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Thermodynamic studies on the interactions between intercalator-neomycin conjugates and a DNA polynucleotide triplex [poly(dA)•2poly(dT)] were conducted. To draw a complete picture of such interactions, naphthalenedimide-neomycin (3) and anthraquinone-neomycin (4) were synthesized and used together with two other analogues, previously synthesized pyrene-neomycin (1) and BQQ-neomycin (2), in our investigations. A combination of experiments including UV denaturation, circular dichroism (CD) titration, differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC) revealed that all four conjugates (1–4) stabilized poly(dA)•2poly(dT) much greater than its parent compound, neomycin. UV melting experiments clearly showed that the temperature (Tm3→2) at which poly(dA)•2poly(dT) dissociated into poly(dA)•poly(dT) and poly(dT) increased dramatically (> 12 °C) in the presence of intercalator-neomycin (1–4) even at a very low concentration (2 µM). In contrast to intercalator-neomycin conjugates, the increment of Tm3→2 of poly(dA)•2poly(dT) induced by neomycin was negligible under the same conditions. The binding preference of intercalator-neomycin (1–4) to poly(dA)•2poly(dT) was also confirmed by competition dialysis and fluorescent intercalator displacement assay. Circular dichroism titration studies revealed that compound 1–4 had slightly larger binding site size (~7–7.5) with poly(dA)•2poly(dT) as compared to neomycin (~6.5). The thermodynamic parameters of these intercalator-neomycin conjugates with poly(dA)•2poly(dT) were derived from an integrated van’t Hoff equation using the Tm3→2 values, the binding site size numbers, and other parameters obtained from DSC and ITC. The binding affinity of all tested ligands with poly(dA)•2poly(dT) increased in the order neomycin < 1 < 3 < 4 < 2. Amongst them, the binding constant [(2.7 ± 0.3) × 108 M−1] of 2 with poly(dA)•2poly(dT) was the highest, almost 1000 fold more than that of neomycin. The binding of compounds 1–4 with poly(dA)•2poly(dT) was mostly enthalpy—driven and gave negative ΔCp values. The results described here suggest that the binding affinity of intercalator-neomycin conjugates to poly(dA)•2poly(dT) increases as a function of the surface area of the intercalator moiety.

Edited by Hans EklundKeywords: RNA recognition Aminoglycoside Neomycin Poly(A) DNA RNA a b s t r a c t Poly(A) is a relevant sequence in cell biology due to its importance in mRNA stability and translation initiation. Neomycin is an aminoglycoside antibiotic that is well known for its ability to target various nucleic acid structures. Here it is reported that neomycin is capable of binding tightly to a single-stranded oligonucleotide (A 30 ) with a K d in the micromolar range. CD melting experiments support complex formation and indicate a melting temperature of 47°C. The poly(A) duplex, which melts at 44°C (pH 5.5), was observed to melt at 61°C in the presence of neomycin, suggesting a strong stabilization of the duplex by the neomycin.

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Multi-membered N-heterocycles R 0690Array Synthesis of Progesterone Receptor Antagonists: 3-Aryl-1,2-diazepines.-A practical, two-step array synthesis of new 3-aryl-1,2-diazepines employs solid-supported reagents. -(WIETHE, R. W.; STEWART, E. L.; DREWRY, D. H.; GRAY, D. W.; MEHBOB, A.; HOEKSTRA*, W.

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Myristoylation is the N-terminal modification of proteins with the fatty acid myristate. This process is mediated by two ubiquitously expressed N-myristoyltransferases, NMT1 and NMT2, and is critical for membrane targeting and cell signaling. Because NMT expression is increased in some cancers, we used three robotic screens to evaluate the potential of the potent pan-NMT inhibitor PCLX-001 on 300 cancer cell lines spanning the spectrum of human cancers. We discovered a marked increase in the sensitivity of hematological cancer cell lines, including B-cell lymphomas, to myristoylation inhibition. PCLX-001 consistently reduced both lymphoma cell proliferation and viability at concentrations lower than those needed to inhibit the growth of or to kill benign immortalized B cells. In lymphoma cell lines, PCLX-001 treatment inhibited early B-cell receptor (BCR) signaling events by disrupting membrane targeting of several myristoylated Src family kinases and promoted their ubiquitin-mediated degradation. Unexpectedly, PCLX-001 also promoted the degradation of non-myristoylated transcriptional activators P-ERK, c-Myc, NFκB and CREB downstream in the BCR signaling cascade, leading to loss of survival signals and apoptosis. Furthermore, compared to clinically approved drugs dasatinib and ibrutinib, PCLX-001 was more potent in vitro at inhibiting B-cell signaling, had a wider breadth of efficacy, and had greater selectivity thus sparing normal B cells. PCLX-001 treatment reduced tumor size in a time and concentration dependent manner in three B-cell lymphoma xenograft models and resulted in complete disease regression in two of these models, including an R-CHOP refractory lymphoma patient-derived xenograft. To investigate the potential mechanisms responsible for the sensitivity of hematological cancers to PCLX-001, we examined the NMT expression levels in cancer cells using publically available databases. Contrary to the reported NMT overexpression in some cancers, we found that hematological cancer cell lines and tumors both display significant reduction in NMT2 expression. The decreased NMT2 expression is significantly correlated with lower EC50 and poorer patient prognosis. Using the CRISPR-based genetic alteration Cancer Dependency Map, we discovered that cancer cells are highly dependent on functional NMT1, and that NMT1 dependency increases with low NMT2 expression. PCLX-001 treatment may mimic the effect of genetic alteration of NMT1 in hematological cancer cells low in NMT2 by pharmacologically inhibiting the remaining NMT1 in these cells. This results in an effect reminiscent of synthetic lethality since the vast majority of normal cells express both NMTs and PCLX-001 selectively kills NMT2-deficient cancer cells while sparing normal cells. Our findings support the ongoing development and eventual clinical trials of PCLX-001 as a therapy for hematological cancers. Citation Format: Erwan Beauchamp, Megan C. Yap, Maneka A. Perinpanayagam, Jay M. Gamma, Krista M. Vincent, Raymond Lai, Wei-Feng Dong, Manikandan Lakshmanan, Anandhkumar Raju, Vinay Tergaonkar, Soo Yong Tan, Soon Thye Lim, Lynne Postovit, Kevin D. Read, David W. Gray, Paul G. Wyatt, John R. Mackey, Luc G. Berthiaume. Targeting N-myristoylation in B-cell lymphomas as a therapeutic strategy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5156.