Knockdown of deoxythymidylate kinase suppresses progression and epithelial-mesenchymal-transition of lung adenocarcinoma via STAT3 signaling.

Current research has shown that inhibiting deoxythymidylate kinase (DTYMK) can significantly reduce development of lung cancer without liver kinase B1. However, its underlying regulatory mechanism is still unclear. We therefore aimed to investigate whether DTYMK inhibitors could suppress lung adenocarcinoma (LUAD) progression. In this study, human tissues, A549 cells, and xenograft tumors were used to explore the regulation and mechanism of DTYMK on LUAD cell proliferation and migration. Meanwhile, YMU1 (a DTYMK inhibitor) was applied to A549 cells and xenograft tumors to investigate its potential as a drug for LUAD. DTYMK was overexpressed in LUAD tissues and correlated with tumor stage. Knockdown of DTYMK suppressed cell viability, migration, and invasion. In addition, the activation of signal transducers and activators of transcription 3 (STAT3) was repressed upon DTYMK inhibition. YMU1 showed the same effect as DTYMK knockdown in vivo and in vitro. DTYMK plays an important role in progression of LUAD through the STAT3 signaling pathway. YMU1 may have the potential to inhibit the development of LUAD.NEW & NOTEWORTHY DTYMK plays an important role in progression of LUAD through the STAT3 signaling pathway. YMU1 may serve as a novel drug to suppress the development of LUAD.

Chemopreventive efficacy of stampidine in a murine breast cancer model.

Background: The purpose of the present study was to examine the chemopreventive effect of stampidine, an aryl phosphate derivative of stavudine, in side by side comparison with the standard anti-breast cancer drug paclitaxel in the well-established 7,12-dimethylbenz(a)anthracene (DMBA)-induced murine breast cancer model.Methods: Groups of 20 female mice were challenged with the DMBA. DMBA-challenged mice were assigned to various chemoprevention treatments, including stampidine, paclitaxel, and stampidine plus paclitaxel according to the same treatment schedules for 25 weeks.Results: Stampidine resulted in substantially reduced numbers of tumors, tumor weight as well as tumor size in DMBA-treated mice. Stampidine was as effective as paclitaxel in the model and their combination exhibited greater chemopreventive activity, as measured by reduced tumor incidence and improved tumor-free survival as well as overall survival of DMBA-treated mice. The length of time for the initial tumor to appear in DMBA-challenged mice treated with stampidine was longer than that of mice treated DMBA-challenged control mice. Tumors from mice treated with stampidine or stampidine plus paclitaxel displayed unique changes of a signature protein cassette comprised BRCA1, p21, Bax, and Bcl-2.Conclusion: Stampidine has potent chemopreventive activity and is as effective as the standard chemotherapy drug paclitaxel in the chemical carcinogenesis.

Drug-Inducible Control of Lethality Genes: A Low Background Destabilizing Domain Architecture Applied to the Gal4-UAS System in Drosophila.

Destabilizing domains (DDs) are genetic tags that conditionally control the level of abundance of proteins-of-interest (POI) with specific stabilizing small-molecule drugs, rapidly and reversibly, in a wide variety of organisms. The amount of the DD-tagged fusion protein directly impacts its molecular function. Hence, it is important that the background levels be tightly regulated in the absence of any drug. This is especially true for classes of proteins that function at extremely low levels, such as lethality genes involved in tissue development and certain transcriptional activator proteins. Here, we establish the uninduced background and induction levels for two widely used DDs (FKBP and DHFR) by developing an accurate quantification method. We show that both DDs exhibit functional background levels in the absence of a drug, but each to a different degree. To overcome this limitation, we systematically test a double architecture for these DDs (DD-POI-DD) that completely suppresses the protein's function in an uninduced state, while allowing tunable functional levels upon adding a drug. As an example, we generate a drug-stabilizable Gal4 transcriptional activator with extremely low background levels. We show that this functions in vivo in the widely used Gal4-UAS bipartite expression system in Drosophila melanogaster. By regulating a cell death gene, we demonstrate that only the low background double architecture enables tight regulation of the lethal phenotype in vivo. These improved tools will enable applications requiring exceptionally tight control of protein function in living cells and organisms.

Synthesis, and in vitro enzymatic and antiviral evaluation of d4T polyphosphate derivatives as chain terminators.

A series of d4T di- or triphosphate derivatives have been synthesized and evaluated as effective substrates for HIV-1 RT, and also tested for their in vitro anti-HIV activity. The steady-state kinetic study of compounds 1-4 in an enzymatic incorporation assay by HIV-1 RT follows Michaelis-Menten profile. In addition, compounds 2-4 are able to inhibit HIV-1 replication to the same extent as d4T and d4TMP in MT-4 cells, as well as in CEM/0 cells and CEM/TK(-) cells. The data suggests that these d4T polyphosphate derivatives are hydrolyzed to d4T and rephosphorylated to d4TTP before exerting their antiviral activity.

Stampidine as a promising antiretroviral drug candidate for pre-exposure prophylaxis against sexually transmitted HIV/AIDS.

INTRODUCTION: Pre-exposure prophylaxis (PrEP) is an evolving new approach to prevention of sexually transmitted HIV-1 that employs antiretroviral (ARV) agents prior to potential HIV-1 exposure in an attempt to reduce the likelihood of HIV-1 infection postexposure. The identification of new ARV agents with potent activity against multidrug-resistant HIV remains an unmet and urgent challenge in the field of PrEP. AREAS COVERED: This article reviews the preclinical and early clinical activity and safety profile of stampidine, a novel antiretroviral (ARV) drug candidate that exhibits remarkable subnanomolar to low nanomolar in vitro antiretroviral potency against genotypically and phenotypically nucleoside reverse transcriptase inhibitor (NRTI)-resistant primary clinical HIV isolates, non-nucleoside RT-resistant HIV-1 isolates. Stampidine has a favorable pharmacokinetic profile in mice, rats, dogs and cats with 25 or 50 mg/kg tolerable dose levels yielding micromolar plasma concentrations that are 1000-fold higher than its in vitro IC(50) value against HIV. Stampidine has a favorable, safety profile in mice, rats, dogs and cats and it showed significant in vivo ARV activity in HIV-infected Hu-PBL-SCID mice as well as FIV-infected domestic cats. Furthermore, it did not cause any maternal toxicity, developmental toxicity or teratogenicity in rabbits treated at 10 - 40 mg/kg/day dose levels. In a recently completed first-in-human Phase I clinical trial, stampidine did not cause dose-limiting toxicity at single dose levels ranging from 5 to 25 mg/kg. EXPERT OPINION: The favorable safety and activity profile of stampidine warrants its further development as a promising next-generation PrEP candidate to prevent the sexual transmission of HIV-1. The discovery of stampidine as a potent antiretroviral agent represents a significant step forward in the development of effective therapeutic as well as preventive strategies against HIV/AIDS.

Radiolabeled cyclosaligenyl monophosphates of 5-iodo-2'-deoxyuridine, 5-iodo-3'-fluoro-2',3'-dideoxyuridine, and 3'-fluorothymidine for molecular radiotherapy of cancer: synthesis and biological evaluation.

Targeted molecular radiotherapy opens unprecedented opportunities to eradicate cancer cells with minimal irradiation of normal tissues. Described in this study are radioactive cyclosaligenyl monophosphates designed to deliver lethal doses of radiation to cancer cells. These compounds can be radiolabeled with SPECT- and PET-compatible radionuclides as well as radionuclides suitable for Auger electron therapies. This characteristic provides an avenue for the personalized and comprehensive treatment strategy that comprises diagnostic imaging to identify sites of disease, followed by the targeted molecular radiotherapy based on the imaging results. The developed radiosynthetic methods produce no-carrier-added products with high radiochemical yield and purity. The interaction of these compounds with their target, butyrylcholinesterase, depends on the stereochemistry around the P atom. IC(50) values are in the nanomolar range. In vitro studies indicate that radiation doses delivered to the cell nucleus are sufficient to kill cells of several difficult to treat malignancies including glioblastoma and ovarian and colorectal cancers.

Selective inhibition of embryonic development in starfish by long-chain alkyl derivatives of UMP, TMP and AMP.

Nucleoside analogs have been evaluated as useful tools for the investigation of the mechanism of cell differentiation. We thus examined the effects of nucleoside 5'-alkylphosphates (1-10) on the morphogenetic development of starfish embryos. These nucleotide derivatives were all permissive for their development up to the blastula stage, but the derivatives with lauryl side chain selectively inhibited one of the following stages into bipinnaria larvae. Among them, uridine 5'-laurylphosphate (2) inhibited gastrulation of the blastula, as is the case with the antibiotic tunicamycin, suggesting its inhibitory activity on sulfated and non-sulfated glycoprotein syntheses. Unexpectedly, adenosine 5'-laurylphosphate (8) was evaluated as a novel class of inhibitor that can arrest the embryos exactly at the late gastrula stage, absolutely inhibiting cell differentiation involved in the development of gastrointestinal tract. This is the first report on the appearance of biological activity due to the structural modification of a naturally-occurring molecule, which is critical to the morphogenetic development of multicellular organisms.

Microbicides for multidrug-resistant and multitropic HIV-1.

The most common mode of acquiring HIV-1 is via sexual transmission across the genital mucosa. Topical microbicides are a promising prevention strategy for the protection against HIV infection and may ultimately have an impact on the global AIDS pandemic. The effectiveness of a microbicide to prevent HIV-1 transmission will depend on the evolutionary and genital transmission dynamics of the viral subtypes, and sexual behavioral characteristics. Contemporary antiretroviral therapy has led to virological failure as a result of HIV-1 reverse transcriptase gene mutations. The transmission of these multidrug-resistant HIV-1 variants, and the superinfection with the same or distinct HIV-1 subtypes and recombination is a formidable hindrance inherent to global microbicide development. Consequently, mechanism-based microbicides targeting both the cell-free and cell-associated HIV-1 variants and subtypes can be expected to have superior clinical efficacy and safety profiles compared with polymeric anionic microbicides. This review describes the discovery of potent anti-HIV-1 agents against multidrug-resistant and multitropic HIV-1 variants with implications for global microbicide development. Stampidine and thiourea non-nucleoside reverse transcriptase inhibitors (NNRTIs) have demonstrated highly potent activity against clinically relevant multidrug-resistant and recombinant HIV-1 isolates spanning different subtypes across several continents. Extensive preclinical studies have shown that stampidine and a candidate thiourea NNRTI (HI-443) have clinical potential as a safe combination microbicide to inhibit, prevent or treat mucosal HIV-1 infections.

Anti-retroviral activity of GMP-grade stampidine against genotypically and phenotypically nucleoside reverse transcriptase inhibitor resistant recombinant human immunodeficiency virus. An in vitro study.

The in vitro potency of GMP-grade stampidine (CAS 217178-62-6) was examined against 3 clinical HIV-1 isolates and 6 recombinant HIV-1 clones with multi-NRTI 'resistance (NRTI: nucleoside reverse transcriptase inhibitors). GMP-grade stampidine active drug substance (Lot #'s MPR-M0008.00-01 and MPR-M0008.01-01) as well as GMP-grade stampidine extracted from the clinical stampidine capsules (GMP-Grade Clinical Batch, Pharmaceutical Service Lot Number 159I0601) were highly potent and exhibited nanomolar IC50 values against clinical HIV-1 isolates as well as recombinant HIV-1 clones with multi-NRTI resistance containing common patterns of reverse transcriptase mutations responsible for NRTI resistance.

The involvement of ATP produced via (ADP-Ribose)n in the maintenance of DNA replication apparatus during DNA repair.

The formation of ATP produced from poly(ADP-ribose) [(ADP-R)n] has been suggested to be required to repair damaged DNA. Here we investigate whether this ATP is involved in DNA replication processes during DNA repair. Poly(ADP-ribosyl)ated mid-S phase cell nuclei, which were isolated from synchronized HeLa S3 cells followed by the treatment with a DNA damaging agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), were revealed to retain DNA replication synthesizing activity during preincubation for de-poly(ADP-ribosyl)ation only in the presence of pyrophosphate (PPi) before DNA synthesis was started by adding 3 mM ATP. This DNA replication activity was not maintained in the presence of a potent and specific inhibitor of poly(ADP-ribose) glycohydrolase (PARG), Oenothein B (Oen B) during the preincubation with PPi. In the preincubation with PPi, muM orders of ATP was produced from (ADP-R)n. These results point to an important function of ATP generated from (ADP-R)n in nuclei for the maintenance of replication apparatus during DNA repair.

Bis-cycloSal-d4T-monophosphates: drugs that deliver two molecules of bioactive nucleotides.

Bis-cycloSal-d4T-monophosphates have been synthesized as potentially anti-HIV active "dimeric" prodrugs of 2',3'-dideoxy-2',3'-didehydrothymidine monophosphate (d4TMP). These pronucleotides display a mask-drug ratio of 1:2, a novelty in the field of pronucleotides. Both bis-cycloSal-d4TMP 6 and bis-5-methyl-cycloSal-d4TMP 7 showed increased hydrolytic stability as compared to their "monomeric" counterparts and a completely selective hydrolytic release of d4TMP. The hydrolysis pathway was investigated via 31P NMR spectroscopy. Moreover, due to the steric bulkiness, compound 6 already displayed strongly reduced inhibitor potency toward human butyrylcholinesterase (BChE), while compound 7 turned out to be devoid of any inhibitory activity against BChE. Partial separation of the diastereomeric mixture of 6 revealed strong dependence of the pronucleotides' properties on the stereochemistry at the phosphorus centers. Both 6 and 7 showed good activity against HIV-1 and HIV-2 in wild-type CEM cells in vitro. These compounds were significantly more potent than the parent nucleoside d4T 1 in HIV-2-infected TK-deficient CEM cells, indicating an efficient TK-bypass.

The Escherichia coli protein YjjG is a house-cleaning nucleotidase in vivo.

House-cleaning enzymes protect cells from the adverse effects of noncanonical metabolic chemical compounds. The Escherichia coli nucleotide phosphatase YjjG (B4374, JW4336) functions as a house-cleaning phosphatase in vivo. YjjG protects the cell against noncanonical pyrimidine derivatives such as 5-fluoro-2'-deoxyuridine (5-FdUridine), 5-fluorouridine, 5-fluoroorotic acid (5-FOA), 5-fluorouracil, and 5-aza-2'-deoxycytidine. YjjG prevents the incorporation of potentially mutagenic nucleotides into DNA as shown for 5-bromo-2'-deoxyuridine (BrdU). Its enzymatic activity in vitro towards noncanonical 5-fluoro-2'-deoxyuridine monophosphate (5-FdUMP) is higher than towards canonical thymidine monophosphate (dTMP). The closest homolog in humans, HDHD4, does not show a protective effect against noncanonical nucleotides, excluding an involvement of HDHD4 in resistance against noncanonical nucleotides used for cancer chemotherapy. The substrate spectrum of YjjG suggests that its in vivo substrates are noncanonical pyrimidine derivatives, which might also include oxidized nucleobases such as 5-formyluracil and 5-hydroxyuracil.

Stampidine as a novel nucleoside reverse transcriptase inhibit with potent anti-HIV activity.

Stavudine (STV, d4T, 2',3'-didehydro-3'-deoxythymidine, CAS 3056-17-5) is a standard anti-HIV drug. Stampidine (STAMP, DDE-113, HI-113, N-[p-(4-bromophenyl)-2',3'-didehydro-3'-deoxy-5'-thymidylyl]-L-alanine methyl ester, CAS 217178-62-6) is a novel aryl phosphate derivative of stavudine with more potent anti-HIV activity and more favorable pharmacodynamic features. The remarkable potency of stampidine against clinical HIV-1 isolates with NRTI- or NNRTI-resistance (NRTI, nucleoside reverse transcriptase inhibitor; NNRTI, non-nucleoside reverse transcriptase inhibitor) warrants the further development of this new anti-HIV agent.

Synthesis, separation and anti-HIV activity of distereoisomers of N-[p-(4-bromophenyl) -2',3'-didehydro-3'-deoxy-5'-thymidylyl]-L-alanine methyl ester (stampidine).

The distereoisomers of stampidine (STAMP, DDE-113, HI-113, N-[p-(4-bromophenyl)-2'3'-didehydro-3'-deoxy-5'-thymidylyl]-L-alanine methyl ester, CAS 217178-62-6) were separated using two different procedures. The first method involved separation of the isomers by fractional crystallization, and the second method utilized a preparative HPLC. Both isomers were active against the HIV-1 strain HTLV(IIIB) and neither isomer was more or less active than distereoisomeric mixture of stampidine.

Site-specific enzymatic activation of the anti-HIV agent stampidine.

Stampidine (STAMP, DDE-113, HI-113, N-[p-(4-bromophenyl)-2',3'-didehydro-3'-deoxy-5'-thymidylyl]-L-alanine methyl ester, CAS 217178-62-6) and two stampidine analogs containing ethyl or t-butyl groups were synthesized and their rates of enzymatic activation were compared side-by-side. Enzymes such as lipase, esterase and protease did not hydrolyze the butyl substituted STAMP analog. These experimental results show that the site of attack for the enzymatic hydrolysis of STAMP is the ester side chain of the molecule.

Potency of stampidine against multi-nucleoside reverse transcriptase inhibitor resistant human immunodeficiency viruses.

The in vitro potency of stampidine (STAMP, DDE-113, HI-113, N-[p-(4-bromophenyl)-2',3'-didehydro-3'-deoxy-5'-thymidylyl]-L-alanine methyl ester, CAS 217178-62-6) was examined against 8 clinical non-B subtype HIV-1 isolates with resistance to stavudine (STV, d4T), adefovir and tenofovir, 19 clinical zidovudine-resistant HIV-1 isolates, and 6 recombinant HIV-1 clones with multi-resistance against nucleoside reverse transcriptase inhibitors. Stampidine exhibited potent anti-HIV activity against each one of these 33 HIV-1 isolates with subnanomolar to nanomolar IC50 values.

3D-QSAR studies on antitubercular thymidine monophosphate kinase inhibitors based on different alignment methods.

Three dimensional quantitative structure-activity relationship (3D-QSAR) studies were carried out on deoxythymidine monophosphate (dTMP) derivatives inhibiting thymidine monophosphate kinase (TMPK) in Mycobacterium tuberculosis. Molecular field analysis (MFA) models with three different alignment techniques, namely, least squares, pharmacophore based and receptor based methods were developed. Receptor based MFA model showed better results when compared with least squares and pharmacophore based models. The results help us to understand the nature of substituents required for activity and thereby provide guidelines to design novel and potent inhibitors as antitubercular agents.

Effect of change in nucleoside structure on the activation and antiviral activity of phosphoramidate derivatives.

Changing the nucleoside group of a series of phosphoramidate derivatives affects the enzyme mediated hydrolysis rate of the compounds. d4T and AZT-substituted analogs were activated by enzymes such as lipases, esterases, and proteases. On the other hand, 3dT-substituted derivatives were comparatively less prone to hydrolysis under similar experimental conditions. From the experimental results, we propose that the most preferable nucleoside group for enzyme activation is d4T rather than AZT or 3dT. Additionally, we also observed that depending on the enzymes used the chiral selectivity of the enzymes for the phosphorus center of these phosphoramidate derivatives differed, demonstrating the importance of the nucleoside structure for this class of compounds.

Stampidine: a selective oculo-genital microbicide.

Adenoviruses (ADVs) are causative agents of severe and extremely contagious ocular and genital infections associated with conjunctivitis, genital ulcers and urethritis. Yet, no functional antiviral compounds are currently available against adenoviral infections. We discovered halogen-substituted phenyl phosphoramidate derivatives of stavudine (STV/d4T) as a new class of dual-function anti-human immunodeficiency virus (HIV) agents with potent and selective anti-ADV activity. The lead compound, stampidine [5'-(4-bromophenyl methoxyalaninylphosphate)-2',3'-didehydro-3'-deoxythymidine], was the most potent non-toxic dual-function antiviral agent. Stampidine displayed remarkable in vitro and in vivo anti-HIV activity against drug-sensitive and drug-resistant HIV strains. Stampidine was non-cytotoxic and nonirritating to mucosal epithelial cells. Several preclinical studies conducted thus far, suggest that stampidine has clinical potential as a dual-function topical agent for the prevention and/or effective treatment of oculo-genital ADV/HIV infections.