Важливе історико-географічне дослідження

Рец. на кн. Темушева В.Н. "Гомельская земля в конце XV первой половине XVI в. Территориальные трансформации в пограничном регионе". — М.: "Квадрига", 2009. — 190 с.Review of the book: Temushev V.N. "Gomel Land in the Late 15th — the 1st half of the 16th Centuries. Territorial Transformations in the Frontier Area". — Moscow: "Kvadriga", 2009. — 190 p

Inhibitors of Guanine and Adenine/Adenosine Deaminases Based On Ring-Expanded Purine Structural Skeleton

The work described in this dissertation is a continuation of decades of research conducted in this laboratory on ring-expanded heterocycles and nucleosides as inhibitors of guanine- and adenine/adenosine deaminases, the key members of the deaminase family of enzymes of nucleic acid metabolism. The dissertation consists of two parts: Part-I: Synthesis and Biochemical Studies of Ring Expanded Purine Analogues Containing the 5:7-Fused Imidazo[4,5-e][1,4]diazepine Ring System as inhibitors of Guanine Deaminase, and Part-II: Development of Synthetic Strategies for Ring-Expanded Purine Analogues Containing the 5:8-Fused Imidazo[4,5-f][1,4]diazocine Ring System as Potential Inhibitors of Adenine/Adenosine Deaminases. Part-I of the dissertation is based on a hypothesis that azepinomycin, a purported transition state analogue inhibitor of guanase, does not represent the transition state of the enzyme-catalyzed reaction as closely as does iso-azepinomycin, wherein the 6-hydroxy group of azepinomycin has been translocated to the 5-position. This is because the hydrolysis involves the C2 and N3 (imine N) atoms rather than C2 and N1 (lactan N) of guanine. Therefore, an aminol intermediate formed by hydrolysis of an imine precursor would be better represented by iso-azepinomycin than azepinomycin. Based on this hypothesis, and assuming that iso-azepinomycin would bind to guanase at the same active site as azepinomycin, several analogues of iso-azepinomycin were designed and successfully synthesized in order to gain a preliminary understanding of the hydrophobic and hydrophilic sites surrounding the guanase binding site of the ligand. Specifically, the analogues were designed to explore the hydrophobic or hydrophilic pockets, if any, in the vicinity of N1, N3, and N4 nitrogen atoms as well as O5 oxygen atom of iso-azepinomycin. Biochemical inhibition studies of these analogues were performed using a mammalian guanase. Our results indicate that (1) increasing the hydrophobicity near O5 results in a negative effect, (2) translocating the hydrophobicity from N3 to N1 also results in decreased inhibition, (3) increasing the hydrophobicity near N4 produces significant enhancement of inhibition, (4) increasing the hydrophobicity at both N4 and O5 considerably brings down the inhibition, and (5) finally, increasing the hydrophilic character near N3 has a deleterious effect on inhibition. Part-II of this dissertation work is geared toward developing synthetic strategies for ring-expanded heterocycles and nucleosides containing the described 5:8-fused ring systems. Most of the analogues investigated in the Hosmane lab so far include ring-expanded heterocycles and nucleosides containing the 5:7-fused ring systems. Little, if anything, is known about the effect of further increasing the size of the 7-membered diazepine ring to the 8-membered diazocine ring to form the 5:8-fused ring systems. Our molecular modeling studies with a hypothetical compound containing the 5:8-fused imidazo[4,5-f][1,4]diazocine ring system revealed that a number of hydrophobic amino acid residues fall near the zinc co-ordination site of ADA. Therefore, ring expansion from the 7-membered diazepine ring to the 8-membered diazocine ring with an extended alkyl chain is anticipated to enhance hydrophobic interactions with the enzyme. Furthermore, the ring expansion from 7 to 8 does not seem to adversely affect the interactions of the heterocycle with the protein. I have successfully synthesized the target parent 5:8-fused heterocyclic ring system as well as its 1-benzyl and 3-benzyl analogues. In addition, the desired nucleoside precursor for the final ring-annulation has also been successfully synthesized. These analogues pave way into the new territory of ring-expanded heterocyclic and nucleoside analogues containing the 5:8-fused imidazodiazocine ring systems

Inhibitors of Guanine and Adenine/Adenosine Deaminases Based On Ring-Expanded Purine Structural Skeleton

The work described in this dissertation is a continuation of decades of research conducted in this laboratory on ring-expanded heterocycles and nucleosides as inhibitors of guanine- and adenine/adenosine deaminases, the key members of the deaminase family of enzymes of nucleic acid metabolism. The dissertation consists of two parts: Part-I: Synthesis and Biochemical Studies of Ring Expanded Purine Analogues Containing the 5:7-Fused Imidazo[4,5-e][1,4]diazepine Ring System as inhibitors of Guanine Deaminase, and Part-II: Development of Synthetic Strategies for Ring-Expanded Purine Analogues Containing the 5:8-Fused Imidazo[4,5-f][1,4]diazocine Ring System as Potential Inhibitors of Adenine/Adenosine Deaminases. Part-I of the dissertation is based on a hypothesis that azepinomycin, a purported transition state analogue inhibitor of guanase, does not represent the transition state of the enzyme-catalyzed reaction as closely as does iso-azepinomycin, wherein the 6-hydroxy group of azepinomycin has been translocated to the 5-position. This is because the hydrolysis involves the C2 and N3 (imine N) atoms rather than C2 and N1 (lactan N) of guanine. Therefore, an aminol intermediate formed by hydrolysis of an imine precursor would be better represented by iso-azepinomycin than azepinomycin. Based on this hypothesis, and assuming that iso-azepinomycin would bind to guanase at the same active site as azepinomycin, several analogues of iso-azepinomycin were designed and successfully synthesized in order to gain a preliminary understanding of the hydrophobic and hydrophilic sites surrounding the guanase binding site of the ligand. Specifically, the analogues were designed to explore the hydrophobic or hydrophilic pockets, if any, in the vicinity of N1, N3, and N4 nitrogen atoms as well as O5 oxygen atom of iso-azepinomycin. Biochemical inhibition studies of these analogues were performed using a mammalian guanase. Our results indicate that (1) increasing the hydrophobicity near O5 results in a negative effect, (2) translocating the hydrophobicity from N3 to N1 also results in decreased inhibition, (3) increasing the hydrophobicity near N4 produces significant enhancement of inhibition, (4) increasing the hydrophobicity at both N4 and O5 considerably brings down the inhibition, and (5) finally, increasing the hydrophilic character near N3 has a deleterious effect on inhibition. Part-II of this dissertation work is geared toward developing synthetic strategies for ring-expanded heterocycles and nucleosides containing the described 5:8-fused ring systems. Most of the analogues investigated in the Hosmane lab so far include ring-expanded heterocycles and nucleosides containing the 5:7-fused ring systems. Little, if anything, is known about the effect of further increasing the size of the 7-membered diazepine ring to the 8-membered diazocine ring to form the 5:8-fused ring systems. Our molecular modeling studies with a hypothetical compound containing the 5:8-fused imidazo[4,5-f][1,4]diazocine ring system revealed that a number of hydrophobic amino acid residues fall near the zinc co-ordination site of ADA. Therefore, ring expansion from the 7-membered diazepine ring to the 8-membered diazocine ring with an extended alkyl chain is anticipated to enhance hydrophobic interactions with the enzyme. Furthermore, the ring expansion from 7 to 8 does not seem to adversely affect the interactions of the heterocycle with the protein. I have successfully synthesized the target parent 5:8-fused heterocyclic ring system as well as its 1-benzyl and 3-benzyl analogues. In addition, the desired nucleoside precursor for the final ring-annulation has also been successfully synthesized. These analogues pave way into the new territory of ring-expanded heterocyclic and nucleoside analogues containing the 5:8-fused imidazodiazocine ring systems

Role of DDX3 in the pathogenesis of inflammatory bowel disease

When crypt stem cells of the gastrointestinal tract become injured, the result is increased synthesis of pro-inflammatory cytokines and matrix metalloproteinases by their progeny - the colonic epithelium. Chronic inflammation of the gastrointestinal tract is a characteristic of inflammatory bowel disease, which includes Crohn's Disease and Ulcerative Colitis. In our ongoing investigation to decipher the characteristic functions of a RNA helicase gene, DDX3, we identified high DDX3 expression by immunohistochemistry of colon biopsy samples, which included chronic/mild Morbus Crohn, active Morbus Crohn, Chronic/mild Colitis Ulcerosa and active Colitis Ulcerosa in epithelium and stromal compartments. We used a small molecule inhibitor of DDX3, RK-33, on two human colonic epithelial cell lines, HCEC1CT and HCEC2CT and found that RK-33 was able to decrease expression of MMP-1, MMP-2, MMP-3, and MMP-10. Moreover, forced differentiation of a human colonic cancer cell line, HT29, resulted in decreased DDX3 levels, indicating that DDX3 contributes to the modulation of colonic epithelium differentiation. In conclusion, our results revealed novel functions of DDX3 in inflammatory bowel disease and indicate a potential for using RK-33 as a systemic therapy to promote not only differentiation of transformed colonic epithelium but also to reduce MMP expression and thus elicit a decreased inflammatory response

Role of DDX3 in the pathogenesis of inflammatory bowel disease

When crypt stem cells of the gastrointestinal tract become injured, the result is increased synthesis of pro-inflammatory cytokines and matrix metalloproteinases by their progeny - the colonic epithelium. Chronic inflammation of the gastrointestinal tract is a characteristic of inflammatory bowel disease, which includes Crohn's Disease and Ulcerative Colitis. In our ongoing investigation to decipher the characteristic functions of a RNA helicase gene, DDX3, we identified high DDX3 expression by immunohistochemistry of colon biopsy samples, which included chronic/mild Morbus Crohn, active Morbus Crohn, Chronic/mild Colitis Ulcerosa and active Colitis Ulcerosa in epithelium and stromal compartments. We used a small molecule inhibitor of DDX3, RK-33, on two human colonic epithelial cell lines, HCEC1CT and HCEC2CT and found that RK-33 was able to decrease expression of MMP-1, MMP-2, MMP-3, and MMP-10. Moreover, forced differentiation of a human colonic cancer cell line, HT29, resulted in decreased DDX3 levels, indicating that DDX3 contributes to the modulation of colonic epithelium differentiation. In conclusion, our results revealed novel functions of DDX3 in inflammatory bowel disease and indicate a potential for using RK-33 as a systemic therapy to promote not only differentiation of transformed colonic epithelium but also to reduce MMP expression and thus elicit a decreased inflammatory response

PLGA nanoparticle formulation of RK-33 an RNA helicase inhibitor against DDX3

BACKGROUND: The DDX3 helicase inhibitor RK-33 is a newly developed anticancer agent that showed promising results in preclinical research (Bol et al. EMBO Mol Med, 7(5):648-649, 2015). However, due to the physicochemical and pharmacological characteristics of RK-33, we initiated development of alternative formulations of RK-33 by preparing sustained release nanoparticles that can be administered intravenously. METHODS: In this study, RK-33 was encapsulated in poly(lactic-co-glycolic acid) (PLGA), one of the most well-developed biodegradable polymers, using the emulsion solvent evaporation method. RESULTS: Hydrodynamic diameter of RK-33-PLGA nanoparticles was about 245 nm with a negative charge, and RK-33-PLGA nanoparticles had a payload of 1.4 % RK-33. RK-33 was released from the PLGA nanoparticles over 7 days (90 ± 5.7 % released by day 7) and exhibited cytotoxicity to human breast carcinoma MCF-7 cells in a time-dependent manner. Moreover, RK-33-PLGA nanoparticles were well tolerated, and systemic retention of RK-33 was markedly improved in normal mice. CONCLUSIONS: PLGA nanoparticles have a potential as a parenteral formulation of RK-33

PLGA nanoparticle formulation of RK-33 an RNA helicase inhibitor against DDX3

BACKGROUND: The DDX3 helicase inhibitor RK-33 is a newly developed anticancer agent that showed promising results in preclinical research (Bol et al. EMBO Mol Med, 7(5):648-649, 2015). However, due to the physicochemical and pharmacological characteristics of RK-33, we initiated development of alternative formulations of RK-33 by preparing sustained release nanoparticles that can be administered intravenously. METHODS: In this study, RK-33 was encapsulated in poly(lactic-co-glycolic acid) (PLGA), one of the most well-developed biodegradable polymers, using the emulsion solvent evaporation method. RESULTS: Hydrodynamic diameter of RK-33-PLGA nanoparticles was about 245 nm with a negative charge, and RK-33-PLGA nanoparticles had a payload of 1.4 % RK-33. RK-33 was released from the PLGA nanoparticles over 7 days (90 ± 5.7 % released by day 7) and exhibited cytotoxicity to human breast carcinoma MCF-7 cells in a time-dependent manner. Moreover, RK-33-PLGA nanoparticles were well tolerated, and systemic retention of RK-33 was markedly improved in normal mice. CONCLUSIONS: PLGA nanoparticles have a potential as a parenteral formulation of RK-33

Targeting DDX3 in Medulloblastoma Using the Small Molecule Inhibitor RK-33

Medulloblastoma is the most common malignant tumor that arises from the cerebellum of the central nervous system. Clinically, medulloblastomas are treated by surgery, radiation, and chemotherapy, all of which result in toxicity and morbidity. Recent reports have identified that DDX3, a member of the RNA helicase family, is mutated in medulloblastoma. In this study, we demonstrate the role of DDX3 in driving medulloblastoma. With the use of a small molecule inhibitor of DDX3, RK-33, we could inhibit growth and promote cell death in two medulloblastoma cell lines, DAOY and UW228, with IC50 values of 2.5 μM and 3.5 μM, respectively. Treatment of DAOY and UW228 cells with RK-33 caused a G1 arrest, resulted in reduced TCF reporter activity, and reduced mRNA expression levels of downstream target genes of the WNT pathway, such as Axin2, CCND1, MYC, and Survivin. In addition, treatment of DAOY and UW228 cells with a combination of RK-33 and radiation exhibited a synergistic effect. Importantly, the combination of RK-33 and 5 Gy radiation caused tumor regression in a mouse xenograft model of medulloblastoma. Using immunohistochemistry, we observed DDX3 expression in both pediatric (55%) and adult (66%) medulloblastoma patients. Based on these results, we conclude that RK-33 is a promising radiosensitizing agent that inhibits DDX3 activity and down-regulates WNT/β-catenin signaling and could be used as a frontline therapeutic strategy for DDX3-expressing medulloblastomas in combination with radiation

Global Effects of DDX3 Inhibition on Cell Cycle Regulation Identified by a Combined Phosphoproteomics and Single Cell Tracking Approach

DDX3 is an RNA helicase with oncogenic properties. The small molecule inhibitor RK-33 is designed to fit into the ATP binding cleft of DDX3 and hereby block its activity. RK-33 has shown potent activity in preclinical cancer models. However, the mechanism behind the antineoplastic activity of RK-33 remains largely unknown. In this study we used a dual phosphoproteomic and single cell tracking approach to evaluate the effect of RK-33 on cancer cells. MDA-MB-435 cells were treated for 24 hours with RK-33 or vehicle control. Changes in phosphopeptide abundance were analyzed with quantitative mass spectrometry using isobaric mass tags (Tandem Mass Tags). At the proteome level we mainly observed changes in mitochondrial translation, cell division pathways and proteins related to cell cycle progression. Analysis of the phosphoproteome indicated decreased CDK1 activity after RK-33 treatment. To further evaluate the effect of DDX3 inhibition on cell cycle progression over time, we performed timelapse microscopy of Fluorescent Ubiquitin Cell Cycle Indicators labeled cells after RK-33 or siDDX3 exposure. Single cell tracking indicated that DDX3 inhibition resulted in a global delay in cell cycle progression in interphase and mitosis. In addition, we observed an increase in endoreduplication. Overall, we conclude that DDX3 inhibition affects cells in all phases and causes a global cell cycle progression delay

RK-33 radiosensitizes prostate cancer cells by blocking the RNA helicase DDX3

Despite advances in diagnosis and treatment, prostate cancer is the most prevalent cancer in males and the second highest cause of cancer-related mortality. We identified an RNA helicase gene, DDX3 (DDX3X), which is overexpressed in prostate cancers, and whose expression is directly correlated with high Gleason scores. Knockdown of DDX3 in the aggressive prostate cancer cell lines DU145 and 22Rv1 resulted in significantly reduced clonogenicity. To target DDX3, we rationally designed a small molecule, RK-33, which docks into the ATP-binding domain of DDX3. Functional studies indicated that RK-33 preferentially bound to DDX3 and perturbed its activity. RK-33 treatment of prostate cancer cell lines DU145, 22Rv1, and LNCaP (which have high DDX3 levels) decreased proliferation and induced a G1 phase cell-cycle arrest. Conversely, the low DDX3-expressing cell line, PC3, exhibited few changes following RK-33 treatment. Importantly, combination studies using RK-33 and radiation exhibited synergistic effects both in vitro and in a xenograft model of prostate cancer demonstrating the role of RK-33 as a radiosensitizer. Taken together, these results indicate that blocking DDX3 by RK-33 in combination with radiation treatment is a viable option for treating locally advanced prostate cancer