Biomedical subjects

Mark Reynolds

Publications and source records attributed to Mark Reynolds.

5 recordsLinked to original sources

PubMed2006

Selective inhibition of fibroblast activation protein protease based on dipeptide substrate specificity.

Fibroblast activation protein (FAP) is a transmembrane serine peptidase that belongs to the prolyl peptidase family. FAP has been implicated in cancer; however, its specific role remains elusive because inhibitors that distinguish FAP from other prolyl peptidases like dipeptidyl peptidase-4 (DPP-4) have not been developed. To identify peptide motifs for FAP-selective inhibitor design, we used P(2)-Pro(1) and acetyl (Ac)-P(2)-Pro(1) dipeptide substrate libraries, where P(2) was varied and substrate hydrolysis occurs between Pro(1) and a fluorescent leaving group. With the P(2)-Pro(1) library, FAP preferred Ile, Pro, or Arg at the P(2) residue; however, DPP-4 showed broad reactivity against this library, precluding selectivity. By contrast, with the Ac-P(2)-Pro(1) library, FAP cleaved only Ac-Gly-Pro, whereas DPP-4 showed little reactivity with all substrates. FAP also cleaved formyl-, benzyloxycarbonyl-, biotinyl-, and peptidyl-Gly-Pro substrates, which DPP-4 cleaved poorly, suggesting an N-acyl-Gly-Pro motif for inhibitor design. Therefore, we synthesized and tested the compound Ac-Gly-prolineboronic acid, which inhibited FAP with a K(i) of 23 +/- 3 nm. This was approximately 9- to approximately 5400-fold lower than the K(i) values for other prolyl peptidases, including DPP-4, DPP-7, DPP-8, DPP-9, prolyl oligopeptidase, and acylpeptide hydrolase. These results identify Ac-Gly-BoroPro as a FAP-selective inhibitor and suggest that N-acyl-Gly-Pro-based inhibitors will allow testing of FAP as a therapeutic target.

Acetylcysteine↗

PubMed2005

Water-soluble polymers for targeted drug delivery to human squamous carcinoma of head and neck.

Human squamous cell carcinoma of the head and neck (SCCHN) is characterized by over expression of a tumor cell surface-specific receptor namely Hsp47/CBP2 that makes it a favorable candidate for targeted delivery of anticancer drugs. Several synthetic peptides have been identified as effective ligands for binding to CBP2. The purpose of this study is to investigate the potential of water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (Dox) conjugates containing a Hsp47/CBP2 binding peptide sequence, namely WHYPWFQNWAMA for targeted delivery to SCCHN. An HPMA copolymer containing Dox and CBP2 targeting peptide conjugated via lysosomally degradable glycylphenylalanylleucylglycine (GFLG) spacer was synthesized by free radical precipitation copolymerization. A control polymer without targeting moiety was also synthesized. The conjugates were characterized for drug content, peptide content, molecular weight and molecular weight distribution. The uptake of polymeric conjugates by both drug resistant and drug sensitive SCCHN cells were determined in vitro by flow cytometry using FACS scan analysis. Cytotoxicity of the conjugates towards drug sensitive as well as multidrug resistant SCCHN cells were evaluated by a clonal survival assay and compared to free Dox. The cytotoxicity of the free peptide was similarly evaluated. The internalization and subcellular fate of the conjugates in drug sensitive SCCHN cells was monitored using confocal microscopy. The new targetable copolymer contained 0.16 mmole peptide/g polymer. Studies on drug sensitive SCCHN cells demonstrated lesser uptake of both targeted and non-targeted conjugates compared to free Dox suggesting a slower endocytic mechanism of uptake for the conjugates as opposed to rapid diffusion of free Dox. At higher Dox equivalent concentrations (>20 microM) the targeted conjugate showed significantly higher uptake (p < or = 0.028) than the non-targeted conjugate. The uptake of the targeted conjugate was inhibited in the presence of an anti Hsp47 antibody suggesting the involvement of active receptor mediated endocytosis in cell entry of the conjugate. Compared to free Dox, the targeted and non-targeted conjugates caused marginally lower inhibition (p < or = 0.01) of the drug sensitive SCCHN cells. In contrast, the same conjugates showed significantly higher uptake (p < or = 0.004) by drug resistant SCCHN cells and caused significantly higher inhibition (p < or = 0.02) of drug resistant SCCHN cells when compared to free Dox. Results suggest that the polymeric conjugates were able to overcome drug resistance. Confocal microscopy studies demonstrated the uptake of the polymeric conjugates, followed by internalization, intralysosomal localization and subsequent release of Dox. HPMA copolymer-Dox-peptide conjugates targeted to SCCHN cells were able to overcome drug resistance and increase efficacy in vitro. The results suggest that targetable polymeric conjugates have potential to improve systemic head and neck cancer chemotherapy by increasing tumor localization and reducing dose-limiting toxicity.

Antibiotics, Antineoplastic↗

PubMed2003

Gap mapping: a paradigm for aligning two sequences.

Pairwise sequence alignment is one of the most essential tools in comparative genomic sequence analysis. It is used to compare the sequences of genes and proteins with the aim of inferring structural, functional and evolutionary relationships. However, current 'mainstream' alignment algorithms have optimisation criteria based primarily on computational efficiency using parameters such as gap penalties, which are not biologically motivated. In addition, current alignment algorithms such as the Smith and Waterman technique provide a single alignment that could be sensitive to rather arbitrary choices in parameters such as gap penalties. This paper explores the range of properties resulting from posing the alignment problem more as a 'mapping gaps in sequences' exercise. We argue that this approach is intuitive and provides greater control over the number of gaps placed within an alignment. This type of approach was proposed by Sankoff (1972), but unfortunately has not received much attention. We report and discuss our findings by comparing this approach to other techniques using structurally confirmed aligned sequences from a benchmark alignment database. Interestingly, this approach consistently provides optimal and near optimal alignments and is thus a viable approach to sequence alignment.

Algorithms↗

PubMed2002

The effect of substituents on the phenyl portion of the imido ligand on the structure and properties of molybdenum(VI) imido complexes.

Anilines with alkyl substituents on the phenyl ring (ArNH2 = 2,4,6-trimethylaniline; 2,3-, 2,4-, 2,6-, and 3,4-dimethylaniline; and 2,6-diisopropylaniline) react with MoO(X)2(dtc)2 (X = Cl or Br; dtc = diethyldithiocarbamate) in methanol in the presence of 2 equiv of triethylamine to form ionic imido complexes of the type [MoNAr(dtc)3]2[Mo6O19] or MoNAr(dtc)3]4[Mo8O26]. The same reaction in THF with butyllithium as base yields imido complexes of the type MoNAr(X)2(dtc)2. The structures of three ionic, five chloro, and two bromo complexes have been determined by X-ray crystallography. In all complexes, the molybenum center is a distorted pentagonal bipyramid. While the structures are similar, the angles of the imido linkages differ. The effect of the substituents on the phenyl ring of the imido ligand on the 95Mo NMR chemical shifts was determined. The Mo nucleus becomes more deshielded with the substituents in the following order: 3,4-Me2 < 2,3-Me2 < 2,4-Me2 < 2,6-Me2 < 2,4,6-Me3 < 2,6 isopropyl. Complexes with more deshielded 95Mo centers tend to have angles of the imido linkage that are closer to 180 degrees.

Journal Article↗

PubMed2002

The encapsulation of ribozymes in biodegradable polymeric matrices.

Ribozymes are catalytic RNA that bind and cleave specific regions of target RNA. Therefore, protein synthesis by the target RNA may be specifically inhibited by ribozymes. However, ribozymes are rapidly cleared from plasma so effective treatment of proliferative diseases may rely on the repeated administration of these agents to maintain therapeutic ribozyme concentrations. Therefore, the objective of this study was to encapsulate ribozymes in injectable polymeric paste and microsphere formulations to allow for the controlled release of these agents over extended periods of time. Ribozymes were effectively encapsulated in poly(L-lactic acid) (PLLA) and poly(lactic-co-glycolic) (PLGA) microspheres in various size ranges using a modified water-in-oil-in-water emulsion system and in poly(epsilon-caprolactone) (PCL) pastes by physical blending. These formulations released non-degraded ribozymes, in vitro, in a controlled manner. PLLA microspheres released the ribozymes rapidly whereas PLGA released drugs more slowly. The release rate of ribozymes from PCL pastes could be effectively controlled by altering the loading concentration of ribozymes in the paste. These polymeric injectable formulations of ribozymes may allow for the extended treatment of localized disease sites, such as cancer and arthritis, without the need for repeated dosing.

Biodegradation, Environmental↗