RAM-589.555 a new Polymerase-1 inhibitor as innovative targeted-treatment for multiple sclerosis

Highlights

• RAM-589.555 is a water soluble, permeable compound, that specifically inhibits POL1.

• RAM-589.555 induces apoptosis and suppresses lymphocytes proliferation and viability.

• Oral administration of RAM589.555 delays EAE onset and reduced disease severity.

Abstract

Targeting Polymerase-1 (POL1) transcription machinery is a new strategy for suppression of multiple sclerosis (MS) disease activity that is based on suppression of ribosomal biogenesis and subsequent activation of apoptosis. We developed an oral POL1 inhibiting compound RAM-589.555, that suppress ribosomal biogenesis as an innovative therapeutic approach to ameliorate MS. RAM-589.555 shows high permeability, specificity to POL1 pathway, ability to induce apoptosis and to inhibit proliferation and viability of activated lymphocytes both in-vitro and in-vivo. Moreover, oral administration of RAM-589.555 blocks ribosomal RNA transcription and significantly suppresses and ameliorates experimental autoimmune encephalomyelitis (EAE).

Introduction

Benign multiple sclerosis (MS) signifies a sub-group of non-active MS patients, in whom in spite of the ongoing disease process patients are protected from long-term disability. Patients with benign MS reflect the capacity to withstand the deteriorating processes of the disease and the ability to endure or recover quickly from the acute demyelinating inflammatory insults. Thus, these patients are not harmed by the ongoing pathologic processes of myelin and neuronal loss that characterize active MS (Gauthier et al., 2009, Glad et al., 2009, Hawkins and McDonnell, 1999, Pittock and Rodriguez, 2008, Poser et al., 1979, Ramsaransing and De Keyser, 2007). Recently, using high throughput peripheral blood gene expression microarray analysis we identified a specific signature that characterizes benign MS (Achiron et al., 2012). This signature is portrayed by decreased expression of RNA Polymerase-1 (POL1) molecular pathway, important for ribosome biogenesis, modulation of cellular stress response and regulation of autoimmune response (Goodfellow and Zomerdijk, 2013, Leary and Huang, 2001, Quin et al., 2014, Sciascia et al., 2007, Stetler et al., 1992).

We have shown that POL1 related genes, RNA Polymerase-1 transcription factor 3 (RRN3), leucine-rich PPR motif containing protein (LRPPRC) and polymerase (RNA)-1 polypeptide D (POLR1D), play a key role in the regulation of apoptosis in MS and their decreased expression is associated with suppression of inflammation by down regulation of NFkB and activation of p53 dependent apoptosis. Moreover, in a previous study (Achiron et al., 2007) we have shown that POL1 related gene RRN3 was the leading gene that predicted good clinical outcome of relapsing-remitting MS patients over a period of two years and appropriately classified 70.4% of the patients. We have further demonstrated that blocking RRN3 expression by siRNA-silencing in PBMC cultures obtained from of RRMS patients resulted in significant increase in apoptosis confirming that signaling through POL1 pathway plays a key role in regulating cell survival in MS (Achiron et al., 2013).

Taken together, our findings provide a basis for direct targeting of POL1 pathway as an innovative strategy for selective induction of apoptosis in active MS. Moreover, the suppression of POL1 pathway can be easily monitored using the platform of POL1 related genes to validate treatment efficacy. As a proof of concept, we have recently reported that a prototype POL1-inhibitor - CX-5461 (developed by Cylene Pharmaceuticals; (Drygin et al., 2014, Drygin et al., 2010, Woods et al., 2015) delayed the appearance and significantly suppressed experimental autoimmune encephalomyelitis (EAE) in mice (Achiron et al., 2013). However, the promising potential of CX-5461 is limited for therapeutic application in MS due to a narrow therapeutic window between the minimal effective and the toxic doses. Hence, we have designed and synthesized a new POL1 inhibitor RAM-589.555. We hereby report its molecular structure, specificity to POL1 pathway inhibition, and efficacy in-vitro and in the EAE animal model. This orally administered small molecule can specifically block ribosomal RNA transcription and selectively induce apoptosis in encephalitogenic cells.