PubMed Health⌕ Search

Biomedical subjects

Craig J Thomas

Publications and source records attributed to Craig J Thomas.

At least 19 recordsLinked to original sources
PubMed2025

Multiple Epigenetic Mechanisms Functionally Cooperate to Silence Expression of Somatostatin Receptor Type 2 in Pancreatic Neuroendocrine Tumors.

Pancreatic neuroendocrine tumors (PNETs) are a rare and understudied set of cancers, with increasing incidence. Neuroendocrine tumors are unique in the fact that they express high levels of the somatostatin receptor type 2 (SSTR2), which represents a target for both tumor imaging and therapeutics. PNET grade inversely correlates with SSTR2 tumor staining and higher tumor grade is associated with poor patient prognosis. With no known mutations, SSTR2 expression is believed to be lost through aberrant epigenetic mechanisms. Enhanced knowledge of the epigenetic biology and players controlling SSTR2 expression may allow for identification of novel PNET imaging and treatment modalities. Through in-depth studies, we found that the specific de novo DNA methyltransferase (DNMT), DNMT3B, is responsible for SSTR2 gene CpG methylation and silencing. Using DNMT3B as a starting point, along with the concept of functional crosstalk between various epigenetic mechanisms, we further discovered that Polycomb Repressor Complexes 1 and 2 (PRC1 and PRC2) play important roles in silencing SSTR2. Moreover, we found several histone lysine demethylases, enzymes that remove activating histone H3K4 methylation marks, to be critical for silencing expression of SSTR2. We additionally identified several chromatin remodeling enzymes/complexes as cellular factors that negatively regulate SSTR2 expression. Finally, using the HiBiT luminescent reporter system, we exploited functional chemo-genomic screens to further expand our knowledge of SSTR2 epigenetic control. These screens both reinforced several of our initial findings and helped to identify additional silencing mechanism potentially regulating SSTR2 expression. A commonality in our findings point to the presence, or necessity, of Class I HDACs in nearly all the epigenetic silencing mechanisms characterized. Overall, our work demonstrates that SSTR2 gene expression is likely silenced through various dynamic and interconnected epigenetic events, resulting in a compacted, transcriptionally repressed chromatin environment. Our study offers novel potential therapeutic targets and combinations to best increase expression of SSTR2, which are currently being tested in pre-clinical studies from our group, with the goal of future clinical trials aimed at increasing SSTR2 expression in high-grade, SSTR2-low NET patients.

Journal Article↗
PubMed2006

Evaluation of small-molecule modulators of the luteinizing hormone/choriogonadotropin and thyroid stimulating hormone receptors: structure-activity relationships and selective binding patterns.

The substituted thieno[2,3-d]pyrimidine 3 (Org 41841), a partial agonist for the luteinizing hormone/choriogonadotropin receptor (LHCGR) and the closely related thyroid-stimulating hormone receptor (TSHR), was fundamentally altered, and the resulting analogues were analyzed for their potencies, efficacies, and specificities at LHCGR and TSHR. Chemical modification of the parent compound combined with prior mutagenesis of TSHR provided compelling experimental evidence in support of computational models of 3 binding to TSHR and LHCGR within their transmembrane cores. Biochemical analysis of a specific modification to the chemical structure of 3 provides additional evidence of a H-bond between the ligand and a glutamate residue in transmembrane helix 3, which is conserved in both receptors. Several key interactions were surveyed to determine their respective biochemical roles in terms of both van der Waals dimensions and hydrogen bond capacity and the respective relationship to biological activity.

Binding Sites↗
PubMed2006

The inhibition and treatment of breast cancer with poly (ADP-ribose) polymerase (PARP-1) inhibitors.

BRCA1 and BRCA2 mutations are responsible for most familial breast carcinomas. Recent reports carried out in non-cancerous mouse BRCA1- or BRCA2-deficient embryonic stem (ES) cells, and hamster BRCA2-deficient cells have demonstrated that the targeted inhibition of poly(ADP-ribose) polymerase (PARP-1) kills BRCA mutant cells with high specificity. Although these studies bring hope for BRCA mutation carriers, the effectiveness of PARP-1 inhibitors for breast cancer remains elusive. Here we present the first in vivo demonstration of PARP-1 activity in BRCA1-deficient mammary tumors and describe the effects of PARP-1 inhibitors (AG14361, NU1025, and 3-aminobenzamide) on BRCA1-deficient ES cells, mouse and human breast cancer cells. AG14361 was highly selective for BRCA1-/- ES cells; however, NU1025 and 3-aminobenzamide were relatively non-selective. In allografts of naïve ES BRCA1-/- cells there was either partial or complete remission of tumors. However, in allografts of mouse, BRCA1-/- mammary tumors, there was no tumor regression or remission although a partial inhibition of tumor growth was observed in both the BRCA1-/- and BRCA1+/+ allografts. In human tumor cells, PARP-1 inhibitors showed no difference in vitro in limiting the growth of mammary tumors irrespective of their BRCA1 status. These results suggest that PARP-1 inhibitors may non-specifically inhibit the growth of mammary tumors.

Aged↗
PubMed2006

An O-GlcNAcase-specific inhibitor and substrate engineered by the extension of the N-acetyl moiety.

A novel analogue of PUGNAc, a potent O-GlcNAcase inhibitor, was synthesized and analyzed as an inhibitor of O-GlcNAcase, hexosaminidase A, and hexosaminidase B. While PUGNAc does not demonstrate selective inhibition of these related enzymes, the extension of the acetyl moiety to the longer butyl chain provided a compound with depressed inhibition of O-GlcNAcase and no observed inhibition of either hexosaminidase A or hexosaminidase B. Further, we applied this knowledge of substrate recognition at the N-acetyl group to our recently reported fluorogenic substrate for monitoring O-GlcNAcase activity. Gratifyingly, this altered small molecule was demonstrated to be a potent substrate for O-GlcNAcase while possessing no activity at hexosaminidase A. This reagent provides, for the first time, a means for monitoring O-GlcNAcase activity independent of the related enzymes hexosaminidase A and hexosaminidase B.

Acetylglucosamine↗
PubMed2006

A low molecular weight agonist signals by binding to the transmembrane domain of thyroid-stimulating hormone receptor (TSHR) and luteinizing hormone/chorionic gonadotropin receptor (LHCGR).

Many cognate low molecular weight (LMW) agonists bind to seven transmembrane-spanning receptors within their transmembrane helices (TMHs). The thienopyrimidine org41841 was identified previously as an agonist for the luteinizing hormone/chorionic gonadotropin receptor (LHCGR) and suggested to bind within its TMHs because it did not compete for LH binding to the LHCGR ectodomain. Because of its high homology with LHCGR, we predicted that thyroid-stimulating hormone receptor (TSHR) might be activated by org41841 also. We show that org41841 is a partial agonist for TSHR but with lower potency than for LHCGR. Analysis of three-dimensional molecular models of TSHR and LHCGR predicted a binding pocket for org41841 in common clefts between TMHs 3, 4, 5, 6, and 7 and extracellular loop 2 in both receptors. Evidence for this binding pocket was obtained in signaling studies with chimeric receptors that exhibited improved responses to org41841. Furthermore, a key receptor-ligand interaction between the highly conserved negatively charged E3.37 and the amino group of org41841 predicted by docking of the ligand into the three-dimensional TSHR model was experimentally confirmed. These findings provide the first evidence that, in contrast to the ectodomain binding of cognate ligands, a LMW agonist can bind to and activate glycoprotein hormone receptors via interaction with their transmembrane domain.

Amino Acid Sequence↗
PubMed2006

Exploring the cellular activity of camptothecin-triple-helix-forming oligonucleotide conjugates.

Topoisomerase I is a ubiquitous DNA-cleaving enzyme and an important therapeutic target in cancer chemotherapy for camptothecins (CPTs). These drugs stimulate DNA cleavage by topoisomerase I but exhibit little sequence preference, inducing toxicity and side effects. A convenient strategy to confer sequence specificity consists of the linkage of topoisomerase poisons to DNA sequence recognition elements. In this context, triple-helix-forming oligonucleotides (TFOs) covalently linked to CPTs were investigated for the capacity to direct topoisomerase I-mediated DNA cleavage in cells. In the first part of our study, we showed that these optimized conjugates were able to regulate gene expression in cells upon the use of a Photinus pyralis luciferase reporter gene system. Furthermore, the formation of covalent topoisomerase I/DNA complexes by the TFO-CPT conjugates was detected in cell nuclei. In the second part, we elucidated the molecular specificity of topoisomerase I cleavage by the conjugates by using modified DNA targets and in vitro cleavage assays. Mutations either in the triplex site or in the DNA duplex receptor are not tolerated; such DNA modifications completely abolished conjugate-induced cleavage all along the DNA. These results indicate that these conjugates may be further developed to improve chemotherapeutic cancer treatments by targeting topoisomerase I-induced DNA cleavage to appropriately chosen genes.

Antineoplastic Agents, Phytogenic↗
PubMed2006

Fluorinated natural products with clinical significance.

The modification of natural products in an effort to alter their biochemical capacity is a common technique utilized by synthetic and medicinal chemists. Fluorine substitution imparts unique and advantageous physiochemical properties that can be shrewdly employed to constructively alter pharmacological agents. The adornment of natural products with fluorine has proven beneficial in several examples. This overview discusses several of the most relevant fluorinated natural products under current examination.

Biological Products↗
PubMed2005

Inhibition of O-GlcNAcase by PUGNAc is dependent upon the oxime stereochemistry.

The potent O-GlcNAcase inhibitor PUGNAc was synthesized and two isomers based on the E and Z stereochemistry of the oxime moiety were separated, defined, and tested for activity. Several lines of evidence were examined in an effort to define the correct stereochemical assignments of each form of PUGNAc. The ability of the Z stereoisomer to undergo the Beckmann rearrangement was ultimately the most definitive proof. It was determined via both in vitro and intact cell experiments that the Z form of PUGNAc was vastly more potent an inhibitor of O-GlcNAcase than the E form.

Acetylglucosamine↗
PubMed2005

"Reversine" and its 2-substituted adenine derivatives as potent and selective A3 adenosine receptor antagonists.

The dedifferentiation agent "reversine" [2-(4-morpholinoanilino)-N(6)-cyclohexyladenine 2] was found to be a moderately potent antagonist for the human A(3) adenosine receptor (AR) with a K(i) value of 0.66 microM. This result prompted an exploration of the structure-activity relationship of related derivatives, synthesized via sequential substitution of 6-chloro-2-fluoropurine with selected nucleophiles. Optimization of substituents at these two positions identified 2-(phenylamino)-N(6)-cyclohexyladenine (12), 2-(phenylamino)-N(6)-cycloheptyladenine (19), and 2-phenylamino-N(6)-endo-norbornyladenine (21) as potent A(3) AR ligands with K(i) values of 51, 42, and 37 nM, respectively, with 30-200-fold selectivity in comparison to A(1) and A(2A) ARs. The most selective A(3) AR antagonist (>200-fold) was 2-(phenyloxy)-N(6)-cyclohexyladenine (22). 9-Methylation of 12, but not 19, was well-tolerated in A(3) AR binding. Extension of the 2-phenylamino group to 2-benzyl- and 2-(2-phenylethylamino) reduced affinity. In the series of 2-(phenylamino), 2-(phenyloxy), and 2-(phenylthio) substitutions, the order of affinity at the A(3) AR was oxy > or = amino > thio. Selected derivatives, including reversine (K(B) value of 466 nM via Schild analysis), competitively antagonized the functional effects of a selective A(3) AR agonist, i.e., inhibition of forskolin-stimulated cAMP production in stably transfected Chinese hamster ovary (CHO) cells. These results are in agreement with other studies suggesting the presence of a lipophilic pocket in the AR binding site that is filled by moderately sized cycloalkyl rings at the N(6) position of both adenine and adenosine derivatives. Thus, the compound series reported herein comprise an important new series of selective A(3) AR antagonists. We were unable to reproduce the dedifferentiation effect of reversine, previously reported, or to demonstrate any connection between A(3) AR antagonist effects and dedifferentiation.

Adenine↗
PubMed2005

Design and synthesis of new bicyclic diketopiperazines as scaffolds for receptor probes of structurally diverse functionality.

Diketopiperazines (DKPs) are a common motif in various biologically active natural products, and hence they may be useful scaffolds for the rational design of receptor probes and therapeutic agents. We constructed a new bicyclic scaffold that combines a DKP bridged with a 10-membered ring. In this way we obtained a three-dimensional molecular skeleton, with several amendable sites that provide a starting point to design a new combinatorial library having diverse substituent groups. Structural variation is based upon the flexibility of alkylation of the nitrogen atoms of the DKP and on the side-chain olefin. We obtained a 10-membered secondary ring through a ring-closure metathesis reaction using the second generation Grubbs catalyst. Rings containing both O-ethers and S-ethers were compared. N-Alkyl or arylalkyl groups were introduced optionally at the two Nalpha-atoms. This is a general scheme that will allow us to test rings of varying sizes, linkages, and stereochemical parameters. The DKP derivatives were tested for activity in astrocytoma cells expressing receptors coupled to phospholipase C. Inhibitory effects were observed for signaling elicited by activation of human nucleotide P2Y receptors but not m3 muscarinic receptors. Compound 20 selectively inhibited calcium mobilization (IC50 value of 486 +/- 16 nM) and phosphoinositide turnover elicited by a selective P2Y1 receptor agonist, but this compound did not compete for binding of a radiolabeled nucleotide-competitive receptor antagonist. Therefore, the new class of DKP derivatives shows utility as pharmacological tools for P2Y receptors.

Bridged Bicyclo Compounds↗
PubMed2005

Activation of camptothecin derivatives by conjugation to triple helix-forming oligonucleotides.

Topoisomerase I (topo I) is a ubiquitous DNA-cleaving enzyme and an important therapeutic target in cancer chemotherapy. Camptothecins (CPTs) reversibly trap topo I in covalent complex with DNA but exhibit limited sequence preference. The utilization of conjugates such as triplex-forming oligonucleotides (TFOs) to target a medicinal agent (like CPT) to a specific genetic sequence and orientation within the DNA has been accomplished successfully. In this study, different attachment points of the TFO to CPT (including positions 7, 9, 10, and 12) were investigated and our findings confirmed and extended previous conclusions. Interestingly, the conjugates induced specific DNA cleavage by topo I at the triplex site even when poorly active or inactive CPT derivatives were used. This suggests that the positioning of the drug in the cleavage complex by the sequence-specific DNA ligand is able to stabilize the ternary complex, even when important interactions between topo I and CPT are disrupted. Finally, certain TFO-CPT conjugates were able to induce sequence-specific DNA cleavage with the topo I mutants R364H and N722S that are resistant to camptothecin. The TFO-CPT conjugates are thus valuable tools to study the interactions involved in the formation of the ternary complex and also to enlarge the family of compounds that poison topo I. The fact that an inactive CPT analogue can act as a topo I poison when appropriately coupled to a TFO provides a new perspective at the level of drug design.

Amino Acid Substitution↗
PubMed2004

Camptothecin: current perspectives.

This review provides a detailed discussion of recent advances in the medicinal chemistry of camptothecin, a potent antitumor antibiotic. Two camptothecin analogues are presently approved for use in the clinic as antitumor agents and several others are in clinical trials. Camptothecin possesses a novel mechanism of action involving the inhibition of DNA relaxation by DNA topoisomerase I, and more specifically the stabilization of a covalent binary complex formed between topoisomerase I and DNA. This review summarizes the current status of studies of the mechanism of action of camptothecin, including topoisomerase I inhibition and additional cellular responses. Modern synthetic approaches to camptothecin and several of the semi-synthetic methods are also discussed. Finally, a systematic evaluation of novel and important analogues of camptothecin and their contribution to the current structure-activity profile are considered.

Animals↗
PubMed2003

Conformationally constrained analogues of bleomycin A5.

The bleomycin (BLM) group antitumor antibiotics are glycopeptide-derived natural products shown to cause sequence selective lesions in DNA. Prior studies have indicated that the linker region, composed of the methylvalerate and threonine residues, may be responsible for a conformational bend in the agent required for efficient DNA cleavage. We have synthesized a number of conformationally constrained methylvalerate analogues and incorporated them into deglycobleomycin A(5) congeners using our recently reported procedure for the solid phase construction of (deglyco)bleomycin and its analogues. These analogues were designed to probe the effects of conformational constraint of the native valerate moiety. Initial experiments indicated that the constrained molecules, none of which mimic the conformation proposed for the natural valerate linker, possessed DNA cleavage activity, albeit with potencies less than that of (deglyco)BLM and lacking sequence selectivity. Further experiments demonstrated that these analogues failed to produce alkali-labile lesions in DNA or sequence selective oxidative damage in RNA. However, two of the conformationally constrained deglycoBLM analogues were shown to mediate RNA cleavage in the absence of added Fe(2+). The ability of the analogues to mediate the oxygenation of small molecules was also assayed, and it was shown that they were as competent in the transfer of oxygen to low molecular weight substrates as the parent compound.

Antibiotics, Antineoplastic↗
PubMed2003

Solid-phase synthesis of bleomycin group antibiotics. Construction of a 108-member deglycobleomycin library.

The bleomycins (BLMs) are structurally related glycopeptide antibiotics isolated from Streptomyces verticillus that mediate the sequence-selective oxidative damage of DNA and RNA. Deglycobleomycin, which lacks the carbohydrate moiety, cleaves DNA analogously to bleomycin itself, albeit less potently, and has been used successfully for analyzing the functional domains of bleomycin. Although structural modifications to bleomycin and deglycobleomycin have been reported, no bleomycin or deglycobleomycin analogue having enhanced DNA cleavage activity has yet been described. The successful synthesis of a deglycobleomycin on a solid support has permitted the facile solid-phase synthesis of 108 unique deglycobleomycin analogues through parallel solid-phase synthesis. Each of the deglycobleomycin analogues was synthesized efficiently; the purity of each crude product was greater than 60%, as determined by HPLC integration. The solid-phase synthesis of the deglycobleomycin library provided near-milligram to milligram quantities of each deglycobleomycin, thereby permitting characterization by (1)H NMR and high-resolution mass spectrometry. Each analogue demonstrated supercoiled plasmid DNA relaxation above background cleavage; the library included two analogues that mediated plasmid relaxation to a greater extent than the parent deglycobleomycin molecule.

Amino Acids↗
PubMed2003

Tuning the optical properties of fluorinated indolylfulgimides.

Photochromic fluorinated indolylfulgides have been identified as potential candidates for a wide range of applications including optical switches, photoregulators of biological processes, and optical memory media. In humid environments or biological systems, hydrolytic stability is essential. In an effort to improve hydrolytic stability, a series of indolylfulgimides has been synthesized from a parent trifluoromethyl-substituted indolylfulgide. The nitrogen of the succinimide moiety is linked to either a dimethyl amino or one of seven substituted phenyl groups. The phenyl groups feature substituents with increasing electron-withdrawing ability. The spectral characteristics of each compound have been examined, revealing that the wavelength absorption maxima of each form increases with increasing electron-withdrawing ability of the substituted N-phenyl ring. The quantum yields of the photoreactions have been determined with the N-(phenyl)fulgimide showing a ring closure value of nearly 0.30 in toluene. In addition, the hydrolytic, thermal, and photochemical stabilities of each compound have been measured. The fulgimides exhibit at least a 200-fold enhancement of hydrolytic stability for the Z-form and over a 1000-fold enhancement for the C-form in comparison to the same form of the parent fulgide. The N-(2,3,5,6-tetrafluoro-4-trifluoromethylphenyl)fulgimide can undergo up to 3000 photochemical cycles (coloration followed by bleaching) before losing 20% of its initial absorbance at photostationary state.

Journal Article↗
PubMed2002

Solid-phase synthesis of bleomycin A(5) and three monosaccharide analogues: exploring the role of the carbohydrate moiety in RNA cleavage.

The solid-phase synthesis of bleomycin A5 (BLM A5) and three monosaccharide analogues is presented. The monosaccharide analogues incorporated alpha-d-mannose, alpha-l-gulose, and alpha-l-rhamnose moieties in lieu of the disaccharide normally present in BLM A5. Also explored were the abilities of each of the monosaccharide congeners to cleave a 53-nt RNA. The elaboration of these carbohydrate-modified bleomycin analogues helps to define the role of the disaccharide moiety during the RNA cleavage event. The relatively facile solid-phase synthesis of bleomycin A5 and each of the carbohydrate analogues constitutes an important advance in the continuing mechanistic studies of bleomycin.

Base Sequence↗
PubMed2002

Structural properties of a series of photochromic fluorinated indolylfulgides.

Fluorinated indolylfulgides are a class of photochromic organic compounds that meet many of the requirements for use as optical memory media and optical switches. The X-ray crystal structures of a series of five photochromic fluorinated indolylfulgides have been determined, namely (3Z)-3-[1-(1,2-dimethyl-1H-indol-3-yl)-2,2,2-trifluoroethylidene]-4-(1-methylethylidene)dihydrofuran-2,5-dione (trifluoromethylisopropylideneindolylfulgide), C(19)H(16)F(3)NO(3), (I), (3Z)-3-[1-(1,2-dimethyl-1H-indol-3-yl)-2,2,3,3,3-pentafluoropropylidene]-4-(1-methylethylidene)dihydrofuran-2,5-dione (pentafluoroethylisopropylideneindolylfulgide), C(20)H(16)F(5)NO(3), (II), (3Z)-3-[1-(1,2-dimethyl-1H-indol-3-yl)-2,2,3,3,4,4,4-heptafluorobutylidene]-4-(1-methylethylidene)dihydrofuran-2,5-dione (heptafluoropropylisopropylideneindolylfulgide), C(21)H(16)F(7)NO(3), (III), (3Z)-3-[1-(1,2-dimethyl-1H-indol-3-yl)-2,2,2-trifluoroethylidene]-4-(tricyclo[3.3.1.1(3,7)]decylidene)dihydrofuran-2,5-dione (trifluoromethyladamantylideneindolylfulgide), C(26)H(24)F(3)NO(3), (IV), and (3Z)-3-[1-(1,2-dimethyl-1H-indol-3-yl)-2,2,3,3,4,4,4-heptafluorobutylidene]-4-(tricyclo[3.3.1.1(3,7)]decylidene)dihydrofuran-2,5-dione (heptafluoropropyladamantylideneindolylfulgide), C(28)H(24)F(7)NO(3), (V). The photochromic property of fulgides is based on the photochemically allowed electrocyclic ring closure of a hexatriene system to form a cyclohexadiene. For each fulgide examined, the bond lengths within the hexatriene system alternate between short and long, as expected. Comparing the structures of the five fulgides with each other demonstrates no significant difference in bond lengths, bond angles or dihedral angles within the hexatriene systems. The distance between the bond-forming C atoms at each end of the hexatriene system does vary. Correlations of structural properties with optical properties are addressed.

Adamantane↗