Malate dehydrogenase as a multi-purpose target for drug discovery.

Malate dehydrogenase (MDH) enzymes play critical roles in cellular metabolism, facilitating the reversible conversion of malate to oxaloacetate using NAD+/NADH as a cofactor. The two human isoforms of MDH have roles in the citric acid cycle and the malate-aspartate shuttle, and thus both are key enzymes in aerobic respiration as well as regenerating the pool of NAD+ used in glycolysis. This review highlights the potential of MDH as a therapeutic drug target in various diseases, including metabolic and neurological disorders, cancer, and infectious diseases. The most promising molecules for targeting MDH have been examined in the context of human malignancies, where MDH is frequently overexpressed. Recent studies have led to the identification of several antagonists, some of which are broad MDH inhibitors while others have selectivity for either of the two human MDH isoforms. Other promising compounds have been studied in the context of parasitic MDH, as inhibiting the function of the enzyme could selectively kill the parasite. Research is ongoing with these chemical scaffolds to develop more effective small-molecule drug leads that would have great potential for clinical applications.

Evolution of a novel adrenal cell type that promotes parental care.

Cell types with specialized functions fundamentally regulate animal behaviour, and yet the genetic mechanisms that underlie the emergence of novel cell types and their consequences for behaviour are not well understood1. Here we show that the monogamous oldfield mouse (Peromyscus polionotus) has recently evolved a novel cell type in the adrenal gland that expresses the enzyme AKR1C18, which converts progesterone into 20α-hydroxyprogesterone. We then demonstrate that 20α-hydroxyprogesterone is more abundant in oldfield mice, where it induces monogamous-typical parental behaviours, than in the closely related promiscuous deer mice (Peromyscus maniculatus). Using quantitative trait locus mapping in a cross between these species, we ultimately find interspecific genetic variation that drives expression of the nuclear protein GADD45A and the glycoprotein tenascin N, which contribute to the emergence and function of this cell type in oldfield mice. Our results provide an example by which the recent evolution of a new cell type in a gland outside the brain contributes to the evolution of social behaviour.

Using transcriptome sequencing to identify mechanisms of drug action and resistance.

Determining mechanisms of drug action in human cells remains a major challenge. Here we describe an approach in which multiple-drug-resistant clones are isolated and transcriptome sequencing is used to find mutations in each clone. Further analysis of mutations common to more than one clone can identify a drug's physiological target and indirect resistance mechanisms, as indicated by our proof-of-concept studies of the cytotoxic anticancer drugs BI 2536 and bortezomib.

Examining the mechanism of action of a kinesin inhibitor using stable isotope labeled inhibitors for cross-linking (SILIC).

It is difficult to determine a chemical inhibitor's binding site in multiprotein mixtures, particularly when high-resolution structural studies are not straightforward. Building upon previous research involving photo-cross-linking and the use of mixtures of stable isotopes, we report a method, Stable Isotope Labeled Inhibitors for Cross-linking (SILIC), for mapping a small molecule inhibitor's binding site in its target protein. In SILIC, structure-activity relationship data is used to design inhibitor analogues that incorporate a photo-cross-linking group along with either natural or 'heavy' stable isotopes. An equimolar mixture of these inhibitor analogues is cross-linked to the target protein to yield a robust signature for identifying inhibitor-modified peptide fragments in complex mass spectrometry data. As a proof of concept, we applied this approach to an ATP-competitive inhibitor of kinesin-5, a widely conserved motor protein required for cell division and an anticancer drug target. This analysis, along with mutagenesis studies, suggests that the inhibitor binds at an allosteric site in the motor protein.

Reconstruction of forearm deformity by distraction osteogenesis in children with relative shortening of the ulna due to multiple cartilaginous exostosis.

BACKGROUND: In patients with multiple cartilaginous exostosis, distal ulnar osteochondromas frequently cause forearm deformities, with relative ulnar shortening, wrist joint deviation, and varus bowing. Progressive deformation often leads to pain, functional impairment, and cosmetic problems. Surgical ulnar lengthening is necessary to restore the carpal balance. The results of fixator-controlled ulnar lengthening were investigated in this study, using appropriate clinical and radiologic parameters and focusing on medium-term functional and structural outcomes. METHODS: Twelve children (3 boys, 9 girls; mean age 9.8 y) with multiple cartilaginous exostosis-induced ulnar shortening treated with fixator-controlled ulnar callotasis were evaluated retrospectively based on clinical and radiographic examinations preoperatively, after fixator removal, and at a follow-up investigation. Subjective symptoms and objective joint function were assessed clinically, whereas the extent of ulnar shortening, radial articular angle, carpal slip, and radial head dislocation were determined radiographically. RESULTS: The average follow-up period was 24.6 months. The mean ulnar shortening and radial articular angle improved significantly, from 14.3 mm or 38.7 degrees preoperatively to 1.7 mm or 25.6 degrees after fixator removal and showed a slight but significant increase to 5.2 mm or 30.1 degrees at the follow-up. Carpal slip and radial head dislocation remained unchanged. With the exception of radial abduction, no notable functional advancement was observed. One unintended ulnar overlengthening with a subsequent ulnocarpal impaction syndrome, one premature callus consolidation, and two fixator dislocations were noted. CONCLUSIONS: In agreement with literature reports, carpal balance can be restored over the medium term. However, mild recurrences of ulnar shortening and radial malformation were observed during further development. To prevent deformity progression in immature patients, surgery should be carried out early. The optimal timing of surgery needs to be calculated precisely to take advantage of the high remodeling potential and an acceptable degree of recurrent deformity. Ulnar lengthening is necessary, but overcorrection is inadvisable due to possible ulnocarpal impaction syndrome. As significant remodeling effects on the radius were observed, simultaneous radial correction procedures are not recommended a priori.

Pyruvate,orthophosphate dikinase in leaves and chloroplasts of C(3) plants undergoes light-/dark-induced reversible phosphorylation.

Pyruvate,orthophosphate (Pi) dikinase (PPDK) is best recognized as a chloroplastic C(4) cycle enzyme. As one of the key regulatory foci for controlling flux through this photosynthetic pathway, it is strictly and reversibly regulated by light. This light/dark modulation is mediated by reversible phosphorylation of a conserved threonine residue in the active-site domain by the PPDK regulatory protein (RP), a bifunctional protein kinase/phosphatase. PPDK is also present in C(3) plants, although it has no known photosynthetic function. Nevertheless, in this report we show that C(3) PPDK in leaves of several angiosperms and in isolated intact spinach (Spinacia oleracea) chloroplasts undergoes light-/dark-induced changes in phosphorylation state in a manner similar to C(4) dikinase. In addition, the kinetics of this process closely resemble the reversible C(4) process, with light-induced dephosphorylation occurring rapidly (< or =15 min) and dark-induced phosphorylation occurring much more slowly (> or =30-60 min). In intact spinach chloroplasts, light-induced dephosphorylation of C(3) PPDK was shown to be dependent on exogenous Pi and photosystem II activity but independent of electron transfer from photosystem I. These in organello results implicate a role for stromal pools of Pi and adenylates in regulating the reversible phosphorylation of C(3)-PPDK. Last, we used an in vitro RP assay to directly demonstrate ADP-dependent PPDK phosphorylation in desalted leaf extracts of the C(3) plants Vicia faba and rice (Oryza sativa). We conclude that an RP-like activity mediates the light/dark modulation of PPDK phosphorylation state in C(3) leaves and chloroplasts and likely represents the ancestral isoform of this unusual and key C(4) pathway regulatory "converter" enzyme.