Jonathan J. Cherry

Enhancement of SMN protein levels in a mouse model of spinal muscular atrophy using novel drug-like compounds

Spinal muscular atrophy (SMA) is a neurodegenerative disease that causes progressive muscle weakness, which primarily targets proximal muscles. About 95% of SMA cases are caused by the loss of both copies of the SMN1 gene. SMN2 is a nearly identical copy of SMN1, which expresses much less functional SMN protein. SMN2 is unable to fully compensate for the loss of SMN1 in motor neurons but does provide an excellent target for therapeutic intervention. Increased expression of functional full‐length SMN protein from the endogenous SMN2 gene should lessen disease severity. We have developed and implemented a new high‐throughput screening assay to identify small molecules that increase the expression of full‐length SMN from a SMN2 reporter gene. Here, we characterize two novel compounds that increased SMN protein levels in both reporter cells and SMA fibroblasts and show that one increases lifespan, motor function, and SMN protein levels in a severe mouse model of SMA.

Binding of Human Papillomavirus Type 16 E6 to E6AP Is Not Required for Activation of hTERT

ABSTRACT The human papillomavirus (HPV) type 16 (HPV16) E6 protein stimulates transcription of the catalytic subunit of telomerase, hTERT, in epithelial cells. It has been reported that binding to the ubiquitin ligase E6AP is required for this E6 activity, with E6 directing E6AP to the hTERT promoter. We previously reported two E6AP binding-defective HPV16 E6 mutations that induced immortalization of human mammary epithelial cells. Because activation of hTERT is proposed to be necessary for epithelial cell immortalization, we sought to further characterize the relationship between E6/E6AP association and telomerase induction. We demonstrate that while these E6 mutants do not bind E6AP, they retain the capability to stimulate the expression of hTERT. Chromatin immunoprecipitation assays confirmed the presence of Myc, wild-type E6, and the E6AP binding-defective E6 mutants, but not E6AP itself, at the endogenous hTERT promoter. Interestingly, an immortalization-defective E6 mutant localized to the hTERT promoter but failed to increase transcription. We conclude that binding to E6AP is not necessary for E6 localization to or activation of the hTERT promoter and that another activity of E6 is involved in hTERT activation.

Discovery, Synthesis and Biological Evaluation of Novel SMN Protein Modulators

Spinal muscular atrophy (SMA) is an autosomal recessive disorder affecting the expression or function of survival motor neuron protein (SMN) due to the homozygous deletion or rare point mutations in the survival motor neuron gene 1 (SMN1). The human genome includes a second nearly identical gene called SMN2 that is retained in SMA. SMN2 transcripts undergo alternative splicing with reduced levels of SMN. Up-regulation of SMN2 expression, modification of its splicing, or inhibition of proteolysis of the truncated protein derived from SMN2 have been discussed as potential therapeutic strategies for SMA. In this manuscript, we detail the discovery of a series of arylpiperidines as novel modulators of SMN protein. Systematic hit-to-lead efforts significantly improved potency and efficacy of the series in the primary and orthogonal assays. Structure-property relationships including microsomal stability, cell permeability, and in vivo pharmacokinetics (PK) studies were also investigated. We anticipate that a lead candidate chosen from this series may serve as a useful probe for exploring the therapeutic benefits of SMN protein up-regulation in SMA animal models and a starting point for clinical development.

Structure Based Identification and Characterization of Flavonoids That Disrupt Human Papillomavirus-16 E6 Function

Expression and function of the human papillomavirus (HPV) early protein 6 (E6) is necessary for viral replication and oncogenesis in cervical cancers. HPV E6 targets the tumor suppressor protein p53 for degradation. To achieve this, “high-risk” HPV E6 proteins bind to and modify the target specificity of the ubiquitin ligase E6AP (E6 associated protein). This E6-dependent loss of p53 enables the virus to bypass host cell defenses and facilitates virally induced activation of the cell cycle progression during viral replication. Disruption of the interaction between E6 and E6AP and stabilization of p53 should decrease viability and proliferation of HPV positive cells. A new in vitro high-throughput binding assay was developed to assay binding between HPV-16 E6 and E6AP and to identify compounds that inhibit this interaction. The compound luteolin emerged from the screen and a library of novel flavones based on its structure was synthesized and characterized using this in vitro binding assay. The compounds identified in this study disrupt the E6/E6AP interaction, increase the levels of p53 and p21Cip1/Waf1, and decrease proliferation of HPV positive cell lines. The new class of flavonoid E6 inhibitors displays a high degree of specificity for HPV positive cells. Docking analyses suggest that these compounds bind in a hydrophobic pocket at the interface between E6 and E6AP and mimic the leucines in the conserved α-helical motif of E6AP. The activity and specificity of these compounds represent a promising new lead for development as an antiviral therapy in the treatment of HPV infection and cervical cancer.

Therapeutic strategies for the treatment of spinal muscular atrophy

Spinal muscular atrophy (SMA) is an inherited neurodegenerative disease that results in progressive dysfunction of motor neurons of the anterior horn of the spinal cord. SMA is caused by the loss of full-length protein expression from the survival of motor neuron 1 (SMN1) gene. The disease has a unique genetic profile as it is autosomal recessive for the loss of SMN1, but a nearly identical homolog, SMN2, acts as a disease modifier whose expression is inversely correlated to clinical severity. Targeted therapeutic approaches primarily focus on increasing the levels of full-length SMN protein, through either gene replacement or regulation of SMN2 expression. There is currently no US FDA approved treatment for SMA. This is an exciting time as multiple efforts from academic and industrial laboratories are reaching the preclinical and clinical testing stages.

Identification of Novel Compounds That Increase SMN Protein Levels Using an Improved SMN2 Reporter Cell Assay

Spinal muscular atrophy (SMA) is a neurodegenerative disorder that is characterized by progressive loss of motor neuron function. It is caused by the homozygous loss of the SMN1 (survival of motor neuron 1) gene and a decrease in full-length SMN protein. SMN2 is a nearly identical homolog of SMN1 that, due to alternative splicing, expresses predominantly truncated SMN protein. SMN2 represents an enticing therapeutic target. Increasing expression of full-length SMN from the SMN2 gene might represent a treatment for SMA. We describe a newly designed cell-based reporter assay that faithfully and reproducibly measures full-length SMN expression from the SMN2 gene. This reporter can detect increases of SMN protein by an array of compounds previously shown to regulate SMN2 expression and by the overexpression of proteins that modulate SMN2 splicing. It also can be used to evaluate changes at both the transcriptional and splicing level. This assay can be a valuable tool for the identification of novel compounds that increase SMN2 protein levels and the optimization of compounds already known to modulate SMN2 expression. We present here preliminary data from a high-throughput screen using this assay to identify novel compounds that increase expression of SMN2.

Differential regulation of the SMN2 gene by individual HDAC proteins

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder that is the leading genetic cause of infantile death. SMA is caused by homozygous deletion or mutation of the survival of motor neuron 1 gene (SMN1). The SMN2 gene is nearly identical to SMN1, however is alternatively spliced. The close relationship to SMN1 results in SMN2 being a very power genetic modifier of SMA disease severity and a target for therapies. We sought to identify the regulatory role individual HDAC proteins use to control expression of full length protein from the SMN2 genes. We used quantitative PCR to determine the effects shRNA silencing of individual HDACs on the steady state levels of a SMN2-luciferase reporter transcripts. We determined that reduction of individual HDAC proteins was sufficient to increase SMN protein levels in a transgenic reporter system. Knockdown of class I HDAC proteins preferentially activated the reporter by increased promoter transcription. Silencing of class II HDAC proteins maintained transcriptional activity; however silencing of HDAC 5 and 6 also appeared to enhance inclusion of an alternatively spliced exon. This work highlights HDAC proteins 2 and 6 as excellent investigative targets. These data are important to the basic understanding of SMN expression regulation and the refinements of current therapeutic compounds as well as the development of novel SMA therapeutics.

Design of Potent mRNA Decapping Scavenger Enzyme (DcpS) Inhibitors with Improved Physicochemical Properties To Investigate the Mechanism of Therapeutic Benefit in Spinal Muscular Atrophy (SMA)

The C-5 substituted 2,4-diaminoquinazoline RG3039 (compound 1), a member of a chemical series that was identified and optimized using an SMN2 promoter screen, prolongs survival and improves motor function in a mouse model of spinal muscular atrophy (SMA). It is a potent inhibitor of the mRNA decapping scavenger enzyme (DcpS), but the mechanism whereby DcpS inhibition leads to therapeutic benefit is unclear. Compound 1 is a dibasic lipophilic molecule that is predicted to accumulate in lysosomes. To understand if the in vivo efficacy is due to DcpS inhibition or other effects resulting from the physicochemical properties of the chemotype, we undertook structure based molecular design to identify DcpS inhibitors with improved physicochemical properties. Herein we describe the design, synthesis, and in vitro pharmacological characterization of these DcpS inhibitors along with the in vivo mouse CNS PK profile of PF-DcpSi (compound 24), one of the analogs found to be efficacious in SMA mouse model.

ML372 blocks SMN ubiquitination and improves spinal muscular atrophy pathology in mice

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease and one of the leading inherited causes of infant mortality. SMA results from insufficient levels of the survival motor neuron (SMN) protein, and studies in animal models of the disease have shown that increasing SMN protein levels ameliorates the disease phenotype. Our group previously identified and optimized a new series of small molecules, with good potency and toxicity profiles and reasonable pharmacokinetics, that were able to increase SMN protein levels in SMA patient-derived cells. We show here that ML372, a representative of this series, almost doubles the half-life of residual SMN protein expressed from the SMN2 locus by blocking its ubiquitination and subsequent degradation by the proteasome. ML372 increased SMN protein levels in muscle, spinal cord, and brain tissue of SMA mice. Importantly, ML372 treatment improved the righting reflex and extended survival of a severe mouse model of SMA. These results demonstrate that slowing SMN degradation by selectively inhibiting its ubiquitination can improve the motor phenotype and lifespan of SMA model mice.

Discovery of a Small Molecule Probe That Post-Translationally Stabilizes the Survival Motor Neuron Protein for the Treatment of Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is the leading genetic cause of infant death. We previously developed a high-throughput assay that employs an SMN2-luciferase reporter allowing identification of compounds that act transcriptionally, enhance exon recognition, or stabilize the SMN protein. We describe optimization and characterization of an analog suitable for in vivo testing. Initially, we identified analog 4m that had good in vitro properties but low plasma and brain exposure in a mouse PK experiment due to short plasma stability; this was overcome by reversing the amide bond and changing the heterocycle. Thiazole 27 showed excellent in vitro properties and a promising mouse PK profile, making it suitable for in vivo testing. This series post-translationally stabilizes the SMN protein, unrelated to global proteasome or autophagy inhibition, revealing a novel therapeutic mechanism that should complement other modalities for treatment of SMA.