Fragment-based lead discovery to identify novel inhibitors that target the ATP binding site of pyruvate dehydrogenase kinases.

A fragment-based lead discovery approach was applied to Pyruvate Dehydrogenase Kinases (PDHKs) to discover inhibitors against the ATP binding site with novel chemotypes. X-ray fragment screening toward PDHK4 provided a fragment hit 1 with a characteristic interaction in a deep pocket of the ATP binding site. While known inhibitors utilize several water molecules in a deep pocket to form water-mediated hydrogen bond interactions, the fragment hit binds deeper in the pocket with a hydrophobic group. Displacement of a remaining water molecule in the pocket led to the identification of lead compound 7 with a notable improvement in inhibition potency. This lead compound possessed high ligand efficiency (LE) and showed decent selectivity profile. Two additional lead compounds 10 and 13 with new scaffolds with tricyclic and bicyclic cores were generated by merging structural information of another fragment hit 2. The characteristic interaction of these novel inhibitors in a deep pocket provides new structural insights about PDHKs ATP binding site and opens a novel direction for the development of PDHKs inhibitors.

Structure-based drug design of novel and highly potent pyruvate dehydrogenase kinase inhibitors.

Pyruvate dehydrogenase kinases (PDHKs) are fascinating drug targets for numerous diseases, including diabetes and cancers. In this report, we describe the result of our structure-based drug design from tricyclic lead compounds that led to the discovery of highly potent PDHK2 and PDHK4 dual inhibitors in enzymatic assay. The C3-position of the tricyclic core was explored, and the PDHK2 X-ray structure with a representative compound revealed a novel ATP lid conformation in which the phenyl ring of Phe326 mediated the interaction of the Arg258 sidechain and the compound. Compounds with amide linkers were designed to release the ATP lid by forming an intramolecular pi-pi interaction, and these compounds showed single-digit nM IC50 values in an enzymatic assay. We also explored the C4-position of the tricyclic core to reproduce the interaction observed with the C3-position substitution, and the pyrrolidine compound showed the same level of IC50 values. By optimizing an interaction with the Asn255 sidechain through a docking simulation, compounds with 2-carboxy pyrrole moiety also showed single-digit nM IC50 values without having a cation-pi interaction with the Arg258 sidechain.

Variation of Extrinsic Tendons of the Thumb in Type IIIA Hypoplastic Thumbs.

PURPOSE: The purpose of this study was to identify the variety of anatomical abnormalities of extrinsic tendons in type IIIA hypoplastic thumbs. METHODS: We reviewed 79 thumbs in 67 patients. Opponensplasty, stabilizing of the thumb metacarpophalangeal joint, and widening of the first web space were performed in all patients. At the time of surgery, we made detailed observations of the anatomical abnormalities of the extrinsic tendons of the thumb. RESULTS: Fifty thumbs (50 of 79; 63%) had an interconnection between the flexor pollicis longus (FPL) and the extensor pollicis longus (EPL) tendons. Twenty-six thumbs (26 of 79; 33%) had bifurcations (25 [32%] bifurcated from the FPL; 1 [1%] bifurcated from the EPL). There were 25 FPL abnormalities (4 [5%] complete absence; 8 [10%] proximal absence; 2 [3%] distal absence; 11 [14%] tendon hypoplasia) and 7 EPL abnormalities (2 [3%] proximal absence; 5 [6%] tendon hypoplasia). CONCLUSIONS: Interconnections between the FPL and the EPL tendons and a duplicated FPL were observed frequently. CLINICAL RELEVANCE: The present study investigates the detailed anatomy of the type IIIA hypoplastic thumbs. The data might help improve the design of surgical procedures.

Identification of a cDNA encoding a novel small secretory protein, neurosecretory protein GL, in the chicken hypothalamic infundibulum.

To find novel neuropeptide and/or peptide hormone precursors in the avian brain, we performed a cDNA subtractive screen of the chicken hypothalamic infundibulum, which contains one of the feeding and neuroendocrine centers. After sequencing 596 clones, we identified a novel cDNA encoding a previously unknown protein. The deduced precursor protein consisted of 182 amino acid residues, including one putative small secretory protein of 80 amino acid residues. This small protein was flanked at the N-terminus by a signal peptide and at the C-terminus by a glycine amidation signal and a dibasic amino acid cleavage site. Because the predicted C-terminal amino acids of the small protein were Gly-Leu-NH2, the small protein was named neurosecretory protein GL (NPGL). Quantitative RT-PCR analysis demonstrated specific expression of the NPGL precursor mRNA in the hypothalamic infundibulum. Furthermore, the mRNA levels in the hypothalamic infundibulum increased during post-hatching development. In situ hybridization analysis showed that the cells containing the NPGL precursor mRNA were localized in the medial mammillary nucleus and infundibular nucleus within the hypothalamic infundibulum of 8- and 15-day-old chicks. Subcutaneous infusion of NPGL in chicks increased body weight gain without affecting food intake. To our knowledge, this is the first report to describe the identification and localization of the NPGL precursor mRNA and the function of its translated product in animals. Our findings indicate that NPGL may participate in the growth process in chicks.

Identification of neurotensin and LANT-6 and localization of mRNA encoding their precursor in the chicken brain.

Neurotensin (NT) and neurotensin-related peptide (Lys(8), Asn(9), NT(8-13): LANT-6) have previously been purified from chicken intestine. However, the presence of these peptides and the localization of their precursor mRNA in the brain were not well understood. In the present study, through a comprehensive analysis of bioactive substances, NT and LANT-6 were identified in the chicken brain using tandem mass spectrometry combined with a bioassay of the colon contraction. The effect of NT and LANT-6 on the colon contraction was assessed, and NT was found to be 10 times more potent than LANT-6. Furthermore, the sites of NT/LANT-6 precursor mRNA expression in the brain were investigated using quantitative RT-PCR. The result showed that the mRNA was expressed most in the telencephalon, followed by the diencephalon. In situ hybridization analysis revealed that cells containing NT/LANT-6 precursor mRNA were widely distributed throughout the brain except for the cerebellum. Additionally, these were highly concentrated in the frontal telencephalon, including the nidopallium, hyperpallium, and hippocampus. Collectively, these results indicate that NT and LANT-6 are produced in the chicken brain, and they may participate in multiple functions.

Characteristics of Cubitus Varus Deformity after Lateral Condylar Fracture of the Humerus.

Background: Lateral humeral condylar fractures often heal with some residual elbow deformity. However, details of angulation or tilting angle of the lateral condyle after the fracture have not been evaluated so far. Methods: Between 2008 and 2016, we followed up 80 mild fractures of the lateral humeral condyle for more than a year. Thirty fractures were treated by open reduction and internal fixation (ORIF) with Kirschner wires. Fifty cases were treated with a long arm splint for 3 weeks (Fig. 1). The average age of the patients at the time of the injury was 5.5 years. The humerus-elbow-wrist angle (HEWA), Baumann's angle (BA), and tilting angle (TA) were measured on the radiographs. The active range of motion (ROM) was clinically assessed at unaffected and affected sides at the final follow-up. Results: No significant differences were detected between the sides about TA or ROM at the final follow-up. However, HEWA/ BA showed more significant loss of correction. There were significant differences in BA at the affected side between the ORIF and splint groups. Conclusions: Cubitus varus deformity after lateral humeral condylar fracture is not accompanied by a change in TA or ROM, unlike the deformity after supracondylar or distal epiphyseal fracture of the humerus (Fig. 2). Operative treatment to precisely correct and fix the lateral condylar fracture still retained some cubitus varus deformity, although it might lessen or prevent the deformity when compared to conservative treatment with a splint.

Treatment for Wassel Types V and VI Thumb Polydactyly.

Background: Polydactyly of the thumb is the most common congenital anomaly of the hand, but there have been few reports regarding Wassel types V and VI. The purpose of this study is to present our surgical strategies and outcomes for cases of Wassel types V and VI polydactyly of the thumb. Methods: Twenty-nine thumbs of 29 patients were included in this study; 17 cases were Wassel type V and 12 cases were type VI. Our strategies for initial surgery were appropriate tendon and muscle relocations. Opponensplasty with the abductor digiti minimi or the flexor digitorum superficialis and osteotomy were not performed in the initial surgery. We evaluated pinch motion ability and the number of additional surgical procedures. The first web space and radial instability of the metacarpophalangeal (MCP) joint were measured by radiography while the patient held a polystyrene foam cone. Results: Twenty-two patients were able to perform a pulp pinch. Narrowing of the first web defined as the angle between the first and second metacarpus (1-2 MCA) < 40° occurred in five cases. Radial instability of the MCP joint defined as the angle between the first metacarpus and thumb proximal phalanx (1 MPA) > 20° occurred in seven cases. Additional surgery was performed in seven cases (24%) to improve insufficient thumb opposition, radial instability of the MCP joint, and narrowing of the first web. Patients in all reoperation cases were able to perform a pulp pinch. Conclusions: Our strategies for initial surgery often had satisfactory outcomes, but careful follow-up observations and appropriate reoperation for cases with poor initial outcomes were more important.

Effect of decrease in radial inclination of distal radius fractures on distal radioulnar joint stability: a biomechanical study.

We investigated the relationship between the radial inclination of the distal radius and distal radioulnar joint stability. Six fresh-frozen upper extremities were used. Radial inclination was decreased by 10° and 20° and increased by 10° from the original radial inclination. Distal radioulnar joint stiffness was assessed with an intact radioulnar ligament and after partial and then complete sectioning of the radioulnar ligament. Radial angulation deformities significantly increased distal radioulnar joint stiffness when the radioulnar ligament is totally or partially attached to the ulnar fovea. After complete sectioning of the radioulnar ligament, distal radioulnar joint stiffness decreased significantly; additional radial angulation deformity slightly increased distal radioulnar joint stiffness, but the distal radioulnar joint never recovered to the original stiffness. Based on the results, radial angulation deformities of the distal radius should be corrected within 10° when the radioulnar ligament is intact, to reduce the risk of symptomatic distal radioulnar joint instability.

Author Correction: Localization and function of neurosecretory protein GM, a novel small secretory protein, in the chicken hypothalamus.

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Localization and function of neurosecretory protein GM, a novel small secretory protein, in the chicken hypothalamus.

Recently, we discovered a novel cDNA encoding the precursor of a small secretory protein, neurosecretory protein GL (NPGL), in the hypothalamic infundibulum of chickens. NPGL plays an important role in the regulation of growth and feeding. A database search indicated that the NPGL gene has a paralogous gene: neurosecretory protein GM (NPGM), also in chickens. We identified cDNA encoding the NPGM precursor in chickens. Morphological analysis showed that NPGM-containing cells are specifically localized in the medial mammillary nucleus (MM) and infundibular nucleus (IN) in the hypothalamus. In addition, we found that NPGM and NPGL are co-localized, especially in the MM. The expression levels of NPGM mRNA gradually decreased during post-hatch development, in contrast to those of NPGL mRNA. Moreover, we investigated the relationship between NPGM and other known factors. NPGM was found to be produced in histaminergic neurons in the MM. NPGM and histidine decarboxylase, a histamine-producing enzyme, displayed similar expression patterns during post-hatch development. Acute intracerebroventricular injection of NPGM decreased food intake, similar to the effect of histamine. To our knowledge, this is the first report of the localization and function of NPGM in the brain of vertebrates. These results will further advance the understanding mechanisms underlying energy homeostasis.

JTP-117968, a novel selective glucocorticoid receptor modulator, exhibits improved transrepression/transactivation dissociation.

Classic glucocorticoids that have outstanding anti-inflammatory effects are still widely prescribed for the treatment of various inflammatory and autoimmune diseases. Conversely, glucocorticoids cause numerous unwanted side effects, particularly systemically dosed glucocorticoids. Therefore, selective glucocorticoid receptor modulator (SGRM), which maintains beneficial anti-inflammatory effects while reducing the occurrence of side effects, is one of the most anticipated drugs. However, there have been no SGRMs marketed to date. The assumption is that there are two major mechanisms of action of glucocorticoids via glucocorticoid receptors, transrepression (TR) and transactivation (TA). In general, the anti-inflammatory effects of glucocorticoids are mostly mediated through TR, while the side effects associated with glucocorticoids are largely caused by TA. We started to evaluate novel orally available SGRMs that maintain anti-inflammatory effects while minimizing adverse effects by favoring TR over TA. Based on this evaluation, we discovered JTP-117968, (4b'S,7'R,8a'S)-4b'-benzyl-7'-hydroxy-N-(2-methylpyridin-3-yl)-7'-(trifluoromethyl)-4b',6',7',8',8a',10'-hexahydro-5'H-spiro[cyclopropane-1,9'-phenanthrene]-2'-carboxamide, a non-steroidal SGRM. JTP-117968 has partial TR activity, but exhibits extremely low TA activity. The maximum TR efficacy of JTP-117968 was comparable to its structural analogue, PF-802, (4bS,7R,8aR)-4b-Benzyl-7-hydroxy-N-(2-methylpyridin-3-yl)-7-(trifluoromethyl)-4b,5,6,7,8,8a,9,10-octahydrophenanthrene-2-carboxamide, which is the active form of Fosdagrocorat that has been developed clinically as a first-in-class orally available SGRM. Remarkably, the TA activity of JTP-117968 was much weaker than PF-802 not only in in vitro assays, but also in in vivo mice experiments. These findings indicate that JTP-117968 exhibits improved TR/TA dissociation because the compound has significantly lower TA activity compared with an already reported SGRM. Therefore, JTP-117968 is expected to be a useful compound for evaluating ideal SGRMs in the future.

Characterization of an avian histidine decarboxylase and localization of histaminergic neurons in the chicken brain.

In mammals, it is established that histamine is a neurotransmitter and/or neuromodulator in the central nervous system. It is produced by the enzyme histidine decarboxylase (HDC) in the tuberomammillary nucleus of the posterior hypothalamus. However, HDC as well as histaminergic neurons have not yet been characterized in the avian brain. We have cloned the cDNA for HDC from the chicken hypothalamus and demonstrated that the chicken HDC sequence is highly homologous to the mammalian counterpart, and that the expressed protein shows high enzymatic activity. The expression of HDC mRNA at various sites in the brain was investigated using quantitative RT-PCR. The results showed that the HDC mRNA was highly expressed in the hypothalamic infundibulum. In situ hybridization analyses revealed that the cells containing HDC mRNA were localized in the medial mammillary nucleus of the hypothalamic infundibulum. Intracerebroventricular injection of histamine in chicks resulted in inhibition of feeding behavior. This is the first report of the characterization of histaminergic neurons in the avian brain, and our findings indicate that neuronal histamine exerts anorexigenic effects in chicks.

Double plate fixation for correction of the malunited distal ulna fracture: a case report.

A 62-year-old woman visited our hospital one year after a motor vehicle accident complaining of ulnar wrist pain and restricted pronation and supination. Radiographs showed a 35° angular deformity at the ulnar neck. Closing wedge osteotomy was performed using two plates for stabilization. Twenty-four months postoperatively, the osteotomy site united without correction loss and the patient gained adequate pronation and supination. To the best of our knowledge, this represents the first report of corrective osteotomy for the treatment of malunited ulnar neck fracture. Although salvage operations such as ulnar head resection and the Sauvé-Kapandji procedure may provide reasonable results, anatomical repair can be considered as an option.