High-throughput platform for design and screening of peptides as inhibitors of calcium oxalate monohydrate crystallization Sahar Farmanesh a , Jihae Chung a , Divya Chandra b , Ricardo D. Sosa a , Pankaj Karande b,n , Jeffrey D. Rimer a,nn a University of Houston, Department of Chemical and Biomolecular Engineering, Houston, TX 77204, USA b Rensselaer Polytechnic Institute, Department of Chemical and Biological Engineering, Troy, NY 12180, USA article info Available online 23 September 2012 Keywords: A1. Biocrystallization A1. Biomaterials B1. Calcium oxalate B1. Peptides B1. Proteins B2. Growth inhibitors abstract Crystal growth modifiers present a versatile tool for controlling crystal shape and size. Our work described here focuses on the design and screening of short peptides as inhibitors of calcium oxalate monohydrate (COM) crystals using high-throughput approaches. We designed a small library of 13 peptides containing Ala and Asp amino acids arranged in varying sequences that mimic ubiquitous motifs in natural calcium-binding proteins. Peptides were screened using a quick assay to measure their efficacy for inhibiting COM crystallization. Our results show that subtle variations in the placement of Ala and Asp residues in the peptide sequence can have a profound effect on their inhibition potential. We were able to discover peptide sequences that inhibit COM crystallization more effectively than some of the well-known COM inhibitors, such as citrate. Our results also demonstrate that peptides can be engineered to bind to specific faces of COM crystals. Peptide sequences identified in this work are promising candidates for further development as therapies for biomineral-related diseases, such as kidney stone disease. Collectively, our work establishes new paradigms for the design, synthesis, and screening of peptides for controlling crystal habit with the potential to impact a variety of fields, including drug discovery, advanced materials, catalysis and separations. & 2012 Elsevier B.V. All rights reserved. 1. Introduction Crystallization is ubiquitous in biological systems where inter- actions between inorganic (salt, ions, etc.) and organic compo- nents (proteins, lipids, etc.) often mediate physiological processes in the human body, such as bone and teeth formation [1,2]. Under abnormal physiological conditions, mineralization can lead to such pathologies as atherosclerotic plaques or vascular calcifi- cations, kidney or gallstones, gout, and osteoarthritis. Small molecules that inhibit abnormal biomineralization are potentially effective therapies against such conditions. Kidney stone disease is a common pathological disorder that affects more than 10–15% of the US population with incidence rates that are on the rise [3, 4]. Kidney stone pathogenesis is a complex process that involves a series of steps operating either singularly or synergistically to produce polycrystalline aggregates in the kidney [5]. Calcium oxalate monohydrate (COM) is the most common compo- nent of human kidney stones. Supersaturated calcium oxalate in urine facilitates COM crystal nucleation and growth. The aggregation of COM crystals and the cumulative retention of crystals and/or aggre- gates in the kidney have adverse effects once stones reach an appreciable size and become dislodged from the epithelial mem- brane. Inhibiting one or more of the critical pathways of COM stone pathogenesis (nucleation, growth, aggregation, and retention) via pathogenesis (nucleation, the addition of external agents can poten- tially serve as an effective therapy for this disease. It has been proposed that crystal growth inhibitors possess two types of moieties, a binder that strongly interacts with crystal surface sites, and a perturber that sterically hinders the attachment of solute to crystal surfaces [6]. A common binder group of COM crystal inhibitors (i.e. urinary proteins and their synthetic analogs discussed later) is carboxylic acid, which binds to oxalate vacancies on COM crystal surfaces via calcium bridges, (COM) COO  yCa 2þ y  OOC (inhibitor) . Weissbuch et al. [7] have proposed mechanisms of crystal growth inhibition that occur through the adsorption of small molecules to surfaces of crystals growing by classical nucleation and spreading of layers (so called layer-by-layer growth). Inhibitors that bind to different sites on a crystal surface (i.e. steps, ledges, and terraces) reduce step advancement normal to that surface. Inhibitors can therefore serve to retard crystal growth, with implications in therapies for biomineralization-based diseases, or alter growth rates Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/jcrysgro Journal of Crystal Growth 0022-0248/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcrysgro.2012.09.018 n Corresponding author. Tel.: þ1 518 276 4459; fax: þ1 518 276 4030. nn Corresponding author. Tel.: þ1 713 743 4131; fax: þ1 713 743 4323. E-mail addresses: karanp@rpi.edu (P. Karande), jrimer@central.uh.edu (J.D. Rimer). Journal of Crystal Growth 373 (2013) 13–19