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1.
J Pept Sci ; 25(9): e3203, 2019 Sep.
Article in English | MEDLINE | ID: mdl-31347248

ABSTRACT

Plasmodium falciparum is the most lethal species of malaria. In infected human red blood cells, P. falciparum digests hemoglobin as a nutrient source, liberating cytotoxic free heme in the process. Sequestration and subsequent conversion of this byproduct into hemozoin, an inert biocrystalline heme aggregate, plays a key role in parasite survival. Hemozoin has been a longstanding target of antimalarials such as chloroquine (CQ), which inhibit the biocrystallization of free heme. In this study, we explore heme-binding interactions with histidine-rich-protein 2 (HRP2), a known malarial biomarker and purported player in free heme sequestration. HRP2 is notoriously challenging to target due to its highly repetitious sequence and irregular secondary structure. We started with three protein-catalyzed capture agents (PCCs) developed against epitopes of HRP2, inclusive of heme-binding motifs, and explored their ability to inhibit heme:HRP2 complex formation. Cocktails of the individual PCCs exhibit an inhibitory potency similar to CQ, while a covalently linked structure built from two separate PCCs provided considerably increased inhibition relative to CQ. Epitope-targeted disruption of heme:HRP2 binding is a novel approach towards disrupting P. falciparum-related hemozoin formation.


Subject(s)
Epitopes/drug effects , Heme/antagonists & inhibitors , Peptides/pharmacology , Protozoan Proteins/antagonists & inhibitors , Amino Acid Sequence , Antigens, Protozoan/genetics , Epitopes/genetics , Heme/genetics , Humans , Molecular Conformation , Peptides/chemistry , Protozoan Proteins/genetics , Recombinant Proteins/genetics
2.
Chem Rev ; 119(17): 9950-9970, 2019 09 11.
Article in English | MEDLINE | ID: mdl-30838853

ABSTRACT

Protein-catalyzed capture agents (PCCs) are synthetic and modular peptide-based affinity agents that are developed through the use of single-generation in situ click chemistry screens against large peptide libraries. In such screens, the target protein, or a synthetic epitope fragment of that protein, provides a template for selectively promoting the noncopper catalyzed azide-alkyne dipolar cycloaddition click reaction between either a library peptide and a known ligand or a library peptide and the synthetic epitope. The development of epitope-targeted PCCs was motivated by the desire to fully generalize pioneering work from the Sharpless and Finn groups in which in situ click screens were used to develop potent, divalent enzymatic inhibitors. In fact, a large degree of generality has now been achieved. Various PCCs have demonstrated utility for selective protein detection, as allosteric or direct inhibitors, as modulators of protein folding, and as tools for in vivo tumor imaging. We provide a historical context for PCCs and place them within the broader scope of biological and synthetic aptamers. The development of PCCs is presented as (i) Generation I PCCs, which are branched ligands engineered through an iterative, nonepitope-targeted process, and (ii) Generation II PCCs, which are typically developed from macrocyclic peptide libraries and are precisely epitope-targeted. We provide statistical comparisons of Generation II PCCs relative to monoclonal antibodies in which the protein target is the same. Finally, we discuss current challenges and future opportunities of PCCs.


Subject(s)
Aptamers, Peptide/chemistry , Amino Acid Sequence , Animals , Aptamers, Peptide/chemical synthesis , Aptamers, Peptide/metabolism , Click Chemistry , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Humans , Ligands , Peptide Library , Peptoids/chemical synthesis , Peptoids/chemistry , Peptoids/metabolism , Protein Binding , Proteins/metabolism
3.
Anal Chem ; 90(15): 8824-8830, 2018 08 07.
Article in English | MEDLINE | ID: mdl-29979578

ABSTRACT

Protein catalyzed capture agents (PCCs) are synthetic antibody surrogates that can target a wide variety of biologically relevant proteins. As a step toward developing a high-throughput PCC pipeline, we report on the preparation of a barcoded rapid assay platform for the analysis of hits from PCC library screens. The platform is constructed by first surface patterning a micrometer scale barcode composed of orthogonal ssDNA strands onto a glass slide. The slide is then partitioned into microwells, each of which contains multiple copies of the full barcode. Biotinylated candidate PCCs from a click screen are assembled onto the barcode stripes using a complementary ssDNA-encoded cysteine-modified streptavidin library. This platform was employed to evaluate candidate PCC ligands identified from an epitope targeted in situ click screen against the two conserved allosteric switch regions of the Kirsten rat sarcoma (KRas) protein. A single microchip was utilized for the simultaneous evaluation of 15 PCC candidate fractions under more than a dozen different assay conditions. The platform also permitted more than a 10-fold savings in time and a more than 100-fold reduction in biological and chemical reagents relative to traditional multiwell plate assays. The best ligand was shown to exhibit an in vitro inhibition constant (IC50) of ∼24 µM.


Subject(s)
Allosteric Regulation/drug effects , DNA, Single-Stranded/chemistry , Enzyme Inhibitors/pharmacology , Microarray Analysis/methods , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Allosteric Site/drug effects , Biotinylation , Drug Evaluation, Preclinical/methods , Enzyme Inhibitors/chemistry , Humans , Proto-Oncogene Proteins p21(ras)/chemistry , Proto-Oncogene Proteins p21(ras)/metabolism , Streptavidin/chemistry
4.
Angew Chem Int Ed Engl ; 54(45): 13219-24, 2015 Nov 02.
Article in English | MEDLINE | ID: mdl-26377818

ABSTRACT

We describe a general synthetic strategy for developing high-affinity peptide binders against specific epitopes of challenging protein biomarkers. The epitope of interest is synthesized as a polypeptide, with a detection biotin tag and a strategically placed azide (or alkyne) presenting amino acid. This synthetic epitope (SynEp) is incubated with a library of complementary alkyne or azide presenting peptides. Library elements that bind the SynEp in the correct orientation undergo the Huisgen cycloaddition, and are covalently linked to the SynEp. Hit peptides are tested against the full-length protein to identify the best binder. We describe development of epitope-targeted linear or macrocycle peptide ligands against 12 different diagnostic or therapeutic analytes. The general epitope targeting capability for these low molecular weight synthetic ligands enables a range of therapeutic and diagnostic applications, similar to those of monoclonal antibodies.


Subject(s)
Drug Design , Epitopes/chemistry , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/pharmacology , Proteins/chemistry , Ligands , Molecular Weight , Peptides, Cyclic/chemistry , Proteins/antagonists & inhibitors
5.
Photochem Photobiol Sci ; 12(8): 1387-400, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23625036

ABSTRACT

Fluorescent analogues of the natural DNA bases are useful in the study of nucleic acids' structure and dynamics. 2-Aminopurine (2AP) is a widely used analogue with environmentally sensitive fluorescence behavior. The quantum yield of 2AP has been found to be significantly decreased when engaged in π-stacking interactions with the native bases. We present a theoretical study on fluorescence quenching mechanisms in dimers of 2AP π-stacked with adenine or guanine as in natural DNA. Relaxation pathways on the potential energy surfaces of the first excited states have been computed and reveal the importance of exciplexes and conical intersections in the fluorescence quenching process.


Subject(s)
2-Aminopurine/chemistry , Adenine/chemistry , Guanine/chemistry , Dimerization , Fluorescence , Models, Molecular
7.
J Am Chem Soc ; 133(17): 6799-808, 2011 May 04.
Article in English | MEDLINE | ID: mdl-21486032

ABSTRACT

Fluorescent analogues of nucleobases are very useful as probes to study DNA dynamics, because natural DNA does not fluoresce significantly. In many of these analogues, such as 2-aminopurine (2AP), the fluorescence is quenched when incorporated into DNA through processes that are not well understood. This work uses theoretical studies to examine fluorescence quenching pathways in 2AP-containing dimers. The singlet excited states of π-stacked dimer systems containing 2AP and a pyrimidine base, thymine or cytosine, have been studied using ab initio computational methods. Computed relaxation pathways along the excited-state surfaces reveal novel mechanisms that can lead to fluorescence quenching in the π-stacked dimers. The placement of 2AP on the 5' or 3' terminus of the dimers has different effects on the excitation energies and the relaxation pathways on the S(1) excited state. Conical intersections between the ground and first excited states exist when 2AP is placed at the 3' side, whereas the placement of 2AP at the 5' side leads to the switching of a bright state to a dark state. Both of these processes can lead to fluorescence quenching and may contribute to the fluorescence quenching observed in 2AP when incorporated in DNA.


Subject(s)
2-Aminopurine/chemistry , DNA/chemistry , Pyrimidines/chemistry , Computer Simulation , Dimerization , Fluorescence , Models, Molecular
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