SMA is an inherited disease that leads to loss of motor function and ambulation and a reduced life expectancy. We have been working to develop orally administrated, systemically distributed small molecules to increase levels of functional SMN protein. Compound 2 was the first SMN2 splicing modifier tested in clinical trials in healthy volunteers and SMA patients. It was safe and well tolerated and increased SMN protein levels up to 2-fold in patients. Nevertheless, its development was stopped as a precautionary measure because retinal toxicity was observed in cynomolgus monkeys after chronic daily oral dosing (39 weeks) at exposures in excess of those investigated in patients. Herein, we describe the discovery of 1 (risdiplam, RG7916, RO7034067) that focused on thorough pharmacology, DMPK and safety characterization and optimization. This compound is undergoing pivotal clinical trials and is a promising medicine for the treatment of patients in all ages and stages with SMA.

Optical melting was used to determine the stabilities of 11 small RNA oligomers of defined secondary structure as a function of magnesium ion concentration. The oligomers included helices composed of Watson-Crick base pairs, GA tandem base pairs, GU tandem base pairs, and loop E motifs (both eubacterial and eukaryotic). The effect of magnesium ion concentration on stability was interpreted in terms of two simple models. The first assumes an uptake of metal ion upon duplex formation. The second assumes nonspecific electrostatic attraction of metal ions to the RNA oligomer. For all oligomers, except the eubacterial loop E, the data could best be interpreted as nonspecific binding of metal ions to the RNAs. The effect of magnesium ions on the stability of the eubacterial loop E was distinct from that seen with the other oligomers in two ways. First, the extent of stabilization by magnesium ions (as measured by either change in melting temperature or free energy) was three times greater than that observed for the other helical oligomers. Second, the presence of magnesium ions produces a doubling of the enthalpy for the melting transition. These results indicate that magnesium ion stabilizes the eubacterial loop E sequence by chelating the RNA specifically. Further, these results on a rather small system shed light on the large enthalpy changes observed upon thermal unfolding of large RNAs like group I introns. It is suggested that parts of those large enthalpy changes observed in the folding of RNAs may be assigned to variations in the hydration states and types of coordinating atoms in some specifically bound magnesium ions and to an increase in the observed cooperativity of the folding transition due to the binding of those magnesium ions coupling the two stems together. Brownian dynamic simulations, carried out to visualize the metal ion binding sites, reveal rather delocalized ionic densities in all oligomers, except for the eubacterial loop E, in which precisely located ion densities were previously calculated.

The case records of 20 horses with tetanus referred to the Ontario Veterinary College-Veterinary Teaching Hospital between 1970 and 1990 were reviewed. The fatality rate was 75%. There was a strong association with previous vaccination and survival (P = .03). Most of the animals had been injured an average of 9 days (range 2 to 21 days) prior to development of clinical signs. Hyperesthesia and prolapse of the third eyelid were the most common clinical signs. Treatment regimens varied during hospitalization; however, all horses received parenteral penicillin, tranquilizers, tetanus toxoid, and antitoxin. Five of the nonsurviving animals were given intrathecal tetanus antitoxin. One animal had seizures as a complication of intrathecal treatment. The prognosis was best for horses that (1) had been vaccinated prior to the injury, (2) responded to the phenothiazine tranquilizers, and (3) did not rapidly (over 24 to 48 hours) become recumbent. Considering the species susceptibility, potential for contaminated wounds, and the increased survival of vaccinated horses, yearly revaccination is recommended.

The preparation of π-rich 2-aryl heterocycles by palladium-catalyzed cross-coupling of sodium heteroarylsilanolates with aryl iodides, bromides and chlorides is described. The cross-coupling process was developed through extensive optimization of the follow key variables: (1) identification of stable, isolable alkali metal silanolates, (2) identification of conditions for preformation and isolation of silanolate salts, (3) judicious choice in the palladium catalyst/ligand combination, and (4) selection of the protecting group on the nitrogen of indole. It was found that the alkali metal silanolates, either isolated or formed in situ, offered a significant rate enhancement and broader substrate scope over the use of silanols activated by Brønsted bases such as NaOt-Bu. In addition, the optimized conditions for the cross-coupling of 2-indolylsilanolates were readily applied to the cross-coupling of 2-pyrrolyl-, 2-furyl-, and 2-thienylsilanolates.

Results suggest that PSSM and shivers are common but unrelated disorders in BDHs.

No abstract

Recent achievements in the genetic correction of keratinocytes isolated from patients with junctional epidermolysis bullosa have paved the way to a gene therapy approach for the disease. Because gene therapy protocols require preclinical validation in animals, we have characterized spontaneous animal models of junctional epidermolysis bullosa. In this study we have elucidated the genetic basis of the hereditary junctional mechanobullous disease in the Belgian horse, a condition characterized by blistering of the skin and mouth epithelia, and exungulation (loss of the hoof). Immunofluorescence analysis associated the condition to the absent expression of the gamma2 chain of laminin 5 and designated Lamc2 as the candidate gene. Comparative analysis of the nucleotide sequence of the full-length gamma2 cDNA isolated by reverse transcription polymerase chain reaction amplification of total RNA purified from the epithelium of a junctional epidermolysis bullosa foal and a healthy control disclosed a homozygous basepair insertion (1368insC) in the affected animal. Mutation 1368insC results in a downstream premature termination codon and is predicted to cause absent expression of the laminin gamma2 polypeptide. Our results also show that: (i) the horse junctional epidermolysis bullosa genetically corresponds to the severe Herlitz form of junctional epidermolysis bullosa in man; (ii) the amino acid sequence and structure of the horse laminin gamma2 chain are virtually identical to the human counterpart; (iii) the moderate eruption of skin blisters in the affected animals with respect to the human Herlitz junctional epidermolysis bullosa patients correlates with the protection provided by hair. Our observations suggest that the affected foals are a convenient source of epithelial cells from tissues that cannot be obtained from human junctional epidermolysis bullosa patients, and imply that hairless strains of animals with recessive skin disorders would be the best models for in vivo gene therapy approaches to skin blistering diseases.

This paper chronicles the conceptual development, proof of principle experiments, and recent advances in the palladium-catalyzed cross-coupling reactions of the conjugate bases of organosilanols. The discovery that led to the design and refinement of this process represents a classical illustration of how mechanistic studies can provide a fertile ground for the invention of new reactions. On the basis of a working hypothesis (which ultimately proved to be incorrect) and the desire to effect silicon-based cross-coupling without the agency of fluoride activation, a mild and practical palladium-catalyzed cross-coupling of alkenyl-, aryl-, and heteroaryl silanolates has been developed. The mechanistic underpinnings, methodological extensions, and the successful applications of this technology to the synthesis of complex molecules are described.