Elucidating substrate and inhibitor binding sites on the surface of GSK-3β and the refinement of a competitive inhibitor

A molecular understanding of substrate recognition of protein kinases provides an important basis for the development of substrate competitive inhibitors. Here, we explored substrate recognition and competitive inhibition of glycogen synthase kinase (GSK)-3β using molecular and computational tools. In previous work, we described Gln89 and Asn95 within GSK-3β as important substrates binding sites. Here, we show that the cavity bordered by loop 89-QDKRFKN-95, located in the vicinity of the GSK-3β catalytic core, is a promiscuous substrate binding subsite. Mutations within this segment highlighted Phe93 as an additional essential contact residue for substrates' recognition. However, unlike Gln89 and Asn95, Phe93 was also important for the binding of our previously described substrate competitive inhibitor, L803 [KEAPPAPPQS(p)P], and its cell-permeable variant L803-mts. The effects of the substitution of charged or polar residues within L803 further suggested that binding to GSK-3β is governed by hydrophobic interactions. Our computational model of GSK-3β bound to L803 was in agreement with the experimental data. It revealed L803 binding with a hydrophobic surface patch and identified interactions between Pro8 (L803) and Phe93 (GSK-3β). Computational modeling of new L803 variants predicted that inhibition would be strengthened by adding contacts with Phe93 or by increasing the hydrophobic content of the peptide. Indeed, the newly designed L803 variants showed improved inhibition. Our study identified different and overlapping elements in GSK-3β substrate and inhibitor recognition and provides a novel example for model-based rational design of substrate competitive inhibitors for GSK-3.     

Monitoring of tumor response to chemotherapy in vivo by a novel small-molecule detector of apoptosis

Utilization of molecular imaging of apoptosis for clinical monitoring of tumor response to anti-cancer treatments in vivo is highly desirable. To address this need, we now present ML-9 (butyl-2-methyl-malonic acid; MW = 173), a rationally designed small-molecule detector of apoptosis, based on a novel alkyl-malonate motif. In proof-of-concept studies, induction of apoptosis in tumor cells by various triggers both in vitro and in vivo was associated with marked uptake of ³H-ML-9 administered in vivo, in correlation with the apoptotic hallmarks of DNA fragmentation, caspase-3 activation and membrane phospholipid scrambling, and with correlative tumor regression. ML-9 uptake following chemotherapy was tumor-specific, with rapid clearance of the tracer from the blood and other non-target organs. Excess of non-labeled “cold” compound competitively blocked ML-9 tumor uptake, thus demonstrating the specificity of ML-9 binding. ML-9 may therefore serve as a platform for a novel class of small-molecule imaging agents for apoptosis, useful for assessment of tumor responsiveness to treatment. H. Grimberg, G. Levin and A. Shirvan contributed equally to this article.     

The protective effect of desferrioxamine on paraquat-treated pea (Pisum sativum)

Paraquat, a widely used herbicide, is photoreduced by photosystem I to the monovalent cation radical, which in turn, can react quickly and efficiently with molecular oxygen to produce superoxide anion radicals. In the presence of redox-active iron (or copper) superoxide radicals can serve as a source for the more active species such as hydroxyl radicals. The present sludv investigated the possible mediatory role of iron in paraquat to xicity. The results demonstrate that desferrioxamme (0–150μM) a highiy specific iron chelator, reduces the loss of proteins (by 34–69%) and lipid peroxidation (by 31–96%) in paraquat treated leaf cuts. Dcsferrioxamine also protects malate dehydrogenase (61–70%) hydroxvpyruvate reductase (54–100%), and Ca²⁺-dependent ATPase (25–34%) against the paraquat-induced loss of their activity. It also induces an increase in glutathione reductase activity (by 188%). These results, together with those from other experiments concerning the effect of desferrioxamine on paraquat uptake by the leaf cuts, suggest that the protection by desferrioxamine arises from its specific iron chelanon properties, and lead to the conclusion that nan-protein-bound and redoxactive forms of iron pluy a role in the manifestation of paraquat toxicity in plants.     

Substrate Competitive GSK-3 Inhibitors strategy and Implications

Glycogen synthase kinase-3 (GSK-3) is a highly conserved protein serine/threonine kinase ubiquitously distributed in eukaryotes as a constitutively active enzyme. Abnormally high GSK-3 activity has been implicated in several pathological disorders, including diabetes and neuron degenerative and affective disorders. This led to the hypothesis that inhibition of GSK-3 may have therapeutic benefit. Most GSK-3 inhibitors developed so far compete with ATP and often show limited specificity. Our goal is to develop inhibitors that compete with GSK-3 substrates, as this type of inhibitor is more specific and may be useful for clinical applications. We have employed computational, biochemical, and molecular analyses to gain in-depth understanding of GSK-3's substrate recognition. Here we argue that GSK-3 is a promising drug discovery target and describe the strategy and practice for developing specific substrate-competitive inhibitors of GSK-3.     

Combining multi-species genomic data for microRNA identification using a Naive Bayes classifier

MOTIVATION: Most computational methodologies for microRNA gene prediction utilize techniques based on sequence conservation and/or structural similarity. In this study we describe a new technique, which is applicable across several species, for predicting miRNA genes. This technique is based on machine learning, using the Naive Bayes classifier. It automatically generates a model from the training data, which consists of sequence and structure information of known miRNAs from a variety of species. RESULTS: Our study shows that the application of machine learning techniques, along with the integration of data from multiple species is a useful and general approach for miRNA gene prediction. Based on our experiments, we believe that this new technique is applicable to an extensive range of eukaryotes' genomes. Specific structure and sequence features are first used to identify miRNAs followed by a comparative analysis to decrease the number of false positives (FPs). The resulting algorithm exhibits higher specificity and similar sensitivity compared to currently used algorithms that rely on conserved genomic regions to decrease the rate of FPs.     

Directed evolution of hydrolases for prevention of G-type nerve agent intoxication

Organophosphate nerve agents are extremely lethal compounds. Rapid in vivo organophosphate clearance requires bioscavenging enzymes with catalytic efficiencies of >10(7) (M(-1) min(-1)). Although serum paraoxonase (PON1) is a leading candidate for such a treatment, it hydrolyzes the toxic S(p) isomers of G-agents with very slow rates. We improved PON1's catalytic efficiency by combining random and targeted mutagenesis with high-throughput screening using fluorogenic analogs in emulsion compartments. We thereby enhanced PON1's activity toward the coumarin analog of S(p)-cyclosarin by ～10(5)-fold. We also developed a direct screen for protection of acetylcholinesterase from inactivation by nerve agents and used it to isolate variants that degrade the toxic isomer of the coumarin analog and cyclosarin itself with k(cat)/K(M) ～ 10(7) M(-1) min(-1). We then demonstrated the in vivo prophylactic activity of an evolved variant. These evolved variants and the newly developed screens provide the basis for engineering PON1 for prophylaxis against other G-type agents.     

Parent-of-Origin Effects of the APOB Gene on Adiposity in Young Adults

Loci identified in genome-wide association studies (GWAS) of cardio-metabolic traits account for a small proportion of the traits'' heritability. To date, most association studies have not considered parent-of-origin effects (POEs). Here we report investigation of POEs on adiposity and glycemic traits in young adults. The Jerusalem Perinatal Family Follow-Up Study (JPS), comprising 1250 young adults and their mothers was used for discovery. Focusing on 18 genes identified by previous GWAS as associated with cardio-metabolic traits, we used linear regression to examine the associations of maternally- and paternally-derived offspring minor alleles with body mass index (BMI), waist circumference (WC), fasting glucose and insulin. We replicated and meta-analyzed JPS findings in individuals of European ancestry aged ≤50 belonging to pedigrees from the Framingham Heart Study, Family Heart Study and Erasmus Rucphen Family study (total N≅4800). We considered p<2.7x10^-4 statistically significant to account for multiple testing. We identified a common coding variant in the 4^th exon of APOB (rs1367117) with a significant maternally-derived effect on BMI (β = 0.8; 95%CI:0.4,1.1; p = 3.1x10^-5) and WC (β = 2.7; 95%CI:1.7,3.7; p = 2.1x10^-7). The corresponding paternally-derived effects were non-significant (p>0.6). Suggestive maternally-derived associations of rs1367117 were observed with fasting glucose (β = 0.9; 95%CI:0.3,1.5; p = 4.0x10^-3) and insulin (ln-transformed, β = 0.06; 95%CI:0.03,0.1; p = 7.4x10^-4). Bioinformatic annotation for rs1367117 revealed a variety of regulatory functions in this region in liver and adipose tissues and a 50% methylation pattern in liver only, consistent with allelic-specific methylation, which may indicate tissue-specific POE. Our findings demonstrate a maternal-specific association between a common APOB variant and adiposity, an association that was not previously detected in GWAS. These results provide evidence for the role of regulatory mechanisms, POEs specifically, in adiposity. In addition this study highlights the benefit of utilizing family studies for deciphering the genetic architecture of complex traits.     

Rac2 regulation of phospholipase C-beta 2 activity and mode of membrane interactions in intact cells

Phospholipase C-beta (PLCbeta) isozymes play important roles in transmembrane signaling. Their activity is regulated by heterotrimeric G proteins. The PLCbeta(2) isozyme is unique in being stimulated also by Rho GTPases (Rac and Cdc42). However, the mechanism(s) of this stimulation are still unclear. Here, we employed fluorescence recovery after photobleaching to investigate the interaction of green fluorescent protein (GFP)-PLCbeta(2) with the plasma membrane. For either GFP-PLCbeta(2) or GFP-PLCbeta(2)Delta, a C-terminal deletion mutant lacking the region required for stimulation by Galpha(q), these interactions were characterized by a mixture of exchange with a cytoplasmic pool and lateral diffusion. Constitutively active Rac2(12V) stimulated the activity of both GFP-PLCbeta(2) and GFP-PLCbeta(2)Delta in live cells, and enhanced their membrane association as evidenced by the marked reduction in their fluorescence recovery rates. Both effects required the putative N-terminal pleckstrin homology (PH) domain of PLCbeta(2). Importantly, Rac2(12V) dramatically increased the contribution of exchange to the fluorescence recovery of GFP-PLCbeta(2), but had the opposite effect on GFP-PLCbeta(2)Delta, where lateral diffusion became dominant. Our results demonstrate for the first time the regulation of membrane association of a PLCbeta isozyme by a GTP-binding protein and assign a novel function to the PLCbeta(2) C-terminal region, regulating its exchange between membrane-bound and cytosolic states.     

Complexes of HLA-G protein on the cell surface are important for leukocyte Ig-like receptor-1 function

The nonclassical class I MHC molecule HLA-G is selectively expressed on extravillous cytotrophoblast cells at the maternal-fetal interface during pregnancy. HLA-G can inhibit the killing mediated by NK cells via interaction with the inhibitory NK cell receptor, leukocyte Ig-like receptor-1 (LIR-1). Comparison of the sequence of the HLA-G molecule to other class I MHC proteins revealed two unique cysteine residues located in positions 42 and 147. Mutating these cysteine residues resulted in a dramatic decrease in LIR-1 Ig binding. Accordingly, the mutated HLA-G transfectants were less effective in the inhibition of NK killing and RBL/LIR-1 induced serotonin release. Immunoprecipitation experiments demonstrated the involvement of the cysteine residues in the formation of HLA-G protein oligomers on the cell surface. The cysteine residue located at position 42 is shown to be critical for the expression of such complexes. These oligomers, unique among the class I MHC proteins, probably bind to LIR-1 with increased avidity, resulting in an enhanced inhibitory function of LIR-1 and an impaired killing function of NK cells.     

Adjuvant Docetaxel and Cyclophosphamide (DC) with Prophylactic Granulocyte Colony-Stimulating Factor (G-CSF) on Days 8 &12 in Breast Cancer Patients: A Retrospective Analysis

Purpose

Four cycles of docetaxel/cyclophosphamide (DC) resulted in superior survival than doxorubicin/cyclophosphamide in the treatment of early breast cancer. The original study reported a 5% incidence of febrile neutropenia (FN) recommending prophylactic antibiotics with no granulocyte colony-stimulating factor (G-CSF) support. The worldwide adoption of this protocol yielded several reports on substantially higher rates of FN events. We explored the use of growth factor (GF) support on days 8 and 12 of the cycle with the original DC protocol.

Methods

Our study included all consecutive patients with stages I–II breast cancer who were treated with the DC protocol at the Institute of Oncology, Davidoff Center (Rabin Medical Center, Petah Tikva, Israel) from April, 2007 to March, 2012. Patient, tumor characteristics, and toxicity were reported. Results: In total, 123 patients received the DC regimen. Median age was 60 years, (range, 25–81 years). Thirty-three patients (26.8%) were aged 65 years and older. Most of the women (87%) adhered to the planned G-CSF protocol (days 8 &12). 96% of the patients completed the 4 planned cycles of chemotherapy. Six patients (5%) had dose reductions, 6 (5%) had treatment delays due to non-medical reasons. Thirteen patients (10.6%) experienced at least one event of FN (3 patients had 2 events), all requiring hospitalization. Eight patients (6.5%) required additional support with G-CSF after the first chemotherapy cycle, 7 because of FN and one due to neutropenia and diarrhea.

In Conclusion

Primary prophylactic G-CSF support on days 8 and 12 of the cycle provides a tolerable option to deliver the DC protocol. Our results are in line with other retrospective protocols using longer schedules of GF support.