New Therapies: Of Kinases and Cascades

In last month’s column we discussed the role of the four Janus kinases, or Jaks, in cytokine signaling, and described how inhibition of this kinase activity has become a major goal of drug discovery efforts in ophthalmology and other specialties. From the Human Genome Project we know there are about 580 kinases in the human body, and many of these function at critical signaling junctions in development, homeostasis and reaction to trauma or other bodily insults.Eglen R, Reisine T. Drug discovery and the human kinome: Recent trends. Pharmacol Ther 2011;130:144-56. In the response to allergen exposure, for example, a series of kinases participates in the initial orchestration of an immunological response to the foreign invasion.MacGlashan DW. IgE-dependent signaling as a therapeutic target for allergies. Trends in Pharm Sci 2012;33:502-509. One of these, spleen tyrosine kinase, is a key target of current drug development efforts aimed at improving therapies, including treatments for ocular allergy and infl ammation. The ever-growing number of biologics and small-molecule drugs designed to interfere with kinase signaling underscores the importance of this cadre of therapeutic agents.

This month, we’ll examine the challenges to development of kinase-targeted drugs such as Syk inhibitors, and describe a handful of other kinase targets of particular relevance to ophthalmologists.

Why Target Kinases?

Kinases are enzymes that transfer phosphate groups from an ATP donor to a target protein, sugar or fat molecule.,,Schwartz PA, Murray BW. Protein kinase biochemistry and drug discovery. Bioorganic Chemistry 2011;39:192–210. In some cases, these transfers are part of a metabolic pathway such as the breakdown of glucose, but in many others the addition of the phosphate molecule acts to change the function of the kinase substrate. Often this change dramatically alters the activity of the substrate target, switching it from an inactive to an active state. Phosphorylation ignites the breakdown of glucose in glycolysis, facilitates muscular cross-bridge formation during contraction and mediates hundreds of hormone and neurotransmitter responses, from epinephrine and insulin to VEGF. All these phosphorylation-based events require the enzymatic participation of one or more kinases.

In terms of intracellular signaling pathways, there are many examples of kinase cascades, which are a succession of enzymes activating each other in turn. This stepwise process in signaling provides opportunities for integration of cell inputs, as well as a degree of signaling redundancy. From our point of view, it presents potential targets for therapeutic intervention and physiologic modulation. As a group, kinases represent the single most important class of enzymes, in terms of their role in cell signal transduction.

One aspect of kinase function that is central to their role is their reversibility: There is a corresponding phosphatase poised to turn off the phosphorylation signal for almost all kinases. There are many examples of physiological processes activated by phosphorylation, and inactivated by dephosphorylation. In some cases, the phosphatases may even be a more attractive alternative to the kinases with regard to therapeutic modulation.Jeffrey KL, Camps M, Rommel C, Mackay CR. Targeting dualspecifi city phosphatases: M anipulating MAP kinase signalling and immune responses. Nat Rev Drug Discov 2007;6:391-403.

Mast cell degranulation pathway

The shared characteristics of different kinases are important because the single biggest hurdle in development of kinase-targeted therapeutics is specifi city. This is particularly true for drugs that compete with ATP binding, since this is a shared function of all kinases. The largest class of kinase inhibitors, termed type I inhibitors, all act by interfering with binding of ATP. Not surprisingly, many kinases share similar binding site characteristics and so the drugs targeted at them often display a lack of kinase specifi city. Future inhibitor development may focus on allosteric sites, or on recognition of sequences surrounding the sites where phosphates are added, to improve drug selectivity.,

The Flow of Allergic Signaling

One of the best examples of a kinase signaling cascade underlies the acute allergic reaction. In the mast cell, cross-linking of the Fcε receptor with antibody-antigen complexes trips a switch eventually leading to calcium entry and degranulation. This path involves at least fi ve kinases acting in concert to convert detection of a foreign antigen into histamine release (See Table, right).Gilfillan AM, Peavy RD, Metcalfe DD. Amplifi cation mechanisms for the enhancement of antigen-mediated mast cell activation. Immunol Res 2009;43:1-3:15-24. By mapping the path of the degranulation signal through the mast cell it’s possible to compare and contrast the benefits and limitations of targeting each step for potential therapeutic intervention.

Following the initial encounter with antigen on the mast cell surface, one of the earliest measurable responses is the activation of Lyn kinase, an enzyme that is physically associated with the antibody-receptor complex on the intracellular side of the cell membrane. Genetic deletions of Lyn kinase eliminate the ability of antigen to trigger histamine release or mast cell degranulation but, paradoxically, drugs that inhibit Lyn kinase activity actually increase mast cell degranulation responses. This is because in addition to being an early step in mast cell activation, Lyn is also part of the “off switch” for degranulation: It starts a separate signal cascade involving the phosphatases that ultimately reverse the kinase-mediated secretion.

Other players in this kinase cascade process are unsuitable therapeutic targets for other reasons. Phosophoinotiside 3-kinase and protein kinase C are each members of kinase families expressed in many cell types, so specifi city is likely to be an issue with any inhibitors of these drugs (inhibitors of these two kinase families are used for advanced stages of some cancers). In contrast, two hematopoietic-cell specifi c kinases, Syk and Bruton’s kinase, are the best targets based upon tissue specifi city and importance to the degranulation pathway. Btk is found primarily in B cells and in T cells, and is an important regulator of B cell maturation. While there is less data on this enzyme, preliminary studies suggest inhibitors may be effective therapeutically against infl ammatory diseases such as rheumatoid arthritis.Chang BY, Huang MM, Francesco M. The Bruton tyrosine kinase inhibitor PCI-32765 ameliorates autoimmune arthritis by inhibition of multiple effector cells. Arthritis Res Ther 2011;13:R115.,Xu D, Kim Y, Postelnek J, et al. RN486, a selective Bruton’s tyrosine kinase inhibitor, abrogates immune hypersensitivity responses and arthritis in rodents. J Pharmacol Exp Ther 2012;341:90-103.

Inhibitors of Syk kinase are in development for a number of diseases, including heparin-induced thrombocytopenia and lymphocytic leukemia. Reilly MP, Sinha U, André P, et al. PRT-060318, a novel Syk inhibitor, prevents heparin-induced thrombocytopenia and thrombosis in a transgenic mouse model. Blood 2011;117:2241- 2246.,Hoellenriegel J, Coffey GP, Sinha U, et al. Selective, novel spleen tyrosine kinase (Syk) inhibitors suppress chronic lymphocytic leukemia B-cell activation and migration. Leukemia 2012; 26:1576-83. These conditions involve antigen or B cell signaling, pathways similar to those seen for mast cell degranulation. The demonstrated efficacy of Syk inhibitors in these studies suggests that these same compounds (or other Syk inhibitors) can be effective when used to disrupt Syk signaling in other settings, including ocular allergy.

An Inhibitor for All Conditions?

In surveying recent drug discovery work on protein kinases, there is a recurring theme in which initial development of inhibitors addresses a specific type of cancer, especially cancers of the blood-forming tissues and those that are relatively intractable to current treatments. Researchers then take candidate therapeutics from these efforts and repurpose them to treat other disorders. Interest in these compounds for ocular diseases is just beginning to gain traction, and it may not be possible to identify the best candidates from existing studies alone. Preclinical models of various ophthalmic disorders will provide key guidance as this process moves forward.

Last month we described the key role of the Jak kinases in cellular inflammatory signaling. Another proinflammatory target for kinase inhibitors is the nuclear factor κB pathway used by cytokines, including tumor necrosis factor-alpha, interleukin-2 and interleukin-6. An inflammatory gene expression profile is activated when kinases such as IκK activate the κB factor, and blockade of this kinase has demonstrated anti-infl ammatory effects in preclinical models of endotoxin- induced uveitis.Lennikov A, Kitaichi N, Noda K et al. Amelioration of endotoxininduced uveitis treated with an IκB kinase β inhibitor in rats. Molecular Vision 2012;18:2586-2597

One area of interest for kinase inhibitors is as alternative anti-angiogenic therapies for ocular diseases. Drugs such as Imatinib, Sorafenib or Sunitinib all inhibit a range of growthpromoting tyrosine kinases including vascular endothelial growth factorreceptor kinases, and were originally developed to treat late-stage renal, liver and lung cancers. Preclinical studies suggest such drugs may be useful, either in combination or as monotherapy, for reducing corneal or retinal neovascularization.Seo JW, Chung SH, Choi JS, Joo CK. Inhibition of Corneal Neovascularization in Rats by Systemic Administration of Sorafenib. Cornea 2012;31:907–912. One or more of these multikinase inhibitors may also be useful in treatment of ocular melanomas.Spagnolo F, Caltabiano G, Queirolo P. Uveal melanoma. Cancer Treat Rev 2012;38:549-53.

Many ophthalmic diseases involve a premature, pathologic degeneration of retinal neurons, and there has been great interest in the role of kinasemediated signaling in apoptotic, or programmed, neuronal cell death. A recent study used genomic profi ling to implicate leucine zipper kinases in retinal cell apoptosis.Welsbie DS, Yanga Z, Gea Y, et al. Functional genomic screening identifi es dual leucine zipper kinase as a key mediator of retinal ganglion cell death. Proc Nat Acad Sci USA 2013;110:4045-4050. Another kinase, fi rst identifi ed as the mammalian target of rapamycin, or mTor, has since been shown to play a key role in cell development and survival.Doonan F, Groeger G, Cotter TG. Preventing retinal apoptosis—is there a common therapeutic theme? Exp Cell Res 2012;318:1278-84. A handful of mTOR inhibitors, such as Everolimus, have been approved as anti-cancer therapies and several of these are currently in clinical trials as therapies for advanced AMD.

Another aspect of mTOR signaling relates to our previous description of kinase cascades, those signaling pathways in which one kinase activates a second, which then activates a third, and so on until the ultimate target is reached. Interestingly, in some cells mTOR functions in a cascade that leads from PI3K to protein kinase B to mTOR. Thus, depending on tissue expression, there is overlap between the cell survival pathway of mTOR and the degranulation pathway described for mast cells, that includes Syk kinase. This type of overlap is the rule, not the exception, in most cell regulatory paradigms.

Many ophthalmic diseases involve a premature, pathologic degeneration of retinal neurons, and there has been great interest in the role of kinasemediated signaling in apoptotic, or programmed, neuronal cell death.

 

As with other new therapeutics, exploring ophthalmic indications for these kinase inhibitors involves new formulation development and optimization of topical or intravitreal delivery methods. Even with established safety profi les, there is still work to be done before any kinase inhibitors will be FDA-approved for ophthalmic use. However, it’s important to realize that Syk inhibition of the degranulation signal cascade represents more than just another new anti-histamine or anti-inflammatory; these drugs would prevent the initial release of histamine and other mast cell allergic mediators, effectively stopping ocular allergic disease before it has a chance to start.

For patients suffering from conjunctivitis, keratoconjunctivitis, vernal keratoconjunctivitis and other chronic, inflammatory ocular surface conditions, halting the fl ow of the allergic kinase cascade may be a gamechanging therapeutic advance.

Dr. Abelson is a clinical professor of ophthalmology at Harvard Medical School and senior clinical scientist at the Schepens Eye Research Institute. Dr. McLaughlin is a medical writer at Ora Inc.