On Press:
Phil LoGrasso's Quartet

At Scripps Florida's Translational Research Institute, the ROCK Hits Just Keep on Coming

By Eric Sauter

Phil LoGrasso, Scripps Florida's senior director of drug discovery and an associate professor in molecular therapeutics, has had four papers published over the last several months, all focused on the discovery of several potent and highly selective Rho Kinase (ROCK-II) inhibitors, not to mention the development of a cell-based assay that allows for the rapid functional assessment of these same inhibitors.

Metaphorically, LoGrasso is four for four in terms of these new studies, but the number of potentially useful protein inhibitors that LoGrasso and his colleagues have created is significantly higher than four.

"In these papers, we're reporting on hundreds of inhibitors but we've actually produced thousands," LoGrasso said. "Which ones might be worth pursuing out of the hundreds identified in these papers? We hope two to three compounds per class will have the properties we want in an effective inhibitor."

Protein kinases like ROCK, a member of the serine/threonine kinase family, are enzymes that add phosphate groups to other proteins and modify their behavior, a process commonly known as phosphorylation. Kinases may modify as many as one-third of all proteins and, since kinases regulate most cellular pathways within the cell, especially signal transduction, this makes them serious contenders for the most favored drug targets of all time.

According to one 2006 report, described in Drugresearcher.com, the kinase-targeted drug market is set to grow from $12.7 billion in 2005 to $58.6 billion in 2010; a 2008 Reuters story pointed out that kinase inhibitors now account for 20-30 percent of all drug discovery programs.

In animal studies, Rho-kinase has been shown to be substantially involved in cerebral vasospasm, arteriosclerosis, ischemia/reperfusion injury, hypertension, pulmonary hypertension, stroke and heart failure, central sympathetic nerve activity, and, last but not least, glaucoma.

None of this surprises LoGrasso, since it's the very same area he's studying himself.

"We're really focusing on glaucoma with these new inhibitors," he said. "I think we might have a compound that we could put into a safety assessment, which is pretty deep in preclinical development, in the next six to eighteen months. From there, you could have an IND [Independent New Drug application] filed within a year."

A New Class of Inhibitor for Glaucoma

Of the four papers, the one published in the Journal of Medicinal Chemistry (2008, Volume 51, No. 21), stands out. This study was a collaborative effort of Scripps Florida and the Duke University School of Medicine.

LoGrasso and his colleagues successfully identified an entirely new class of potent and highly selective ROCK-II inhibitors, one of which, labeled compound 5 (originally SR3677), turned out to be very potent indeed.

"Compound 5 is the most selective ROCK inhibitor ever published," LoGrasso said. "There might be another one in a laboratory somewhere, but this is most selective we've ever seen. Selectivity is critical because there are 518 other kinases that are ATP competitive inhibitors—ATP is the energy transport vehicle within the cell—and you want to make certain that you don't inhibit any of these. If you do, you may develop significant safety issues."

In terms of glaucoma, the study showed that compound 5 was efficacious both in increasing aqueous humor outflow and in modulating or reducing phosphorylated-myosin light chain, making it a good candidate for treating glaucoma. Myosin light chain phosphorylation, an event important for cellular contraction, is involved in the development of diseases like glaucoma, as well as hypertension and cancer.

"Generally speaking, the potency of a compound depends on how tight it binds to the target protein," said Yangbo Feng, the first author of the study. "We're delighted that the Rho kinase protein obviously likes the molecules we designed as its inhibitors. This is mainly due to several special structural elements we designed and built into our novel scaffolds, which differentiate our ROCK inhibitors from others."

Further optimization of the novel compound, plus additional pharmacology studies, are already under way.

More to Come

Another study, published in Biochemical and Biophysical Research Communications, describes a cell-based myosin light chain phosphorylation assay for the rapid functional assessment of novel ROCK inhibitors. The results showed that the measurement of changes in myosin light chain phosphorylation can be successfully used to screen a larger set of small molecule ROCK inhibitors.

"The throughput and ease of this functional assay enables it to be used for medicinal chemistry efforts, potentially not just ROCK but for other proteins and pathways regulating myosin light chain phosphorylation," LoGrasso said.

Finally, in a pair of studies, both published last October in the journal Biooganic & Medicinal Chemistry Letters, LoGrasso and his colleagues identified a slew of new ROCK inhibitors, in two distinct classes.

In the first study, the scientists synthesized a series of chroman-3-amides with remarkable inhibition of ROCK-II, all of which favor further study. Assessment of the in vivo efficacy of these inhibitors, specific absorption rate studies and a detailed analysis of factors affecting affinity for both ROCK-I and ROCK-II are all under way.

In the second study, LoGrasso and his colleagues discovered an early lead compound, which they gave the name "amide 1," with a high affinity for ROCK-II, good selectivity over a related kinase, plus high potency. Of course, amide 1 is just the beginning; further work added a potent series of benzimidazole- and benzoxazole-based ROCK inhibitors from that initial amide lead.

"Ultimately all these inhibitors will have the same properties for drug development," LoGrasso said, "but since they're from different chemical series, they could later be differentiated by toxicities that we aren't aware of now but that may pop up during further testing."

As with the other discoveries, the group has already embarked on a effort to optimize the compounds with an eye on developing selective ROCK (I and II) inhibitors and the development of pharmacokinetic properties tailored to specific therapeutic applications.

"The compounds in the last two studies are companions in the sense that the same biological tests were conducted for both," he said, "but they're different structurally. I wouldn't even call them cousins, really; they're just friends. The chrom-3-amides hold more promise for glaucoma, while the benzimidazole and benzoxazole based inhibitors are probably betterfor peripheral applications."

If all of this seems to have a kind of Neverending Story quality to it, LoGrasso is cheerfully aware of how the journey continues.

"These discovery searches go on and on," he said, laughing. "I think that's why I like cutting my grass at home so much. I spend an hour, do it once, and I can see the finished product. I don't have to do it a thousand more times to get results."

The first author of the study, "Discovery of Substituted 4-(Pyrazol-4-yl)-phenylbenzodioxane-2-carboxamides as Potent and Highly Selective Rho Kinase (ROCK-II) Inhibitors," published in the Journal of Medicinal Chemistry (2008, Volume 51, No. 21), is Yangbo Feng of The Scripps Research Institute; other authors from Scripps Research included Yan Yin, Amiee Weiser, Evelyn Griffin, Michael D. Cameron, Li Lin, Claudia Ruiz, Stephan C. Schürer, and Thomas Schröter; authors from Duke University include Toshihiro Inoue and P. Vasanth Rao.

The first author of the study, "Detection of Myosin Light Chain Phosphorylation—A Cell-Based Assay For Screening Rho-Kinase Inhibitors," published in an advance on-line edition of the journal, Biochemical and Biophysical Research Communications, is Thomas Schröter of the Translational Research Institute at Scripps Florida. Other authors include Evelyn Griffin, Amiee Weiser, and Yangbo Feng.

The first author of the study, "Chroman-3-Amides As Potent Rho Kinase Inhibitors," which was published in Biooganic & Medicinal Chemistry Letters, is Yen Ting Chen of The Scripps Research Institute; other authors include Thomas D. Bannister, Amiee Weiser, Evelyn Griffin, Li Lin, Claudia Ruiz, Michael D. Cameron, Stephan Schürer, Derek Duckett, Thomas Schröter, and Yangbo Feng.

The first author of the study, Benzimidazole- And Benzoxazole-Based Inhibitors Of Rho Kinase, which was published in Biooganic & Medicinal Chemistry Letters, is E. Hampton Sessions of The Scripps Research Institute; other authors include Yan Yin, Thomas D. Bannister, Amiee Weiser, Evelyn Griffin, Jennifer Pocas, Michael D. Cameron, Claudia Ruiz, Li Lin, Stephan C. Schürer, Thomas Schröter, and Yangbo Feng.

The studies were supported by the National Institutes of Health (Journal of Medicinal Chemistry paper), the State of Florida, and The Scripps Research Institute.

Send comments to: mikaono[at]scripps.edu

Phil LoGrasso is developing inhibitors that might be used to treat glaucoma, atherosclerosis, cancer, and other conditions.