs11643593 and rs8048866 of CPPED1 were associated with duration of term pregnancy. Finally, post‐transcriptional silencing of CPPED1 in cultured HTR8/SVneo cells by siRNAs affected gene expression in pathways associated with inflammation and blood vessel development. We postulate that functions regulated by CPPED1 in trophoblasts at choriodecidual interphase have a role in the induction of term labour, but it may be independent of AKT1.