Discovery of Therapeutic Deubiquitylase Effector Molecules: Current Perspectives.

The approval of proteasome inhibitors bortezomib and carfilzomib and the E3 ligase antagonist thalidomide and its analogs, lenalidomide and pomalidomide, validates the ubiquitin-proteasome pathway as a source of novel drugs for treating cancer and, potentially, a variety of devastating illnesses, including inflammation, cardiovascular disease, and neurodegenerative disease. All elements of this critical regulatory pathway-the proteasome itself, E3 ligases (which conjugate ubiquitin to target proteins), and deubiquitylating enzymes (which deconjugate ubiquitin, reversing ligase action)-are potential therapeutic targets, and all have been worked on extensively during the past decade. No deubiquitylase inhibitors or activators have yet progressed to clinical trial, however, despite compelling target validation and several years of high-throughput screening and preclinical development of hits by numerous pharmaceutical companies, biotechnology organizations, and academic groups. The appropriateness of deubiquitylases as therapeutic targets in many disease areas is reviewed, followed by evidence that selective inhibitors of these cysteine proteases can be discovered. Because the lack of progress in drug-discovery efforts with deubiquitylases suggests a need for improved discovery methodologies, currently available platforms and strategies are analyzed, and improved or completely novel, unrelated approaches are considered in terms of their likelihood of producing clinically viable effectors of deubiquitylases.

Novel class of thiourea compounds that inhibit herpes simplex virus type 1 DNA cleavage and encapsidation: resistance maps to the UL6 gene.

In our search for novel inhibitors of herpes simplex virus type 1 (HSV-1), a new class of thiourea inhibitors was discovered. N-(4-[3-(5-Chloro-2,4-dimethoxyphenyl)-thioureido]-phenyl)-acetamide and its 2-fluoro-benzamide derivative inhibited HSV-1 replication. HSV-2, human cytomegalovirus, and varicella-zoster virus were inhibited to a lesser extent. The compounds acted late in the replication cycle by impairing both the cleavage of concatameric viral DNA into progeny genome length and the packaging of the DNA into capsids, indicative of a defect in the encapsidation process. To uncover the molecular target of the inhibition, resistant HSV-1 isolates were generated, and the mutation responsible for the resistance was mapped using marker transfer techniques. Each of three independent isolates had point mutations in the UL6 gene which resulted in independent single-amino-acid changes. One mutation was located in the N terminus of the protein (E121D), while two were located close together in the C terminus (A618V and Q621R). Each of these point mutations was sufficient to confer drug resistance when introduced into wild-type virus. The UL6 gene is one of the seven HSV-1 genes known to play a role in DNA packaging. This novel class of inhibitors has provided a new tool for dissection of HSV-1 encapsidation mechanisms and has uncovered a new viable target for the treatment of herpesviral diseases.

Inhibition of influenza A virus replication by compounds interfering with the fusogenic function of the viral hemagglutinin.

Several compounds that specifically inhibited replication of the H1 and H2 subtypes of influenza virus type A were identified by screening a chemical library for antiviral activity. In single-cycle infections, the compounds inhibited virus-specific protein synthesis when added before or immediately after infection but were ineffective when added 30 min later, suggesting that an uncoating step was blocked. Sequencing of hemagglutinin (HA) genes of several independent mutant viruses resistant to the compounds revealed single amino acid changes that clustered in the stem region of the HA trimer in and near the HA2 fusion peptide. One of the compounds, an N-substituted piperidine, could be docked in a pocket in this region by computer-assisted molecular modeling. This compound blocked the fusogenic activity of HA, as evidenced by its inhibition of low-pH-induced cell-cell fusion in infected cell monolayers. An analog which was more effective than the parent compound in inhibiting virus replication was synthesized. It was also more effective in blocking other manifestations of the low-pH-induced conformational change in HA, including virus inactivation, virus-induced hemolysis of erythrocytes, and susceptibility of the HA to proteolytic degradation. Both compounds inhibited viral protein synthesis and replication more effectively in cells infected with a virus mutated in its M2 protein than with wild-type virus. The possible functional relationship between M2 and HA suggested by these results is discussed.

Suppression of cell-mediated immunity in hypothyroidism.

A 71-year-old man had severe hypothyroidism, chronic autoimmune thyroiditis, and bacteremia due to Edwardsiella tarda. Review of the literature identified the hypothesis that E tarda infections may occur more frequently in immunocompromised patients. Previous animal studies have shown decreases in lymphocyte function during hypothyroidism, with return of normal lymphocyte function during euthyroid states. Therefore, lymphocyte transformation studies were obtained, demonstrating severe decreases in our patient's lymphocyte function. Except for chronic autoimmune thyroiditis, other immune system abnormalities were excluded. Serial lymphocyte transformation studies showed gradual improvement in lymphocyte function during gradual return to euthyroid state. Possible pathophysiologic mechanisms for these findings will be reviewed.

Ovarian vein sampling in rapidly progressing virilization. A case report.

A woman presented with progressive hirsutism, deepening of the voice, clitoromegaly and increased libido. The preoperative serum testosterone level was 2,042 ng/dL. The intraoperative ovarian venous blood testosterone levels were 56,327 ng/dL on the left and 1,417 on the right. After a bilateral salpingo-oophorectomy, the serum testosterone level was 20.7 ng/dL. Initial pathologic examination of the ovaries revealed no potential source of testosterone, but reexamination revealed a pure lipoid cell tumor. Intraoperative ovarian vein androgen measurements can be helpful in establishing a histopathologic diagnosis in cases of microscopic virilizing ovarian neoplasms. Ovarian vein sampling for androgen was essential in locating this patient's microscopic tumor.