In our efforts to identify molecules that selectively reduce the expression of BMI1, a stem cell gene, we discovered and characterized the first-in-class series of small molecules that modulate the expression of BMI1 protein in cancer cells. Structure-activity and structure-property relationships associated with this series were investigated through medicinal chemistry efforts. These studies revealed important structural features required for achieving anti-tumor activity and acceptable pharmacokinetic properties within this series. The 4-CF3-Ph at the left-side of the molecule, a proper placement of the N-atom on the six-membered heterocycle in the middle, combined with a properly substituted C(2)-methyl benzimidazole on the right-hand side were required to achieve potency, microsomal stability, and exposure upon oral dosing. A compound (PTC-02) with acceptable pharmacological properties and efficacious in vivo in several tumor animal models was identified. image

Supplementary Figures S1-S7: Supplementary Figure S1 shows chemical Synthesis and biological characterization of PTC299. Supplementary Figure S2 shows generation and characterization of PTC299-resistant HT1080 cells. Supplementary Figure 3 shows that PTC299 interacts with and inhibits the activity of DHODH. Supplementary Figure S4 shows that PTC299 inhibits de novo UMP production in both sensitive and insensitive cell lines. Supplementary Figure S5 shows that PTC299 has broad activity against leukemia in vitro and in vivo. Supplementary Figure S6 shows that PTC299 inhibits production of VEGF protein in tumor cells more potently than does brequinar or teriflunomide. Supplementary Figure S7 shows the comparison of GSK983 and PTC299 in pharmacokinetic study in mice; Supplementary Tables S1-S7: Supplementary Table S1 shows the information on the source of cells and when they were obtained. Supplementary Table S2 shows that the metabolism of 15N-glutamine in cells treated with PTC299 is similar to that measured in cells treated with brequinar, a known DHODH inhibitor. Supplementary Table S3 shows the summary of enriched proteins identified in PTC299 pull-down samples. Supplementary Table S4 shows PTC299 species selectivity: inhibition of de novo UMP. Supplementary Table S5 shows the preclinical safety profile of PTC299. Supplementary Table S6 shows the demographic information for patients in the NF-2 clinical trial. Supplementary Table S7 shows the differences in expression of genes related to pyrimidine nucleotide synthesis and salvage pathway in hematopoietic cancers versus solid tumors.

contains the raw data from the Microarrays study in the PTC299-resistant HT1080 cells

Spinal muscular atrophy (SMA) is caused by the loss of the survival motor neuron 1 (SMN1) gene function. The related SMN2 gene partially compensates but produces insufficient levels of SMN protein due to alternative splicing of exon 7. Evrysdi (TM) (risdiplam), recently approved for the treatment of SMA, and related compounds promote exon 7 inclusion to generate full-length SMN2 mRNA and increase SMN protein levels. SMN Delta 7 type I SMA mice survive without treatment for similar to 17 days. SMN2 mRNA splicing modulators increase survival of SMN Delta 7 mice with treatment initiated at postnatal day 3 (PND3). To define SMN requirements for adult mice, SMN Delta 7 mice were dosed with an SMN2 mRNA splicing modifier from PND3 to PND40, then dosing was stopped. Mice not treated after PND40 showed progressive weight loss, necrosis, and muscle atrophy after similar to 20 days. Male mice presented a more severe phenotype than female mice. Mice dosed continuously did not show disease symptoms. The estimated half-life of SMN protein is 2 days indicating that the SMA phenotype reappeared after SMN protein levels returned to baseline. Although SMN protein levels decreased with age in mice and SMN protein levels were higher in brain than in muscle, our studies suggest that SMN protein is required throughout the life of the mouse and is especially essential in adult peripheral tissues including muscle. These studies indicate that drugs such as risdiplam will be optimally therapeutic when given as early as possible after diagnosis and potentially will be required for the life of an SMA patient.

Emvododstat was identified as a potent inhibitor of dihydroorotate dehydrogenase and is now in clinical development for the treatment of acute myeloid leukaemia and COVID-19. The objective of this paper is to evaluate the metabolism, pharmacokinetics, and drug interaction potentials of emvododstat.Emvododstat showed high binding to plasma protein with minimal distribution into blood cells in mouse, rat, dog, monkey, and human whole blood.O-Demethylation followed by glucuronidation appeared to be the major metabolic pathway in rat, dog, monkey, and human hepatocytes. CYP2C8, 2C19, 2D6, and 3A4 were involved in O-desmethyl emvododstat metabolite formation. Both emvododstat and O-desmethyl emvododstat inhibited CYP2D6 activity and induced CYP expression to different extents in vitro.Emvododstat and O-desmethyl emvododstat inhibited BCRP transporter activity but did not inhibit bile salt transporters and other efflux or uptake transporters. Neither emvododstat nor O-desmethyl emvododstat was a substrate for common efflux or uptake transporters investigated.Emvododstat is bioavailable in mice, rats, dogs, and monkeys following a single oral dose. The absorption was generally slow with the mean plasma Tmax ranging from 2 to 5 h; plasma exposure of O-desmethyl emvododstat was lower in rodents, but relatively higher in dogs and monkeys.

Huntington’s disease (HD) is a hereditary neurodegenerative disorder caused by expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats in the huntingtin ( HTT ) gene. Consequently, the mutant protein is ubiquitously expressed and drives pathogenesis of HD through a toxic gain-of-function mechanism. Animal models of HD have demonstrated that reducing huntingtin (HTT) protein levels alleviates motor and neuropathological abnormalities. Investigational drugs aim to reduce HTT levels by repressing HTT transcription, stability or translation. These drugs require invasive procedures to reach the central nervous system (CNS) and do not achieve broad CNS distribution. Here, we describe the identification of orally bioavailable small molecules with broad distribution throughout the CNS, which lower HTT expression consistently throughout the CNS and periphery through selective modulation of pre-messenger RNA splicing. These compounds act by promoting the inclusion of a pseudoexon containing a premature termination codon (stop-codon psiExon), leading to HTT mRNA degradation and reduction of HTT levels.

Background Huntington’s disease (HD) is a hereditary neurodegenerative disorder caused by expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats in the huntingtin gene (HTT). Consequently, the mutant protein is ubiquitously expressed and drives HD pathogenesis via a toxic gain-of-function mechanism.1–3 HD animal models demonstrate that reducing huntingtin protein (HTT) levels alleviates motor and neuropathological abnormalities, supporting HTT lowering as a therapeutic approach2. Clinical and preclinical stage modalities including antisense oligonucleotides, virally delivered microRNAs, and zinc finger transcription factors, reduce HTT levels by repressing HTT transcription, stability and/or translation.1 2 Such modalities require invasive procedures to reach the central nervous system (CNS) and are not evenly distributed. These compounds act via a novel mechanism promoting the inclusion of a pseudoexon containing a premature termination codon, leading to HTT messenger RNA (mRNA) degradation and reduction of HTT levels. Aims We aimed to develop a class of small molecule splicing modifiers specifically synthesised to promote selective splicing and ultimately reduction in huntingtin mRNA and protein levels. Methods/Techniques Here, we describe the identification of small molecule splicing modifiers lowering HTT expression by selective modulation of the critical recognition step of pre-mRNA splicing. Results/Outcome These data demonstrate the potential of small molecules that effectively lower HTT consistently throughout the CNS and periphery to be a non-invasive effective treatment option for patients. Conclusions This work represents the first example of the identification and optimisation of orally bioavailable splicing modifiers that penetrate all tissues and lower HTT evenly throughout the body. References Wild EJ, Tabrizi SJ. Therapies targeting DNA and RNA in huntington’s disease. Lancet Neurol 2017;16:837–847. Tabrizi SJ, Ghosh R, Leavitt BR. Huntingtin lowering strategies for disease modification in huntington’s disease. Neuron 2019;101:801–819. Nopoulos PC. Huntington disease: a single-gene degenerative disorder of the striatum. Dialogues Clin Neurosci 2016;18:91–98.

Ataluren is an aromatic acid derivative with a 1,2,4-oxodiazole moiety. Ataluren-O-1β-acyl glucuronide is a prominent circulatory metabolite in mice, rats, dogs, and humans following oral administration of ataluren. The objective of this paper was to evaluate the stability in vitro and in vivo of ataluren-O-1β-acyl glucuronide metabolite. Ultrahigh performance liquid chromatography-mass spectrometry methods were developed to separate and monitor ataluren-O-1β-acyl glucuronide and its possible migration isomers. In vitro stability was assessed in phosphate buffered saline as well as in control rat and human plasma. The disappearance of ataluren-O-1β-acyl glucuronide and the formation of migration isomers were monitored by the ultrahigh performance liquid chromatography-mass spectrometry methods. In vitro, ataluren-O-1β-acyl glucuronide underwent isomerization with an estimated half-life of approximately 1 h. However, ataluren-O-1β-acyl glucuronide was stable and was the only detectable acyl glucuronide following oral administration of ataluren in mice, rats, dogs, and humans using the same analytical methods. Ataluren acyl glucuronide in mouse, rat, dog, and human plasma could be hydrolyzed by β-glucuronidase, further confirming the structure of O-1β-acyl glucuronide. These results demonstrated that ataluren-O-1β-acyl glucuronide did not undergo migration in vivo. No clinical safety concern related to ataluren-O-1β-acyl glucuronide migration has been detected.

Abstract PTC299 was identified as an inhibitor of VEGFA mRNA translation in a phenotypic screen and evaluated in the clinic for treatment of solid tumors. To guide precision cancer treatment, we performed extensive biological characterization of the activity of PTC299 and demonstrated that inhibition of VEGF production and cell proliferation by PTC299 is linked to a decrease in uridine nucleotides by targeting dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme for de novo pyrimidine nucleotide synthesis. Unlike previously reported DHODH inhibitors that were identified using in vitro enzyme assays, PTC299 is a more potent inhibitor of DHODH in isolated mitochondria suggesting that mitochondrial membrane lipid engagement in the DHODH conformation in situ is required for its optimal activity. PTC299 has broad and potent activity against hematologic cancer cells in preclinical models, reflecting a reduced pyrimidine nucleotide salvage pathway in leukemia cells. Archived serum samples from patients treated with PTC299 demonstrated increased levels of dihydroorotate, the substrate of DHODH, indicating target engagement in patients. PTC299 has advantages over previously reported DHODH inhibitors, including greater potency, good oral bioavailability, and lack of off-target kinase inhibition and myelosuppression, and thus may be useful for the targeted treatment of hematologic malignancies.

Current anti-VEGF (Vascular Endothelial Growth Factor A) therapies to treat various cancers indiscriminately block VEGF function in the patient resulting in the global loss of VEGF signaling which has been linked to dose-limiting toxicities as well as treatment failures due to acquired resistance. Accumulating evidence suggests that this resistance is at least partially due to increased production of compensatory tumor angiogenic factors/cytokines. VEGF protein production is differentially controlled depending on whether cells are in the normal “homeostatic” state or in a stressed state, such as hypoxia, by post-transcriptional regulation imparted by elements in the 5’ and 3’ untranslated regions (UTR) of the VEGF mRNA. Using the Gene Expression Modulation by Small molecules (GEMS™) phenotypic assay system, we performed a high throughput screen to identify low molecular weight compounds that target the VEGF mRNA UTR-mediated regulation of stress-induced VEGF production in tumor cells. We identified a number of compounds that potently and selectively reduce endogenous VEGF production under hypoxia in HeLa cells. Medicinal chemistry efforts improved the potency and pharmaceutical properties of one series of compounds resulting in the discovery of PTC-510 which inhibits hypoxia-induced VEGF expression in HeLa cells at low nanomolar concentration. In mouse xenograft studies, oral administration of PTC-510 results in marked reduction of intratumor VEGF production and single agent control of tumor growth without any evident toxicity. Here, we show that selective suppression of stress-induced VEGF production within tumor cells effectively controls tumor growth. Therefore, this approach may minimize the liabilities of current global anti-VEGF therapies.

The underlying cause of spinal muscular atrophy (SMA) is a deficiency of the survival motor neuron (SMN) protein. Starting from hits identified in a high-throughput screening campaign and through structure-activity relationship investigations, we have developed small molecules that potently shift the alternative splicing of the SMN2 exon 7, resulting in increased production of the full-length SMN mRNA and protein. Three novel chemical series, represented by compounds 9, 14, and 20, have been optimized to increase the level of SMN protein by >50% in SMA patient-derived fibroblasts at concentrations of <160 nM. Daily administration of these compounds to severe SMA Δ7 mice results in an increased production of SMN protein in disease-relevant tissues and a significant increase in median survival time in a dose-dependent manner. Our work supports the development of an orally administered small molecule for the treatment of patients with SMA.

Spinal muscular atrophy (SMA) is caused by defects in the survival motor neuron 1 (SMN1) gene that encodes survival motor neuron (SMN) protein. The majority of therapeutic approaches currently in clinical development for SMA aim to increase SMN protein expression and there is a need for sensitive methods able to quantify increases in SMN protein levels in accessible tissues. We have developed a sensitive electrochemiluminescence (ECL)-based immunoassay for measuring SMN protein in whole blood with a minimum volume requirement of 5μL. The SMN-ECL immunoassay enables accurate measurement of SMN in whole blood and other tissues. Using the assay, we measured SMN protein in whole blood from SMA patients and healthy controls and found that SMN protein levels were associated with SMN2 copy number and were greater in SMA patients with 4 copies, relative to those with 2 and 3 copies. SMN protein levels did not vary significantly in healthy individuals over a four-week period and were not affected by circadian rhythms. Almost half of the SMN protein was found in platelets. We show that SMN protein levels in C/C-allele mice, which model a mild form of SMA, were high in neonatal stage, decreased in the first few weeks after birth, and then remained stable throughout the adult stage. Importantly, SMN protein levels in the CNS correlated with SMN levels measured in whole blood of the C/C-allele mice. These findings have implications for the measurement of SMN protein induction in whole blood in response to SMN-upregulating therapy.

Abstract The Polycomb group (PcG) transcription repressor BMI1 is highly expressed in human cancers and is required for the clonogenic self-renewal and tumorigenesis of human cancer cells including those in hematological cancer and neuroblastoma. PTC596 is efficacious in vivo across a range of xenograft tumor models, including models of glioblastoma, fibrosarcoma and leukemia as well as orthotopic models of GBM. With EC50 values of 30-200 nM in a variety of tumor cell lines, PTC596 selectively reduces the level of functional BMI1 protein resulting in the depletion of the tumor stem cell fraction. PTC596 induces the hyper-phosphorylation of BMI1 leading to its degradation and the reduction of polycomb repressive complex 1 (PRC1) activity. Mechanistic studies suggest that PTC596 inhibits APC/CCDC20 activity resulting in the persistent activation of CDK1 and CDK2 which mediate the hyperphosphorylation of BMI1. Studies are ongoing to elucidate the mechanism of PTC596 inhibition of APC/CCDC20 and its preferential depletion of the tumor stem cell fraction. Citation Format: Min Jung Kim, Liangxian Cao, Josephine Sheedy, Nicole Risher, Melissa Dumble, Chang-Sun Lee, Nadiya Sydorenko, Ramil Baiazitov, Wu Du, Young-Choon Moon, Marla L. Weetall, Joseph Colacino, Thomas W. Davis. PTC596-induced Bmi1 hyper-phosphorylation via Cdk1/2 activation resulting in tumor stem cell depletion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5517. doi:10.1158/1538-7445.AM2014-5517

Spinal muscular atrophy (SMA) is a genetic disease caused by mutation or deletion of the survival of motor neuron 1 (SMN1) gene. A paralogous gene in humans, SMN2, produces low, insufficient levels of functional SMN protein due to alternative splicing that truncates the transcript. The decreased levels of SMN protein lead to progressive neuromuscular degeneration and high rates of mortality. Through chemical screening and optimization, we identified orally available small molecules that shift the balance of SMN2 splicing toward the production of full-length SMN2 messenger RNA with high selectivity. Administration of these compounds to Δ7 mice, a model of severe SMA, led to an increase in SMN protein levels, improvement of motor function, and protection of the neuromuscular circuit. These compounds also extended the life span of the mice. Selective SMN2 splicing modifiers may have therapeutic potential for patients with SMA.

A novel series of 6-(indol-2-yl)pyridine-3-sulfonamides was prepared and evaluated for their ability to inhibit HCV RNA replication in the HCV replicon cell culture assay. Preliminary optimization of this series furnished compounds with low nanomolar potency against the HCV genotype 1b replicon. Among these, compound 8c has identified as a potent HCV replicon inhibitor (EC50=4 nM) with a selectivity index with respect to cellular GAPDH of more than 2500. Further, compound 8c had a good pharmacokinetic profile in rats with an IV half-life of 6h and oral bioavailability (F) of 62%. Selection of HCV replicon resistance identified an amino acid substitution in HCV NS4B that confers resistance to these compounds. These compounds hold promise as a new chemotype with anti-HCV activity mediated through an underexploited viral target.

Spinal muscular atrophy (SMA) is caused by the reduced expression of the survival of motor neuron (SMN) protein due to the loss of functional SMN1 gene and alternative splicing of exon 7 in the SMN2 gene. We are pursuing innovative drug discovery strategies aimed at restoring the production of the SMN protein by modulating SMN2 alternative splicing. Panels of cell based assays and animal models have been established and optimized to assess the effects of compounds on the splicing of SMN mRNA and the production and function of SMN protein. Using these assays, small molecules have been identified and developed that increase the inclusion of exon 7 into SMN2 mRNA and efficiently correct the splicing defect of SMN2. As a result of the increase of exon 7 inclusion, SMN protein level in SMA patient cell lines and mouse models is elevated by several fold and can even exceed that in healthy SMA carriers. These small molecules extend the lifespan of severely affected delta7 SMA mice tenfold (>150days) and result in striking gains in motor function relative to untreated mice that live an average of 14days. Moreover, these molecules demonstrate efficacy when treatment of delta7 SMA mice is initiated after disease onset. Lead compounds from this program are undergoing further characterization and chemical optimization with the ultimate goal of identifying molecules for preclinical and clinical development.

Hepatocellular carcinoma (HCC) is a major health burden worldwide for its high incidence and mortality. Osteopontin (OPN) is a chemokine-like, matricellular phosphoglycoprotein whose expression is elevated in various types of cancer including HCC. OPN has been shown to be involved in tumorigenesis, chemo-resistance, metastasis and sustaining stem-like properties of cancer cells. Autophagy is a cellular process by which cytoplasmic components are degraded and recycled for maintaining cellular homeostasis. There is increasing evidence supports that autophagy plays a critical role for stem-like properties and chemo-resistance of cancer cells. However, the relationship between OPN and autophagy in maintaining cancer stem-like properties and chemo-resistance is yet to be clarified. Herein, we found that secreted OPN induced autophagy via binding with its receptor integrin αvβ3 and sustaining FoxO3a stability. OPN-elicited autophagy could promote cancer cell survival and resistance to chemotherapy drugs, as well as stem-like properties. Our findings indicated that OPN was capable of promoting chemo-resistance of HCCs via autophagy, which might provide a new strategy for the treatment of HCC.

PTC124: a no-nonsense drug Many inherited diseases result from premature termination during translation of a messenger RNA into protein; one such disease is muscular dystrophy. Welch et al . now report that a small molecule, PTC124, enables the translation machinery to bypass sites that cause premature termination, but still terminate normally at the end of the mRNA. In human and mouse cells, this drug restores normal translation of the gene that is mutated in muscular dystrophy, and it restores muscle function in the mdx mouse model for the human disease. This work offers the hope that similar drugs might be used to target nonsense mutations and restore protein function in a wide variety of diseases. PTC124 is now undergoing clinical trials in muscular dystrophy and cystic fibrosis patients.