Covalent inhibition of NSD1 histone methyltransferase.

The nuclear receptor-binding SET domain (NSD) family of histone methyltransferases is associated with various malignancies, including aggressive acute leukemia with NUP98-NSD1 translocation. While NSD proteins represent attractive drug targets, their catalytic SET domains exist in autoinhibited conformation, presenting notable challenges for inhibitor development. Here, we employed a fragment-based screening strategy followed by chemical optimization, which resulted in the development of the first-in-class irreversible small-molecule inhibitors of the nuclear receptor-binding SET domain protein 1 (NSD1) SET domain. The crystal structure of NSD1 in complex with covalently bound ligand reveals a conformational change in the autoinhibitory loop of the SET domain and formation of a channel-like pocket suitable for targeting with small molecules. Our covalent lead-compound BT5-demonstrates on-target activity in NUP98-NSD1 leukemia cells, including inhibition of histone H3 lysine 36 dimethylation and downregulation of target genes, and impaired colony formation in an NUP98-NSD1 patient sample. This study will facilitate the development of the next generation of potent and selective inhibitors of the NSD histone methyltransferases.

Synthesis, structure and cytotoxic activity of new acetylenic derivatives of betulin.

A new series of betulin derivatives containing one or two pharmacophores bearing an acetylenic and carbonyl function at the C-3 and/or C-28 positions has been synthesized and characterized by ¹H- and ¹³C-NMR, IR, MS and elemental analyses. The crystal structure of 28-O-propynoylbetulin was determined by X-ray structural analysis. All new compounds, as well as betulin, were tested in vitro for their antiproliferative activity against human SW707 colorectal, CCRF/CEM leukemia, T47D breast cancer, and against murine P388 leukemia and Balb3T3 normal fibroblasts cell lines. Most of the compounds showed better cytotoxicity than betulin and cisplatin used as reference agent. 28-O-Propynoylbetulin was the most potent derivative, being over 500 times more potent than betulin and about 100 times more cytotoxic than cisplatin against the human leukemia (CCRF/CEM) cell line, with an ID50 value of 0.02 µg/mL.

Anti-GD2 induced allodynia in rats can be reduced by pretreatment with DFMO.

BACKGROUND: Anti-GD2 therapy with dinutuximab is effective in improving the survival of high-risk neuroblastoma patients in remission and after relapse. However, allodynia is the major dose-limiting side effect, hindering its use for neuroblastoma patients at higher doses and for other GD2-expressing malignancies. As polyamines can enhance neuronal sensitization, including development of allodynia and other forms of pathological pain, we hypothesized that polyamine depletion might prove an effective strategy for relief of anti-GD2 induced allodynia. METHOD: Sprague-Dawley rats were allowed to drink water containing various concentrations of difluoromethylornithine (DFMO) for several days prior to behavioral testing. Anti-GD2 (14G2a) was injected into the tail vein of lightly sedated animals and basal mechanical hindpaw withdrawal threshold assessed by von Frey filaments. Endpoint serum DFMO and polyamines, assessed 24h after 14G2a injection, were measured by HPLC and mass spectrometry. RESULTS: An i.v. injection of 14G2a causes increased paw sensitivity to light touch in this model, a response that closely mimics patient allodynia. Animals allowed to drink water containing 1% DFMO exhibited a significant reduction of 14G2a-induced pain sensitivity (allodynia). Increasing the dosage of the immunotherapeutic increased the magnitude (intensity and duration) of the pain behavior. Administration of DFMO attenuated the enhanced sensitivity. Consistent with the known actions of DFMO on ornithine decarboxylase (ODC), serum putrescene and spermidine levels were significantly reduced by DFMO, though the decrease in endpoint polyamine levels did not directly correlate with the behavioral changes. CONCLUSIONS: Our results demonstrate that DFMO is an effective agent for reducing anti-GD2 -induced allodynia. Using DFMO in conjunction with dinutuximab may allow for dose escalation in neuroblastoma patients. The reduction in pain may be sufficient to allow new patient populations to utilize this therapy given the more acceptable side effect profile. Thus, DFMO may be an important adjunct to anti-GD2 immunotherapy in addition to a role as a potential anti-cancer therapeutic.

Menin inhibitor MI-3454 induces remission in MLL1-rearranged and NPM1-mutated models of leukemia.

The protein-protein interaction between menin and mixed lineage leukemia 1 (MLL1) plays a critical role in acute leukemias with translocations of the MLL1 gene or with mutations in the nucleophosmin 1 (NPM1) gene. As a step toward clinical translation of menin-MLL1 inhibitors, we report development of MI-3454, a highly potent and orally bioavailable inhibitor of the menin-MLL1 interaction. MI-3454 profoundly inhibited proliferation and induced differentiation in acute leukemia cells and primary patient samples with MLL1 translocations or NPM1 mutations. When applied as a single agent, MI-3454 induced complete remission or regression of leukemia in mouse models of MLL1-rearranged or NPM1-mutated leukemia, including patient-derived xenograft models, through downregulation of key genes involved in leukemogenesis. We also identified MEIS1 as a potential pharmacodynamic biomarker of treatment response with MI-3454 in leukemia, and demonstrated that this compound is well tolerated and did not impair normal hematopoiesis in mice. Overall, this study demonstrates, for the first time to our knowledge, profound activity of the menin-MLL1 inhibitor as a single agent in clinically relevant PDX models of leukemia. These data provide a strong rationale for clinical translation of MI-3454 or its analogs for leukemia patients with MLL1 rearrangements or NPM1 mutations.

Comparison of bead array and glass nanoreactor multi-analyte platforms for the evaluation of CNS and peripheral inflammatory markers during HIV infection.

While human immunodeficiency virus (HIV) infection has become a treatable disease with the development of combination antiretroviral therapy (cART), chronic inflammation that affects the central nervous system and other organs is still common. Reliable methods are needed to study HIV-associated inflammatory biomarkers. In this study involving both plasma and cerebrospinal fluid (CSF), we compared multiplex bead array (MBA) to a relatively new technology based on microfluidics and glass nanoreactor (GNR) technology for the measurement of three commonly studied markers from HIV-infected individuals. We found that results correlated between the two platforms for MCP-1 in both fluids as well as for plasma TNFα (all p < .005). However, results between the two platforms did not correlate for CSF TNFα or fractalkine from plasma or CSF. A statistically significant decrease in CSF TNFα over time (p < .0001) was only detectable with the MBA platform, and TNFα on the MBA was the only CSF biomarker to correlate with CSF HIV RNA (rho = 0.71, p < .0001). Meanwhile, the GNR platform was superior in terms of intra-assay fractalkine (FKN) variability and the detection of a significant FKN decrease over time. Additionally, the only significant correlation between blood biomarkers and plasma HIV RNA was with FKN on the GNR platform (rho = 0.38, p = .015). Given the variability in results between platforms, more research is needed on methods to quantitate HIV-associated inflammation.

Pharmacologic Inhibition of the Menin-MLL Interaction Leads to Transcriptional Repression of PEG10 and Blocks Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) accounts for approximately 85% of malignant liver tumors and results in 600,000 deaths each year, emphasizing the need for new therapies. Upregulation of menin was reported in HCC patients and high levels of menin correlate with poor patient prognosis. The protein-protein interaction between menin and histone methyltransferase mixed lineage leukemia 1 (MLL1) plays an important role in the development of HCC, implying that pharmacologic inhibition of this interaction could lead to new therapeutic strategy for the HCC patients. Here, we demonstrate that the menin-MLL inhibitor MI-503 shows antitumor activity in in vitro and in vivo models of HCC and reveals the potential mechanism of menin contribution to HCC. Treatment with MI-503 selectively kills various HCC cell lines and this effect is significantly enhanced by a combination of MI-503 with sorafenib, the standard-of-care therapy for HCC. Furthermore, MI-503 reduces sphere formation and cell migration in in vitro HCC models. When applied in vivo, MI-503 gives a strong antitumor effect both as a single agent and in combination with sorafenib in mice xenograft models of HCC. Mechanistically, treatment with MI-503 downregulates expression of several genes known to play a critical role in proliferation and migration of HCC cells, including PEG10, and displaces the menin-MLL1 complex from the PEG10 promoter, resulting in reduced H3K4 methylation and transcriptional repression. Overall, our studies reveal a mechanistic link between menin and genes involved in HCC and demonstrate that pharmacologic inhibition of the menin-MLL interaction might represent a promising therapeutic approach for HCC. Mol Cancer Ther; 17(1); 26-38. ©2017 AACR.

Complexity of Blocking Bivalent Protein-Protein Interactions: Development of a Highly Potent Inhibitor of the Menin-Mixed-Lineage Leukemia Interaction.

The protein-protein interaction between menin and mixed-lineage leukemia 1 (MLL1) plays an important role in development of acute leukemia with translocations of the MLL1 gene and in solid tumors. Here, we report the development of a new generation of menin-MLL1 inhibitors identified by structure-based optimization of the thienopyrimidine class of compounds. This work resulted in compound 28 (MI-1481), which showed very potent inhibition of the menin-MLL1 interaction (IC50 = 3.6 nM), representing the most potent reversible menin-MLL1 inhibitor reported to date. The crystal structure of the menin-28 complex revealed a hydrogen bond with Glu366 and hydrophobic interactions, which contributed to strong inhibitory activity of 28. Compound 28 also demonstrates pronounced activity in MLL leukemia cells and in vivo in MLL leukemia models. Thus, 28 is a valuable menin-MLL1 inhibitor that can be used for potential therapeutic applications and in further studies regarding the role of menin in cancer.

Oral Administration of Polymyxin B Modulates the Activity of Lipooligosaccharide E. coli B against Lung Metastases in Murine Tumor Models.

INTRODUCTION: Polymyxin B (PmB) belongs to the group of cyclic peptide antibiotics, which neutralize the activity of LPS by binding to lipid A. The aim of this study was to analyze the effect of PmB on the biological activity of lipooligosaccharide (LOS E. coli B,rough form of LPS) in vitro and in experimental metastasis models. RESULTS: Cultures of murine macrophage J774A.1 cells and murine bone marrow-derived dendritic cells (BM-DC) stimulated in vitro with LOS and supplemented with PmB demonstrated a decrease in inflammatory cytokine production (IL-6, IL-10, TNF-α) and down-regulation of CD40, CD80, CD86 and MHC class II molecule expression. Additionally, PmB suspended in drinking water was given to the C57BL/6 mice seven or five days prior to the intravenous injection of B16 or LLC cells and intraperitoneal application of LOS. This strategy of PmB administration was continued throughout the duration of the experiments (29 or 21 days). In B16 model, statistically significant decrease in the number of metastases in mice treated with PmB and LOS (p<0.01) was found on the 14th day of the experiments, whereas the most intensive changes in surface-antigen expression and ex vivo production of IL-6, IL-1ß and TNF-α by peritoneal cells were observed 7 days earlier. By contrast, antigen expression and ex vivo production of IL-6, IL-10, IFN-γ by splenocytes remained relatively high and stable. Statistically significant decrease in LLC metastases number was observed after the application of LOS (p<0.01) and in the group of mice preconditioned by PmB and subsequently treated with LOS (LOS + PmB, p<0.01). CONCLUSIONS: In conclusion, prolonged in vivo application of PmB was not able to neutralize the LOS-induced immune cell activity but its presence in the organism of treated mice was important in modulation of the LOS-mediated response against the development of metastases.

Property Focused Structure-Based Optimization of Small Molecule Inhibitors of the Protein-Protein Interaction between Menin and Mixed Lineage Leukemia (MLL).

Development of potent small molecule inhibitors of protein-protein interactions with optimized druglike properties represents a challenging task in lead optimization process. Here, we report synthesis and structure-based optimization of new thienopyrimidine class of compounds, which block the protein-protein interaction between menin and MLL fusion proteins that plays an important role in acute leukemias with MLL translocations. We performed simultaneous optimization of both activity and druglike properties through systematic exploration of substituents introduced to the indole ring of lead compound 1 (MI-136) to identify compounds suitable for in vivo studies in mice. This work resulted in the identification of compound 27 (MI-538), which showed significantly increased activity, selectivity, polarity, and pharmacokinetic profile over 1 and demonstrated a pronounced effect in a mouse model of MLL leukemia. This study, which reports detailed structure-activity and structure-property relationships for the menin-MLL inhibitors, demonstrates challenges in optimizing inhibitors of protein-protein interactions for potential therapeutic applications.

Pharmacologic inhibition of the Menin-MLL interaction blocks progression of MLL leukemia in vivo.

Chromosomal translocations affecting mixed lineage leukemia gene (MLL) result in acute leukemias resistant to therapy. The leukemogenic activity of MLL fusion proteins is dependent on their interaction with menin, providing basis for therapeutic intervention. Here we report the development of highly potent and orally bioavailable small-molecule inhibitors of the menin-MLL interaction, MI-463 and MI-503, and show their profound effects in MLL leukemia cells and substantial survival benefit in mouse models of MLL leukemia. Finally, we demonstrate the efficacy of these compounds in primary samples derived from MLL leukemia patients. Overall, we demonstrate that pharmacologic inhibition of the menin-MLL interaction represents an effective treatment for MLL leukemias in vivo and provide advanced molecular scaffold for clinical lead identification.

Efficient human breast cancer xenograft regression after a single treatment with a novel liposomal formulation of epirubicin prepared using the EDTA ion gradient method.

Liposomes act as efficient drug carriers. Recently, epirubicin (EPI) formulation was developed using a novel EDTA ion gradient method for drug encapsulation. This formulation displayed very good stability and drug retention in vitro in a two-year long-term stability experiment. The cryo-TEM images show drug precipitate structures different than ones formed with ammonium sulfate method, which is usually used to encapsulate anthracyclines. Its pharmacokinetic properties and its efficacy in the human breast MDA-MB-231 cancer xenograft model were also determined. The liposomal EPI formulation is eliminated slowly with an AUC of 7.6487, while the free drug has an AUC of only 0.0097. The formulation also had a much higher overall antitumor efficacy than the free drug.

Synthesis and cytotoxic activity of new betulin and betulinic acid esters with conjugated linoleic acid (CLA).

The synthesis of new ester derivatives of betulin (3a-c) and betulinic acid (4) with conjugated linoleic acid isomers (CLA; in a mixture of 43.4% 9c, 11t; 49.5% 10t, 12c; 7.1% other isomers) is presented. Esterification was carried out with N,N'-dicyclohexylcarbodiimide (DCC) as the coupling agent in the presence of 4-dimethylamino-pyridine (DMAP) in dichloromethane (or pyridine). The in vitro cytotoxic effect of betulin (1), betulinic acid (2), a mixture of CLA isomers and their derivatives (3a-c, 4) was examined using the MTT assay against four cancer cell lines (P388, CEM/C2, CCRF/CEM and HL-60) and the SRB assay on the HT-29 cell line. Ester 4 was the most active among the esters synthesized against the CEM/C2 cell line with an ID50 value 16.9 +/- 6.5 microg/mL. Betulin (1), betulinic acid (2) and CLA were the most active agents against the cancer cell lines studied.