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The recognition of protease inhibitors with insecticidal activity is important as a basis for the development of mimetic peptides with potential use as biorational insecticides. We sprayed benzamidine on soybean plants and assessed whether this potent synthetic trypsin-inhibitor has protease inhibitory, insecticidal and deterrent effects on the velvetbean caterpillar Anticarsia gemmatalis Hübner (Lepidoptera: Erebidae). Activity of trypsin inhibition in soybean leaves was increased and total proteolytic activity in the midgut extract from larvae fed on these leaves was reduced by benzamidine. Different concentrations of benzamidine sprayed on the plant caused approximately 50 % of larval mortality, and larval choice and moth preference and oviposition were all negatively affected. Low concentrations of benzamidine increased mortality and hindered insect choice and oviposition as well as higher doses. Since many synthetic protease inhibitors are usually expensive, small doses of benzamidine may be effective to protect soybean against A. gemmatalis attack. Our results highlight the potential of synthetic protease inhibitors for insecticidal and deterrent purposes in insect pest management.

Background and Purpose: Human parathyroid hormone (PTH) is critical for maintaining physiological calcium homeostasis and plays an important role in the formation and maintenance of the bone. Full-length PTH and a truncated peptide form are approved for treatment of hypoparathyroidism and osteoporosis respectively. Our initial goal was to develop an improved PTH therapy for osteoporosis, but clinical development was halted. The novel compound was then repurposed as an improved therapy for hypoparathyroidism., Experimental Approach: A longer-acting form of PTH was synthesised by altering the peptide to increase cell surface residence time of the bound ligand to its receptor. In vitro screening identified a compound, which was tested in an animal model of osteoporosis before entering human trials. This compound was subsequently tested in two independent animal models of hypoparathyroidism., Key Results: The peptide identified, LY627-2K, exhibited delayed internalization kinetics. In an ovariectomy-induced bone loss rat model, LY627-2K demonstrated improved vertebral bone mineral density and biomechanical properties at skeletal sites and a modest increase in serum calcium. In a Phase I clinical study, dose-dependent increases in serum calcium were reproduced. These observations prompted us to explore a second indication, hypoparathyroidism. In animal models of this disease, LY627-2K restored serum calcium, comparing favourably to treatment with wild-type PTH., Conclusions and Implications: We summarize the repositioning of a therapeutic candidate with substantial preclinical and clinical data. Our results support its repurposing and continued development, from a common indication (osteoporosis) to a rare disease (hypoparathyroidism) by exploiting a shared molecular target., Linked Articles: This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc., (© 2017 The British Pharmacological Society.)

While there are approximately 7,000 identified human diseases considered as "rare" based on population prevalence or incidence, the cumulative impact runs into the millions of patients globally. Although the genetic underpinnings of more than 2,000 rare diseases have been elucidated, there remains a paucity of therapeutic options, frequently due to lack of commercial interest. Development programs suffer high attrition within the so-called "Valley of Death," in which the risks of scientific failure are still too high to justify the increasing development costs. This problem is common to any drug development campaign, but it is particularly exacerbated in the rare diseases, many of which arise in childhood. To stimulate development of therapeutics for these otherwise underserved patient populations, a number of regulatory incentives and research initiatives have been established. Extended patent protections, expedited regulatory reviews for qualified drug sponsors, and clinical trial grant support aim to foster interest in completing development programs. To stimulate researchers to embark on rare disease drug development campaigns, earlier-stage preclinical research resources have been created, as well, such as the Therapeutics for Rare and Neglected Diseases (TRND) program at the U.S. National Institutes of Health (NIH). TRND is a unique NIH program created to support drug development through formation of public-private partnerships. These partnerships leverage the robust biopharmaceutical industry experience of the TRND staff scientists and the deep disease area expertise of the collaborating partners. Each project adopted into the TRND portfolio aims to satisfy two broad goals: developing a novel therapy for a rare or otherwise neglected disease, and exploring ways to accelerate the drug development process overall so that lessons learned can be disseminated to the wider community undertaking translational research. This article discusses common obstacles and opportunities for therapeutic development, and provides examples of the types of projects TRND has undertaken across a broad range of pediatric rare disorders.

Erythropoiesis is dependent on the activity of transcription factors, including the erythroid-specific erythroid Kruppel-like factor (EKLF). ChIP followed by massively parallel sequencing (ChIP-Seq) is a powerful, unbiased method to map trans-factor occupancy. We used ChIP-Seq to study the interactome of EKLF in mouse erythroid progenitor cells and more differentiated erythroblasts. We correlated these results with the nuclear distribution of EKLF, RNA-Seq analysis of the transcriptome, and the occupancy of other erythroid transcription factors. In progenitor cells, EKLF is found predominantly at the periphery of the nucleus, where EKLF primarily occupies the promoter regions of genes and acts as a transcriptional activator. In erythroblasts, EKLF is distributed throughout the nucleus, and erythroblast-specific EKLF occupancy is predominantly in intragenic regions. In progenitor cells, EKLF modulates general cell growth and cell cycle regulatory pathways, whereas in erythroblasts EKLF is associated with repression of these pathways. The EKLF interactome shows very little overlap with the interactomes of GATA1, GATA2, or TAL1, leading to a model in which EKLF directs programs that are independent of those regulated by the GATA factors or TAL1.

Erythroid Krüppel-like factor (EKLF) is a Krüppel-like transcription factor identified as a transcriptional activator and chromatin modifier in erythroid cells. EKLF-deficient (Eklf(-/-)) mice die at day 14.5 of gestation from severe anemia. In this study, we demonstrate that early progenitor cells fail to undergo terminal erythroid differentiation in Eklf(-/-) embryos. To discover potential EKLF target genes responsible for the failure of erythropoiesis, transcriptional profiling was performed with RNA from wild-type and Eklf(-/-) early erythroid progenitor cells. These analyses identified significant perturbation of a network of genes involved in cell cycle regulation, with the critical regulator of the cell cycle, E2f2, at a hub. E2f2 mRNA and protein levels were markedly decreased in Eklf(-/-) early erythroid progenitor cells, which showed a delay in the G(1)-to-S-phase transition. Chromatin immunoprecipitation analysis demonstrated EKLF occupancy at the proximal E2f2 promoter in vivo. Consistent with the role of EKLF as a chromatin modifier, EKLF binding sites in the E2f2 promoter were located in a region of EKLF-dependent DNase I sensitivity in early erythroid progenitor cells. We propose a model in which EKLF-dependent activation and modification of the E2f2 locus is required for cell cycle progression preceding terminal erythroid differentiation.

Erythroid Krüppel-like factor (EKLF) is an erythroid zinc finger protein identified by its interaction with a CACCC sequence in the beta-globin promoter, where it establishes local chromatin structure permitting beta-globin gene transcription. We sought to identify other EKLF target genes and determine the chromatin status of these genes in the presence and absence of EKLF. We identified alpha hemoglobin-stabilizing protein (AHSP) by subtractive hybridization and demonstrated a 95 to 99.9% reduction in AHSP mRNA and the absence of AHSP in EKLF-deficient cells. Chromatin at the AHSP promoter from EKLF-deficient cells lacked a DNase I hypersensitive site and exhibited histone hypoacetylation across the locus compared to hyperacetylation of wild-type chromatin. Wild-type chromatin demonstrated a peak of EKLF binding over a promoter region CACCC box that differs from the EKLF consensus by a nucleotide. In mobility shift assays, the AHSP promoter CACCC site bound EKLF in a manner comparable to the beta-globin promoter CACCC site, indicating a broader recognition sequence for the EKLF consensus binding site. The AHSP promoter was transactivated by EKLF in K562 cells, which lack EKLF. These results support the hypothesis that EKLF acts as a transcription factor and a chromatin modulator for the AHSP and beta-globin genes and indicate that EKLF may play similar roles for other erythroid genes.

In previous studies amphotropic MFGS-gp91phox (murine onco-retrovirus vector) was used in a clinical trial of X-linked chronic granulomatous disease (X-CGD) gene therapy to achieve transient correction of oxidase activity in 0.1% of neutrophils. We later showed that transduced CD34+ peripheral blood stem cells (CD34+ PBSCs) from this trial transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in correction of only 2.5% of human neutrophils. However, higher rates of transduction into stem cells are required. In the current study we demonstrate that the same vector (MFGS-gp91phox) pseudo-typed with RD114 envelope in a 4-day culture/transduction regimen results in a 7-fold increase in correction of NOD/SCID mouse repopulating X-CGD CD34+ PBSCs (14%-22% corrected human neutrophils; human cell engraftment 13%-67%). This increase may result from high expression of receptor for RD114 that we demonstrate on CD34+CD38- stem cells. Using RD114-MFGS encoding cyan fluorescent protein to allow similar studies of normal CD34+ PBSCs, we show that progressively higher levels of gene marking of human neutrophils (67%-77%) can be achieved by prolongation of culture/transduction to 6 days, but with lower rates of human cell engraftment. Our data demonstrate the highest reported level of functional correction of any inherited metabolic disorder in human cells in vivo with the NOD/SCID mouse system using onco-retrovirus vector.

We have analyzed the 1 alpha, 25-dihydroxycholecalciferol [1,25(OH)2D3] receptor content of cultured cells from human amniotic fluid. Six cell lines were grown to confluence in a minimum essential medium containing 20% fetal calf serum. All had a normal karyotype, five were male and one was female. Hypertonic cytosol extracts were prepared by sonication followed by centrifugation at 200,000 X g 30 min. Saturation analysis was performed by incubating the extracts with [3H]-1,25(OH)2D3 (20-500 pM, 160 Ci/mmol) with and without 100-fold molar excess of unlabeled 1,25(OH)2D3. Linear sucrose gradient (5-20% w/v) analysis was performed with 1.5 nM [3H]-1,25(OH)2D3 alone or in presence of 100-fold molar excess, 1,25(OH)2D3. Functional responsiveness was measured by induction of 25-hydroxycholecalciferol-24R-hydroxylase with 1 and 10 nM 1,25(OH)2D3. The six cell lines studied had receptors with dissociation constant of 44 +/- 6 pM (mean +/- SEM). The binding capacity was 10,200 +/- 1,750 sites/ng protein (mean +/- SEM) with extreme values of 4,700 and 15,500. A single peak for specific binding migrating at approximately 3S was observed by sucrose gradient centrifugation. 25-Hydroxycholecalciferol-24R-hydroxylase was induced by 1 and 10 nM 1,25(OH)2D3 in a dose-dependent fashion. The data show that receptors for 1,25(OH)2D3 are present in cultured amniotic fibroblast-like cells early in pregnancy. These cells may thus prove to be useful for further characterization of 1,25(OH)2D3 receptors in fetal tissue.