Synthetic benzamide derivatives were investigated for their ability to inhibit histone deacetylase (HDA). In this study, one of the most active benzamide derivatives, MS-27-275, was examined with regard to its biological properties and antitumor efficacy. MS-27-275 inhibited partially purified human HDA and caused hyperacetylation of nuclear histones in various tumor cell lines. It behaved in a manner similar to other HDA inhibitors, such as sodium butyrate and trichostatin A; MS-27-275 induced p21 WAF1͞CIP1 and gelsolin and changed the cell cycle distribution, decrease of S-phase cells, and increase of G 1 -phase cells. The in vitro sensitivity spectrum of MS-27-275 against various human tumor cell lines showed a pattern different than that of a commonly used antitumor agent, 5-f luorouracil, and, of interest, the accumulation of p21 WAF1͞CIP1 tended to be faster and greater in the cell lines sensitive to MS-27-275. MS-27-275 administered orally strongly inhibited the growth in seven of eight tumor lines implanted into nude mice, although most of these did not respond to 5-f luorouracil. A structurally analogous compound to MS-27-275 without HDA-inhibiting activity showed neither the biological effects in cell culture nor the in vivo therapeutic efficacy. These results suggest that MS-27-275 acts as an antitumor agent through HDA inhibition and may provide a novel chemotherapeutic strategy for cancers insensitive to traditional antitumor agents.

Newly synthesized benzamide derivatives were evaluated for their inhibitory activity against histone deacetylase. The structure of these derivatives was unrelated to the known inhibitors, and IC 50 values of the active compounds were in the range of 2-50 µM. Structure-activity relationship on the benzanilide moiety showed that the 2′-substituent, an amino or hydroxy group, was indispensable for inhibitory activity. Although the electronic influence of the substituent in the anilide moiety showed only a small effect on inhibitory activity, the steric factor in the anilide moiety, especially at positions 3′and 4′, played an important role in interaction with the enzyme. Among these benzamide derivatives, MS-275 (1), which showed significant antitumor activity in vivo, has been selected for further investigation.

Secondary trigeminal neuralgia (STN) follows an injury to the trigeminal nerve or one of its branches. Although rare, this condition results in great suffering and it is notoriously difficult to treat. The experimental analysis of painful neuropathy due to damage to the innervation of the limbs (e.g., the sciatic nerve) has progressed rapidly in recent years, but very few reports have appeared concerning experimental neuropathy in the trigemenial region. We report here an experimental rat model of trigeminal neuropathic pain produced by a chronic constriction injury to the infraorbital nerve (CCI-ION), and on a method that detects heat-evoked pain-related behavior. Rats with the CCI-ION have clear signs of heat-hyperalgesia when stimulated on the snout (the vibrissal pad). The hyperalgesia is seen both ipsi- and contralateral to the side of nerve injury, but is significantly more severe ipsilaterally, and lasts about 12 days.

Accumulating evidence has demonstrated the importance of alternative splicing in various physiological processes, including the development of different diseases. CDC-like kinases (CLKs) and serine-arginine protein kinases (SRPKs) are components of the splicing machinery that are crucial for exon selection. The discovery of small molecule inhibitors against these kinases is of significant value, not only to delineate the molecular mechanisms of splicing, but also to identify potential therapeutic opportunities. Here we describe a series of small molecules that inhibit CLKs and SRPKs and thereby modulate pre-mRNA splicing. Treatment with these small molecules (Cpd-1, Cpd-2, or Cpd-3) significantly reduced the levels of endogenous phosphorylated SR proteins and caused enlargement of nuclear speckles in MDA-MB-468 cells. Additionally, the compounds resulted in splicing alterations of RPS6KB1 (S6K), and subsequent depletion of S6K protein. Interestingly, the activity of compounds selective for CLKs was well correlated with the activity for modulating S6K splicing as well as growth inhibition of cancer cells. A comprehensive mRNA sequencing approach revealed that the inhibitors induced splicing alterations and protein depletion for multiple genes, including those involved in growth and survival pathways such as S6K, EGFR, EIF3D, and PARP. Fluorescence pulse-chase labeling analyses demonstrated that isoforms with premature termination codons generated after treatment with the CLK inhibitors were degraded much faster than canonical mRNAs. Taken together, these results suggest that CLK inhibitors exhibit growth suppression and apoptosis induction through splicing alterations in genes involved in growth and survival. These small molecule inhibitors may be valuable tools for elucidating the molecular machinery of splicing and for the potential development of a novel class of antitumor agents.