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Biomedical subjects

Amit K Galande

Publications and source records attributed to Amit K Galande.

6 recordsLinked to original sources
PubMed2006

Enzyme-targeted fluorescent imaging probes on a multiple antigenic peptide core.

Peptide dendrimers have a variety of applications in biology such as the vehicles for drug and gene delivery, molecular inhibitors, protein mimics, and synthetic vaccines. The multiple antigenic peptide (MAP) system is a well-known example of a discrete, dendrimeric scaffold. We explored a novel application of the MAP-based scaffold by designing molecular probes that fluoresce only after enzymatic treatment. The probes, which were synthesized on solid support, incorporate a cathepsin S dipeptide substrate (Leu-Arg), and a poly(ethylene glycol) (PEG) chain in their dendritic arms. The fluorescence emission of the near-infrared fluorochromes attached to the N-termini of the dendritic arms was quenched. Mechanistic studies revealed formation of H-type dye aggregates within the tetravalent MAP system. By varying the length of the PEG chain, three probes were synthesized, CyPEG-1, CyPEG-2, and CyPEG-3 with 4, 8, and 12 ethylene oxide units, respectively. CyPEG-2 showed optimum aqueous solubility and quenching efficiency for imaging applications. Upon proteolytic activation with cathepsin S (EC 3.4.22.27), CyPEG-2 showed greater than 70-fold increase and more than 95% recovery in fluorescence emission.

Cathepsins↗
PubMed2005

Potent inhibitors of LXXLL-based protein-protein interactions.

Protein-protein interactions between estrogen receptors, ERalpha and ERbeta, and their coactivators (CoAs) are an attractive target for drug intervention. This interaction is mediated by a small pentapeptide motif (LXXLL), termed the NR box. Based on this motif, a variety of cyclic and linear peptides were synthesized in order to gain a better understanding of the association of CoA proteins with the ER isoforms. Utilizing a time-resolved florescence-based coactivator interaction assay, we determined the abilities of these peptides to inhibit this interaction. Using molecular modeling and CD spectroscopy, we have examined the structural basis of their bioactivities with both hormone receptor isoforms. Either homocysteine or penicillamine was utilized as a substitute for cysteine in the disulfide-bridged peptides, while tertiary leucine and neopentyl glycine were used as the surrogates for the NR box leucines. The most potent disufide-bridged peptide (K(i)= 70 pM, with ERalpha) incorporates neopentyl glycine in the NR box, while the most active peptide in this series with ERbeta (K(i)=350 pM) incorporates tertiary leucine. Surprisingly, several linear peptides containing a single cysteine residue showed activities with low nanomolar K(i) values. Collectively, our results suggest a synthetic approach for designing potent and selective peptidomimetics for ERalpha and ERbeta interactions with CoA proteins effecting estrogen action.

Acetate-CoA Ligase↗
PubMed2003

Solid-phase synthesis of disulfide heterodimers of peptides.

[structure: see text] The synthesis of the orthogonal disulfide template 1 and its use to synthesize unsymmetrical intermolecular disulfide bond peptides on a solid support are described. Application of template 1 to synthesize bioconjugates of cell permeable moieties based on the disulfide bond is demonstrated.

Amino Acid Sequence↗
PubMed2003

Understanding base-assisted desulfurization using a variety of disulfide-bridged peptides.

A recently rediscovered reaction of base-assisted lanthionine formation has been applied to several systems of disulfide-bridged peptides. In addition to previously described nonapeptides consisting of i, i+3 cystine linkages, the reaction has now been extended to systems consisting of shorter (i, i+2) and longer (i, i+4) disulfide bridges. The desulfurization reaction is also compatible with disulfide bridges formed through homocysteines and penicillamines, yielding unusual amino acids such as cystathionine and beta,beta-dimethyl lanthionine (referred to as "penthionine") in a peptide chain, respectively. Systematic study of this transformation has provided several new insights into its mechanism. We have observed formation of dehydroalanine and dehydrovaline residues resulting from i, i+2-bridged cysteines and i, i+3-bridged cysteine/penicillamine peptides, respectively, thereby supporting a beta-elimination/Michael-addition mechanism for this transformation. Amino acid analysis and NMR data from total correlation spectroscopy (TOCSY) and (1)H-(13)C heteronuclear single quantum correlation (HSQC) experiments show three diastereomeric lanthionine-bridged peptides in the product mixture. But in the case of desulfurization of a cysteine/homocysteine containing disulfide-bridged peptide, Michael addition appears to be stereoselective, yielding a single stereoisomer of cystathionine within the peptide. According to molecular modeling and CD spectroscopy, constrained peptides such as those containing penicillamine are less likely to undergo facile desulfurization. Flexibility of the torsional angles (C(alpha)H-C(alpha)-C(beta)-S) corresponding to the cysteine residues and temperature appear to be contributing factors determining the extent of desulfurization.

Cystine↗
PubMedDate not supplied

Fluorescence probe with a pH-sensitive trigger.

Acid-catalyzed hydrolysis was used as the mechanism to design a new type of environmentally sensitive fluorescence probe. A mild and selective periodate oxidation of the 2-amino alcohol of serine in the presence of a disulfide bond was developed to prepare dialdehyde peptides. Two identical fluorochrome hydrazide derivatives were then linked to the dialdehyde peptide forming an acid-labile hydrazone linkage. This self-quenched probe is weakly fluorescent at a physiological pH of 7.4 but shows more than 3-fold fluorescence enhancement at pH 4.5.

Amino Acid Sequence↗