RESUMEN
This retrospective case series describes the radiographic features of suspected suture-associated cystic calculi in six dogs with a history of at least one or multiple prior cystotomies. One of the dogs presented twice. Suspected suture-associated cystic calculi were multifocal, short, predominantly linear mineral opacities localized in the center of the urinary bladder on abdominal radiographs. One patient (n = 1) presented with multifocal round, pin point, and linear radiopaque calculi. The calculi were all calcium oxalate in composition. On gross examination, the calculi had a hollow center. Six cystotomies used monofilament absorbable suture material (polydioxanone [n = 4] or poliglecaprone 25 [n = 1]) in prior cystotomies. Suture material in two of the cases was unknown. Suspected suture-associated cystic calculi are a rare occurrence in veterinary medicine but should be considered in dogs that have a history of prior cystotomy, hollow core on gross analysis, and radiographic evidence of mineral opaque, predominantly linear, cystic calculi.
Asunto(s)
Cistotomía/veterinaria , Enfermedades de los Perros/etiología , Suturas/veterinaria , Cálculos de la Vejiga Urinaria/veterinaria , Animales , Cistotomía/efectos adversos , Enfermedades de los Perros/diagnóstico por imagen , Enfermedades de los Perros/cirugía , Perros , Estudios Retrospectivos , Suturas/efectos adversos , Cálculos de la Vejiga Urinaria/etiologíaRESUMEN
ß-Sheets account for over 30 % of all secondary structural conformations found in proteins. The intramolecular hydrogen bonding that exists between the two peptide strands is imperative in maintaining this secondary structure. With the proper design, cyclic peptides may act as scaffolds emulating active ß-sheet regions, enabling investigation of their importance in molecular recognition and protein aggregation. Starting from Fmoc-Lys(Fmoc)-OH, macrocyclic peptides were synthesized on a solid support, with peptide-chain elongation extending from both the alpha and epsilon amines of the lysine. The branching peptides were cyclized with a pyridyl tridentate chelation core followed by coordination using [(99m) Tc/Re(CO)3 (H2 O)3 ](+) . Variable temperature (1) Hâ NMR spectroscopy studies were performed, demonstrating that intramolecular hydrogen bonding exists between the two sides of the uncoordinated macrocyclic peptide scaffolds. Additionally, computational modelling and circular dichroism spectroscopic analysis revealed that the peptide backbone exists in a similar conformation both before and after metal coordination. The ability to seamlessly incorporate a tridentate chelation core into the backbone of a macrocyclic peptide, without disrupting the secondary structure, can greatly assist in the design of metal-centric peptidomimetic imaging agents. This novel integrated imaging probe approach may facilitate the investigation into protein-protein interactions using macrocyclic ß-sheet scaffolds.
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Compuestos Macrocíclicos/química , Péptidos Cíclicos/química , Renio/química , Tecnecio/química , Compuestos Macrocíclicos/síntesis química , Modelos Moleculares , Sondas Moleculares/síntesis química , Sondas Moleculares/química , Péptidos Cíclicos/síntesis química , Estructura Secundaria de ProteínaRESUMEN
The first solid-phase parallel synthesis of macrocyclic peptides using three-component coupling driven by aziridine aldehyde dimers is described. The method supports the synthesis of 9- to 18-membered aziridine-containing macrocycles, which are then functionalized by nucleophilic opening of the aziridine ring. This constitutes a robust approach for the rapid parallel synthesis of macrocyclic peptides.
RESUMEN
There is an ever-increasing interest in synthetic methods that not only enable peptide macrocyclization, but also facilitate downstream application of the synthesized molecules. We have found that aziridine amides are stereoelectronically attenuated in a macrocyclic environment such that non-specific interactions with biological nucleophiles are reduced or even shut down. The electrophilic reactivity, revealed at high pH, enables peptide sequencing by mass spectrometry, which will further broaden the utility of aziridine amide-containing libraries of macrocycles.
Asunto(s)
Amidas/química , Electrones , Péptidos Cíclicos/química , Análisis de Secuencia de Proteína , Aziridinas/química , Hidrólisis , Cetonas/química , Espectrometría de MasasRESUMEN
The concept of site-specific integration of fragments into macrocyclic entities has not yet found application in the realm of synthetic chemistry. Here we show that the reduced amidicity of aziridine amide bonds provides an entry point for the site-specific integration of amino acids and peptide fragments into the homodetic cyclic peptide architecture. This new synthetic operation improves both the convergence and divergence of cyclic peptide synthesis.
Asunto(s)
Amidas/química , Aminoácidos/química , Aziridinas/química , Fragmentos de Péptidos/química , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/químicaRESUMEN
The factors determining diastereoselectivity observed in the multicomponent conversion of amino acids, aziridine aldehyde dimers, and isocyanides into chiral piperazinones have been investigated. Amino acid-dependent selectivity for either trans- or cis-substituted piperazinone products has been achieved. An experimentally determined diastereoselectivity model for the three-component reaction driven by aziridine aldehyde dimers has predictive value for different substrate classes. Moreover, this model is useful in reconciling the previously reported observations in multicomponent reactions between isocyanides, α-amino acids, and monofunctional aldehydes.
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Aldehídos/química , Aminoácidos/química , Aziridinas/química , Cianuros/química , Dicetopiperazinas/química , Estructura Molecular , EstereoisomerismoRESUMEN
Life is constructed primarily using a toolbox of 20 canonical amino acids-relying upon these building blocks for the assembly of proteins and peptides that regulate nearly every cellular task, including cell structure, function, and maintenance. While Nature continues to be a source of inspiration for drug discovery, medicinal chemists are not beholden to only 20 canonical amino acids and have begun to explore non-canonical amino acids (ncAAs) for the construction of designer peptides with improved drug-like properties. However, as our toolbox of ncAAs expands, drug hunters are encountering new challenges in approaching the iterative peptide design-make-test-analyze cycle with a seemingly boundless set of building blocks. This Microperspective focuses on new technologies that are accelerating ncAA interrogation in peptide drug discovery (including HELM notation, late-stage functionalization, and biocatalysis) while shedding light on areas where further investment could not only accelerate the discovery of new medicines but also improve downstream development.
RESUMEN
Nucleic acid assays are not typically deployable in point-of-care settings because they require costly and sophisticated equipment for the control of the reaction temperature and for the detection of the signal. Here we report an instrument-free assay for the accurate and multiplexed detection of nucleic acids at ambient temperature. The assay, which we named INSPECTR (for internal splint-pairing expression-cassette translation reaction), leverages the target-specific splinted ligation of DNA probes to generate expression cassettes that can be flexibly designed for the cell-free synthesis of reporter proteins, with enzymatic reporters allowing for a linear detection range spanning four orders of magnitude and peptide reporters (which can be mapped to unique targets) enabling highly multiplexed visual detection. We used INSPECTR to detect a panel of five respiratory viral targets in a single reaction via a lateral-flow readout and ~4,000 copies of viral RNA via additional ambient-temperature rolling circle amplification of the expression cassette. Leveraging synthetic biology to simplify workflows for nucleic acid diagnostics may facilitate their broader applicability at the point of care.
Asunto(s)
Ácidos Nucleicos , ARN Viral , ARN Viral/genética , Temperatura , Férulas (Fijadores) , Sondas de ADNRESUMEN
Molecular scaffolds have been shown to facilitate and stabilise secondary structural turn elements, with a central core-arranging functionality in a defined three-dimensional orientation. In a peptide-based molecular imaging probe, this approach is of particular value as it would essentially "hide" a metal radioisotope within the ligand framework, making the labelling element a critical component of the receptor-bound structure. Starting from a 1,2-diaminoethane loaded 2-chlorotrityl resin, a versatile set of triamine ligand systems were synthesised by using solid-phase Fmoc-based peptide chemistry. The resultant resin-bound peptides then underwent amide reduction by treatment with borane-THF at 65 °C. This provided complete conversion to the corresponding polyamine entities in high purity for the majority of the amino acids utilised. The triamines were then coordinated on solid support by using [NEt(4)](2)[Re(CO)(3)(Br)(3)] followed by resin cleavage and HPLC purification, to give the desired rhenium coordinated species. We have shown that amino acid sequences can be assembled, reduced and coordinated on-resin, resulting in a versatile set of metal-ligand constructs. These studies could be expanded to generate libraries of turn-based peptidomimetics containing Re/Tc(I) organometallic scaffolds, with the intention of developing an improved approach for finding new diagnostic and therapeutic radiopharmaceutical entities.
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Metales/química , Compuestos Organometálicos/química , Compuestos Organometálicos/síntesis química , Péptidos/química , Radioisótopos/química , Radiofármacos/química , Radiofármacos/síntesis química , Renio/química , Cromatografía Líquida de Alta Presión , Ligandos , Estructura MolecularRESUMEN
Multivalent nanoparticles have several key advantages in terms of solubility, binding avidity, and uptake, making them particularly well suited to molecular imaging applications. Herein is reported the stepwise synthesis and characterization of NIR viral nanoparticles targeted to gastrin-releasing peptide receptors that are over-expressed in human prostate cancers. The pan-bombesin analogue, [ß-Ala11, Phe13, Nle14]bombesin-(7-14), is conjugated to cowpea mosaic virus particles functionalized with an NIR dye (Alexa Fluor 647) and polyethylene glycol (PEG) using the copper(I)-catalyzed azide-alkyne cycloaddition reaction. Targeting and uptake in human PC-3 prostate cells is demonstrated in vitro. Tumor homing is observed using human prostate tumor xenografts on the chicken chorioallantoic membrane model using intravital imaging. Further development of this viral nanoparticle platform may open the door to potential clinical noninvasive molecular imaging strategies.
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Nanopartículas/química , Neoplasias de la Próstata/patología , Receptores de Bombesina/metabolismo , Animales , Bombesina/química , Línea Celular Tumoral , Pollos , Comovirus/química , Humanos , Masculino , Polietilenglicoles/químicaRESUMEN
At the forefront of synthetic endeavors in the pharmaceutical industry, including drug discovery and high-throughput screening, timelines are tight and large quantities of pure chemical targets are rarely available. In this regard, the development of novel and increasingly challenging chemistries requires a commensurate level of innovation to develop reliable analytical assays and purification workflows with rapid turnaround that enables accelerated pharmacological evaluation. A small-scale automation platform enabling high-throughput analysis and purification to streamline the selection of candidate leads would be a transformative advance. Herein, we introduce an automation-friendly solid-phase extraction-matrix-assisted laser desorption/ionization (SPE-MALDI) platform applied to the high-throughput purification and analysis of peptide libraries. This advance enabled us to purify peptides from microgram levels in less than a day with results comparable to traditional high-performance liquid chromatography-diode array detection-mass spectrometry (HPLC-DAD-MS).
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Biblioteca de Péptidos , Péptidos , Ensayos Analíticos de Alto Rendimiento , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Flujo de TrabajoRESUMEN
Identification of proteins that couple kinetochores to spindle microtubules is critical for understanding how accurate chromosome segregation is achieved in mitosis. Here we show that the protein hNuf2 specifically functions at kinetochores for stable microtubule attachment in HeLa cells. When hNuf2 is depleted by RNA interference, spindle formation occurs normally as cells enter mitosis, but kinetochores fail to form their attachments to spindle microtubules and cells block in prometaphase with an active spindle checkpoint. Kinetochores depleted of hNuf2 retain the microtubule motors CENP-E and cytoplasmic dynein, proteins previously implicated in recruiting kinetochore microtubules. Kinetochores also retain detectable levels of the spindle checkpoint proteins Mad2 and BubR1, as expected for activation of the spindle checkpoint by unattached kinetochores. In addition, the cell cycle block produced by hNuf2 depletion induces mitotic cells to undergo cell death. These data highlight a specific role for hNuf2 in kinetochore-microtubule attachment and suggest that hNuf2 is part of a molecular linker between the kinetochore attachment site and tubulin subunits within the lattice of attached plus ends.
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Muerte Celular/fisiología , Proteínas Fúngicas/metabolismo , Células HeLa/metabolismo , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Mitosis/fisiología , Proteínas Nucleares/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Dineínas/metabolismo , Citometría de Flujo , Proteínas Fúngicas/genética , Células HeLa/citología , Humanos , Proteínas Mad2 , Proteínas Nucleares/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas Represoras , Huso Acromático/metabolismoRESUMEN
OBJECTIVES: Ideal long-term vocal fold augmentation materials should be biocompatible, easily administered, allow tissue integration for long-term effect, and remain at the site of injection. A novel silk protein particle suspended in hyaluronic acid (Silk-HA) has been developed specifically for vocal fold augmentation to address this unmet need. This article presents the 6-month, preclinical findings of a canine vocal fold injection trial for Silk-HA. METHODS: Twelve beagle dogs were injected transorally in the lateral/deep aspect of their right thyroarytenoid muscles with 0.3 cc of Silk-HA or calcium hydroxylapatite in carboxymethyl cellulose (CaHA-CMC). The Silk-HA particle injectable was delivered via a custom catheter, whereas CaHA-CMC was delivered through a commercially available malleable needle. The six dogs from each material group were sacrificed 6 months from the injection date for the evaluation of implant longevity, immune response, and material migration. RESULTS: Silk-HA provides immediate medialization of the right vocal fold, lasting for a minimum of 6 months in a canine model. Silk-HA and CaHA-CMC both demonstrate similar inflammatory responses. The Silk-HA was shown to remain without migration at the site of injection in all six canine subjects, whereas CaHA-CMC demonstrated migration in four of the six canines. In two canines implanted with CaHA-CMC, material was discovered to migrate to the retropharyngeal lymph nodes. CONCLUSION: In a canine subject model, the Silk-HA material compares favorably in terms of longevity and immune response to CaHA-CMC. The lack of migration of the Silk-HA material demonstrates a promising potential for vocal fold injection in the clinic. LEVEL OF EVIDENCE: NA Laryngoscope, 129:1856-1862, 2019.
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Materiales Biocompatibles/administración & dosificación , Ácido Hialurónico/administración & dosificación , Seda/administración & dosificación , Animales , Carboximetilcelulosa de Sodio/administración & dosificación , Perros , Durapatita/administración & dosificación , Inyecciones Intramusculares , Músculos Laríngeos , Modelos Animales , Factores de Tiempo , Parálisis de los Pliegues Vocales/terapia , Pliegues VocalesRESUMEN
A major goal in the study of vertebrate mitosis is to identify proteins that create the kinetochore-microtubule attachment site. Attachment sites within the kinetochore outer plate generate microtubule dependent forces for chromosome movement and regulate spindle checkpoint protein assembly at the kinetochore. The Ndc80 complex, comprised of Ndc80 (Hec1), Nuf2, Spc24, and Spc25, is essential for metaphase chromosome alignment and anaphase chromosome segregation. It has also been suggested to have roles in kinetochore microtubule formation, production of kinetochore tension, and the spindle checkpoint. Here we show that Nuf2 and Hec1 localize throughout the outer plate, and not the corona, of the vertebrate kinetochore. They are part of a stable "core" region whose assembly dynamics are distinct from other outer domain spindle checkpoint and motor proteins. Furthermore, Nuf2 and Hec1 are required for formation and/or maintenance of the outer plate structure itself. Fluorescence light microscopy, live cell imaging, and electron microscopy provide quantitative data demonstrating that Nuf2 and Hec1 are essential for normal kinetochore microtubule attachment. Our results indicate that Nuf2 and Hec1 are required for organization of stable microtubule plus-end binding sites in the outer plate that are needed for the sustained poleward forces required for biorientation at kinetochores.
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Cinetocoros/química , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Anticuerpos Monoclonales/metabolismo , Proteínas de Ciclo Celular , Proteínas del Citoesqueleto , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Cinetocoros/efectos de los fármacos , Cinetocoros/ultraestructura , Microscopía Fluorescente , Microtúbulos/ultraestructura , Mitosis , Nocodazol/farmacología , Proteínas Nucleares/ultraestructura , ARN Interferente Pequeño/metabolismo , Tinción con Nitrato de PlataAsunto(s)
Ácidos Borónicos/química , Cianuros/química , Péptidos/química , Humanos , Estructura MolecularRESUMEN
BACKGROUND: Pregnancy resource centers (PRCs) are nonprofit organizations with a primary mission of promoting childbirth among pregnant women. Given a new state grant program to publicly fund PRCs, we analyzed Georgia PRC websites to describe advertised services and related health information. METHODS: We systematically identified all accessible Georgia PRC websites available from April to June 2016. Entire websites were obtained and coded using defined protocols. RESULTS: Of 64 reviewed websites, pregnancy tests and testing (98%) and options counseling (84%) were most frequently advertised. However, 58% of sites did not provide notice that PRCs do not provide or refer for abortion, and 53% included false or misleading statements regarding the need to make a decision about abortion or links between abortion and mental health problems or breast cancer. Advertised contraceptive services were limited to counseling about natural family planning (3%) and emergency contraception (14%). Most sites (89%) did not provide notice that PRCs do not provide or refer for contraceptives. Two sites (3%) advertised unproven "abortion reversal" services. Approximately 63% advertised ultrasound examinations, 22% sexually transmitted infection testing, and 5% sexually transmitted infection treatment. None promoted consistent and correct condom use; 78% with content about condoms included statements that seemed to be designed to undermine confidence in condom effectiveness. Approximately 84% advertised educational programs, and 61% material resources. CONCLUSIONS: Georgia PRC websites contain high levels of false and misleading health information; the advertised services do not seem to align with prevailing medical guidelines. Public funding for PRCs, an increasing national trend, should be rigorously examined. Increased regulation may be warranted to ensure quality health information and services.
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Publicidad , Decepción , Servicios de Planificación Familiar , Internet , Organizaciones sin Fines de Lucro , Servicios de Salud Reproductiva , Aborto Inducido , Acceso a la Información , Condones , Anticoncepción/métodos , Anticonceptivos , Consejo , Servicios de Planificación Familiar/ética , Servicios de Planificación Familiar/normas , Femenino , Financiación Gubernamental , Georgia , Educación en Salud , Recursos en Salud , Humanos , Organizaciones sin Fines de Lucro/ética , Organizaciones sin Fines de Lucro/normas , Embarazo , Servicios de Salud Reproductiva/ética , Servicios de Salud Reproductiva/normas , Conducta Sexual , Enfermedades de Transmisión Sexual/diagnóstico , Enfermedades de Transmisión Sexual/terapia , Ultrasonografía PrenatalRESUMEN
Members of the Ndc80/Nuf2 complex have been shown in several systems to be important in formation of stable kinetochore-microtubule attachments and chromosome alignment in mitosis. In HeLa cells, we have shown that depletion of Nuf2 by RNA interference (RNAi) results in a strong prometaphase block with an active spindle checkpoint, which correlates with low but detectable Mad2 at kinetochores that have no or few stable kinetochore microtubules. Another RNAi study in HeLa cells reported that Hec1 (the human Ndc80 homolog) is required for Mad1 and Mad2 binding to kinetochores and that kinetochore bound Mad2 does not play a role in generating and maintaining the spindle assembly checkpoint. Here, we show that depletion of either Nuf2 or Hec1 by RNAi in HeLa cells results in reduction of both proteins at kinetochores and in the cytoplasm. Mad1 and Mad2 concentrate at kinetochores in late prophase/early prometaphase but become depleted by 5-fold or more over the course of the prometaphase block, which is Mad2 dependent. The reduction of Mad1 and Mad2 is reversible upon spindle depolymerization. Our observations support a model in which Nuf2 and Hec1 function to prevent microtubule-dependent stripping of Mad1 and Mad2 from kinetochores that have not yet formed stable kinetochore-microtubule attachments.
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Proteínas de Unión al Calcio/metabolismo , Cinetocoros/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas Represoras/metabolismo , Huso Acromático/fisiología , Proteínas de Ciclo Celular , Proteínas del Citoesqueleto , Técnica del Anticuerpo Fluorescente , Expresión Génica , Células HeLa , Humanos , Proteínas Mad2 , Interferencia de ARNRESUMEN
Thrombospondin-1 (TSP-1) and transforming growth factor-beta1 (TGF-beta1) are both implicated in the pathogenesis of in-stent restenosis. This study evaluated the hypothesis that the HMG-CoA reductase inhibitor fluvastatin inhibits TGF-beta1 induced TSP-1 expression via inhibition of p38 mitogen activated protein kinase (MAPK) phosphorylation in human coronary artery smooth muscle cells (HCASMC) and may therefore have anti-restenosis potential. Fluvastatin significantly reduced TSP-1 mRNA and protein expression in HCASMC in a concentration-dependent manner with a significant reduction in expression observed after treatment with 0.25 microM fluvastatin. TGF-beta1 (5 ng/ml) induced phosphorylation of p38 MAPK and induced TSP-1 mRNA and protein expression in HCASMC. Fluvastatin abolished TGF-beta1-induced phosphorylation of p38 MAPK and TGF-beta1-induced TSP-1 expression. Blockade of the p38 MAPK pathway with the upstream inhibitor SB-203580 also abolished TGF-beta1-induced TSP-1 expression. We conclude that fluvastatin decreases expression of TSP-1 and abolishes the ability of TGF-beta1 to induce TSP-1 expression in HCASMC; this may be achieved by preventing signalling through the p38 MAPK pathway. Targeted delivery of fluvastatin may therefore be a useful therapeutic objective for prevention of the intimal hyperplasia associated with in-stent restenosis.
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Ácidos Grasos Monoinsaturados/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Indoles/farmacología , Músculo Liso Vascular/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Adulto , Células Cultivadas , Reestenosis Coronaria/prevención & control , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/enzimología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Ácidos Grasos Monoinsaturados/uso terapéutico , Fluvastatina , Regulación de la Expresión Génica , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Imidazoles/farmacología , Indoles/uso terapéutico , Sistema de Señalización de MAP Quinasas , Masculino , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/enzimología , Fosforilación , Piridinas/farmacología , ARN Mensajero/metabolismo , Trombospondina 1/genética , Trombospondina 1/metabolismo , Factor de Crecimiento Transformador beta1RESUMEN
We have developed a strategy for synthesizing passively permeable peptidomimetic macrocycles. The cyclization chemistry centers on using aziridine aldehydes in a multicomponent reaction with peptides and isocyanides. The linker region in the resulting product contains an exocyclic amide positioned α to the peptide backbone, an arrangement that is not found among natural amino acids. This amide provides structural rigidity within the cyclic peptidomimetic and promotes the creation of a stabilizing intramolecular hydrogen bonding network. This exocyclic control element also contributes to the increased membrane permeability exhibited by multicomponent-derived macrocycles with respect to their homodetic counterparts. The exocyclic control element is employed along with a strategic placement of N-methyl and d-amino acids to produce passively permeable peptides, which contain multiple polar residues. This strategy should be applicable in the pursuit of synthesizing therapeutically relevant macrocycles.
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Amidas/química , Compuestos Macrocíclicos/química , Amidas/síntesis química , Compuestos Macrocíclicos/síntesis química , Conformación MolecularRESUMEN
Our previously reported structures of calpain bound to its endogenous inhibitor calpastatin have motivated the use of aziridine aldehyde-mediated peptide macrocyclization toward the design of cyclic peptides and peptidomimetics as calpain inhibitors. Inspired by nature's hint that a ß-turn loop within calpastatin forms a broad interaction around calpain's active site cysteine, we have constructed and tested a library of 45 peptidic compounds based on this loop sequence. Four molecules have shown reproducibly low micromolar inhibition of calpain-2. Further systematic sequence changes led to the development of probes that displayed increased potency and specificity of inhibition against calpain over other cysteine proteases. Calculated Ki values were in the low micromolar range, rivaling other peptidomimetic calpain inhibitors and presenting an improved selectivity profile against other therapeutically relevant proteases. Competitive and mixed inhibition against calpain-2 was observed, and an allosteric inhibition site on the enzyme was identified for a noncompetitive inhibitor.