Mirror-Image Random Nonstandard Peptide Integrated Discovery (MI-RaPID) Technology Yields Highly Stable and Selective Macrocyclic Peptide Inhibitors for Matrix Metallopeptidase 7.

Matrix metallopeptidase 7 (MMP7) plays a crucial role in cancer metastasis and progression, making it an attractive target for therapeutic development. However, the development of selective MMP7 inhibitors is challenging due to the conservation of active sites across various matrix metalloproteinases (MMPs). Here, we have developed mirror-image random nonstandard peptides integrated discovery (MI-RaPID) technology to discover innate protease-resistant macrocyclic peptides that specifically bind to and inhibit human MMP7. One identified macrocyclic peptide against D-MMP7, termed D20, was synthesized in its mirror-image form, D'20, consisting of 12 D-amino acids, one cyclic b-amino acid, and a thioether bond. Notably, it potently inhibited MMP7 with an IC50 value of 90 nM, and showed excellent selectivity over other MMPs with similar substrate specificity. Moreover, D'20 inhibited the migration of pancreatic cell line CFPAC-1, but had no effect on the cell proliferation and viability. D'20 exhibited excellent stability in human serum, as well as in simulated gastric and intestinal fluids. This study highlights that MI-RaPID technology can serve as a powerful tool to develop in vivo stable macrocyclic peptides for therapeutic applications.

A Case of a Mirror Image Artifact in the Forearm.

Teaching point: To emphasize the importance of recognizing mirror image artifacts in musculoskeletal ultrasound to avoid misdiagnosis, unnecessary interventions, and additional diagnostic procedures that can lead to patient anxiety, increased healthcare costs, and potential harm.

TSAT: Efficient evaluation software for NGS data of phage/mirror-image phage display selections.

Phage display and mirror-image phage display are commonly used techniques for the identification of binders that are specific to predefined targets. Recent studies demonstrated the effectiveness of next-generation sequencing (NGS) by increasing the amount of information extracted from selections. This allows for a better analysis and increases the possibility to select effective binders. A potential downside to NGS analysis of phage display selections is the increased workload that is needed to analyze the obtained information. Here, we report on the development of TSAT (target-specific analysis tool), software for user-friendly and efficient analysis of peptide sequence data from NGS of phage display selections.

Navigating the Unexpected: Right Aortic Arch Unveiled During Left Heart Catheterization.

A right aortic arch (RAA) is an extremely rare congenital anomaly with seven identified variants. While most variants are asymptomatic, those with a vascular ring can be associated with severe symptoms. We present an incidental RAA finding during left heart catheterization (LHC) in a 68-year-old female with multiple morbidities presented with worsening angina. Her echocardiogram was significant for inferolateral wall akinesia, prompting LHC. The procedure was challenging with an unexpected course of the guide wire distally behind the cardiac shadow. Pressure tracing confirmed arterial access and contrast injection revealed RAA. A subsequent aortic computed tomography angiography (CTA) confirmed RAA with mirror-image branching. Abnormal origin and angle of aortic arch branches pose challenges in choosing the proper access. We used the right radial artery approach, but the left radial approach may be superior in providing a more proximal access and avoiding the abnormal origin of the right subclavian artery (RSA). Choosing the appropriate angiographic view is also of utmost importance, and the right anterior oblique view provided better visualization in our case. Aortic arch anomalies are confirmed by a CTA or magnetic resonance angiography (MRA) of the aorta. This case underscores the importance of identifying the aortic arch anomalies and the imposed challenges during the LHC.

Prenatal echocardiography diagnosis of a novel combination of bilateral ductus arteriosus and cardiovascular anomalies: a case report and literature review.

Background: Bilateral ductus arteriosus (BDA) is a relatively rare vascular malformation. According to the double arch theory, BDA is formed when the distal ends of the sixth pairs of primitive arches on the left and right sides have not regressed. We describe a fetus with prenatal echocardiographic findings of BDA and right aortic arch mirror-image branching (RAA-MIB) combined with congenital heart disease. Furthermore, to gain a deeper understanding of the embryological mechanism of BDA, we review the literature on all combinations of BDA present in 40 fetuses/infants. Case summary: A 22-year-old female patient underwent fetal echocardiography at 23 weeks of gestation. Both the two-dimensional (2D) grayscale image and color Doppler flow imaging (CDFI) revealed dextro-transposition of the great arteries combined with a ventricular septal defect and RAA-MIB. The following scan revealed a rare vascular ring, which was identified as BDA extending from the confluent of the left pulmonary artery and right pulmonary artery, completely encircling the trachea to form an "O"-shaped vascular ring before finally converging into the descending aorta. A persistent left superior vena cava was also observed. We subsequently used four-dimensional (4D) color Doppler imaging with the spatiotemporal image correlation (STIC) HD live flow and STIC HD live flow silhouette mode to clearly display ventricular arterial connectivity and the direction of vessel travel. Adjusting the image quality and display angle is very important when applying STIC. The 4D images confirmed our diagnosis. After multidisciplinary counseling and discussion with her family, this female patient decided to terminate the pregnancy. Conclusion: Our review of the literature summarized nine combinations classified into three types of BDA and aortic arch pathology. However, our case differs because it is a novel combination of intracardiac structural abnormalities and vascular rings in a fetus. Prenatal ultrasound diagnosis of BDA is important and requires a combination of 2D grayscale, CDFI, and STIC images to assist in scanning.

Comparative Study of the Efficacy and Side Effects of Brand-Name and Generic Clozapine for Long-Term Maintenance Treatment Among Korean Patients With Schizophrenia: A Retrospective Naturalistic Mirror-Image Study.

OBJECTIVE: Clozapine is considered the most reliable drug for treatment-resistant schizophrenia. In 2014, a generic formulation of clozapine (Clzapine) was introduced in Korea. This study was performed to provide clinical information regarding the use of clozapine and to compare efficacy and tolerability when converting from the brand-name formulation (Clozaril) to the generic formulation during longterm maintenance treatment among Korean patients with schizophrenia. METHODS: This mirror-image study retrospectively investigated the electronic medical records of patients who had switched from Clozaril to Clzapine with a ≥1-year duration for each formulation. Clinical data were collected, including information regarding clozapine use, psychiatric hospitalization, co-medications, and blood test findings. Data before and after the switch were compared using paired t-tests. RESULTS: Among 332 patients, the mean 1-year dosages were 233.32±149.35 mg/day for Clozaril and 217.36±136.66 mg/day for Clzapine. The mean clozapine concentration-to-dose ratios were similar before and after the switch (Clozaril, 1.33±0.68; Clzapine, 1.26±0.80). Switching from Clozaril to Clzapine resulted in no significant differences in the hospitalization rate, hospitalization duration, or laboratory findings (liver function parameters, serum cholesterol level, and serum glucose level). Equivalent doses of co-prescribed antidepressants were decreased, but concomitant medications otherwise showed no significant differences. CONCLUSION: Clinical efficacy and tolerability appear comparable when switching to Clzapine during clozapine maintenance treatment. This study offers descriptive real-world clinical insights into clozapine maintenance treatment in Korea, thereby providing patients with more treatment options and contributing to the development of maintenance guidelines tailored to the Korean population.

An interesting electrocardiogram caused by lead reversal.

BACKGROUND: During normal sinus rhythm, atrial depolarization is conducted from right atrium to left atrium through Bachmann's bundle, and a normal P wave axis which is measured on the frontal plane is between 0º and + 75º. The change of P wave polarity is helpful for the analysis of origin point. CASE PRESENTATION: We report a patient with negative P wave in lead I. The characteristics of QRS complex in leads V1 to V6 are helpful to preliminarily differential diagnosis. The 12-lead electrocardiogram (ECG) with correct limb leads (right arm-left arm) placement shows sinus rhythm with complete right bundle branch block (RBBB). CONCLUSIONS: The change of P wave polarity as well as characteristics of QRS complex can help identify limb-lead reversals.

Improvement in Pain, Quality of Life, and Urinary Dysfunction following Correction of Lumbar Lordosis and Reduction in Lumbar Spondylolistheses Using Chiropractic BioPhysics® Structural Spinal Rehabilitation: A Case Series with >1-Year Long-Term Follow-Up Exams.

(1) Background: Lumbar spondylolisthesis affects ~20% of the US population and causes spine-related pain and disability. (2) Methods: This series reports on three patients (two females and one male) aged 68-71 years showing improvements in back pain, quality of life (QOL), and urinary dysfunction following correction of lumbar spondylolistheses using CBP® spinal rehabilitation. Pre-treatment radiographs showed lumbar hyperlordosis (-49.6°, ideal is -40°) and anterolisthesis (14.5 mm, ideal is 0 mm). Pre-treatment patient-reported outcome measures (PROMs) included a numeric rating scale (NRS) for back pain (7.3/10, ideal is 0), urinary urgency (8/10, ideal is 0), and SF-36 physical (PCS) and mental component score (MCS) (29.8 and 46.6, ideal is 46.8 and 52.8). Patients underwent 2-3 CBP® sessions per week to correct lumbar hyperlordosis and lumbar anterolistheses. (3) Results: Post-treatment radiographs showed improvements in lumbar curvature (-42.8°) and anterolisthesis (4.2 mm). Post-treatment PROMs showed improvements in NRS for back pain (1/10), urinary urgency (2.3/10), and SF-36 PCS and MCS (50.2 and 57.7). Long-term follow-up radiographs and PROMs showed maintained improvements. (4) Conclusions: This series documents the first-recorded long-term corrections of lumbar spondylolisthesis and concomitant improvements in back pain, urinary urgency, and QOL using CBP®. This series provides evidence for CBP® as a non-surgical approach to lumbar spinal rehabilitation and the possible impacts of spinal alignment on pain, urinary dysfunction, and QOL.

Mirror-Image Pain Update: Complex Interactions Between Central and Peripheral Mechanisms.

The appearance of contralateral effects after unilateral injury has been shown in various experimental pain models, as well as in clinics. They consist of a diversity of phenomena in contralateral peripheral nerves, sensory ganglia, or spinal cord: from structural changes and altered gene or protein expression to functional consequences such as the development of mirror-image pain (MP). Although MP is a well-documented phenomenon, the exact molecular mechanism underlying the induction and maintenance of mirror-like spread of pain is still an unresolved challenge. MP has generally been explained by central sensitization mechanisms leading to facilitation of pain impulse transfer through neural connections between the two sides of the central nervous system. On the contrary, the peripheral nervous system (PNS) was usually regarded unlikely to evoke such a symmetrical phenomenon. However, recent findings provided evidence that events in the PNS could play a significant role in MP induction. This manuscript provides an updated and comprehensive synthesis of the MP phenomenon and summarizes the available data on the mechanisms. A more detailed focus is placed on reported evidence for peripheral mechanisms behind the MP phenomenon, which were not reviewed up to now.

Matching-to-Sample Task Training of a Killer Whale (Orcinus orca).

Matching-to-sample tasks have been a useful method in visual cognitive studies on non-human animals. The use of touch panels in matching-to-sample tasks has contributed to cognitive studies on terrestrial animals; however, there has been a difficulty in using these devices underwater, which is one of the factors that has slowed the progress of visual studies on underwater animals. Cetaceans (e.g., dolphins and whales) are highly adapted to underwater environments, and further studies on their cognitive abilities are needed to advance our understanding of the interactions between environmental factors and the evolution of cognitive abilities. In this study, we aimed to develop a new experimental method in which a captive killer whale performed a matching-to-sample task using a monitor shown through an underwater window as if a touch panel were used. In order to confirm the usefulness of this method, one simple experiment on mirror image discrimination was conducted, and the pairs with mirror images were shown to be more difficult to identify than the pairs with other normal images. The advantages of using this method include (1) simplicity in the devices and stimuli used in the experiments, (2) appropriate and rigorous experimental control, (3) the possibility of increasing the number of individuals to be tested and interspecies comparisons, and (4) contributions to animal welfare. The use of this method solves some of the problems in previous visual cognitive studies on cetaceans, and it suggests the further possibility of future comparative cognitive studies. It is also expected to contribute to animal welfare in terms of cognitive enrichment, and it could help with the proposal of new exhibition methods in zoos and aquariums.

Unidirectional Lasing from Mirror-Coupled Dielectric Lattices.

This paper reports how a hybrid system composed of transparent dielectric lattices over a metal mirror can produce high-quality lattice resonances for unidirectional lasing. The enhanced electromagnetic fields are concentrated in the cladding of the periodic dielectric structures and away from the metal. Based on a mirror-image model, we reveal that such high-quality lattice resonances are governed by bound states in the continuum resulting from destructive interference. Using hexagonal arrays of titanium dioxide nanoparticles on a silica-coated silver mirror, we observed lattice resonances with quality factors of up to 2750 in the visible regime. With the lattice resonances as optical feedback and dye solution as the gain medium, we demonstrated unidirectional lasing under optical pumping, where the array size was down to 100 µm × 100 µm. Our scheme can be extended to other spectral regimes to simultaneously achieve strongly enhanced surface fields and high quality factors.

An Enzyme-Cleavable Solubilizing-Tag Facilitates the Chemical Synthesis of Mirror-Image Proteins.

Mirror-image proteins (D-proteins) are useful in biomedical research for purposes such as mirror-image screening for D-peptide drug discovery, but the chemical synthesis of many D-proteins is often low yielding due to the poor solubility or aggregation of their constituent peptide segments. Here, we report a Lys-C protease-cleavable solubilizing tag and its use to synthesize difficult-to-obtain D-proteins. Our tag is easily installed onto multiple amino acids such as DLys, DSer, DThr, and/or the N-terminal amino acid of hydrophobic D-peptides, is impervious to various reaction conditions, such as peptide synthesis, ligation, desulfurization, and transition metal-mediated deprotection, and yet can be completely removed by Lys-C protease under denaturing conditions to give the desired D-protein. The efficacy and practicality of the new method were exemplified in the synthesis of two challenging D-proteins: D-enantiomers of programmed cell death protein 1 IgV domain and SARS-CoV-2 envelope protein, in high yield. This work demonstrates that the enzymatic cleavage of solubilizing tags under denaturing conditions is feasible, thus paving the way for the production of more D-proteins.

Three-dimensional high-definition live tissue virtual dissection of mirror-image dextrocardia with thoracic-abdominal discordance in a fetus.

Three-dimensional virtual dissection using high-definition live tissue rendering ultrasound tool of a 23-week gestation fetus with situs solitus, mirror image dextrocardia, ventricular septal defect, aortic override, and pulmonary atresia.

Mirror-Image Phage Display for the Selection of D-Amino Acid Peptide Ligands as Potential Therapeutics.

In neurodegenerative diseases like Alzheimer's disease (AD), endogenous proteins or peptides aggregate with themselves. These proteins may lose their function or aggregates and/or oligomers can obtain toxicity, causing injury or death to cells. Aggregation of two major proteins characterizes AD. Amyloid-ß peptide (Aß) is deposited in amyloid plaques within the extracellular space of the brain and Tau in so-called neurofibrillary tangles in neurons. Finding peptide ligands to halt protein aggregation is a promising therapeutical approach. Using mirror-image phage display with a commercially available, randomized 12-mer peptide library, we have selected D-amino acid peptides, which bind to the Tau protein and modulate its aggregation in vitro. Peptides can bind specifically and selectively to a target molecule, but natural L-amino acid peptides may have crucial disadvantages for in vivo applications, as they are sensitive to protease degradation and may elicit immune responses. One strategy to circumvent these disadvantages is the use of non-naturally occurring D-amino acid peptides as they exhibit increased protease resistance and generally do not activate the immune system. To perform mirror-image phage display, the target protein needs to be synthesized as D-amino acid version. If the target protein sequence is too long to be synthesized properly, smaller peptides derived from the full length protein can be used for the selection process. This also offers the possibility to influence the binding region of the selected D-peptides in the full-length target protein. Here we provide the protocols for mirror-image phage display selection on the PHF6* peptide of Tau, based on the commercially available Ph.D.™-12 Phage Display Peptide Library Kit, leading to D-peptides that also bind the full length Tau protein (Tau441), next to PHF6*. In addition, we provide protocols and data for the first characterization of those D-peptides that inhibit Tau aggregation in vitro. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Mirror image phage display selection against D-PHF6* fibrils Support Protocol 1: Single phage ELISA Basic Protocol 2: Sequencing and D-peptide generation Basic Protocol 3: Thioflavin-T (ThT) test to control inhibition of Tau aggregation Support Protocol 2: Purification of full-length Tau protein Basic Protocol 4: ELISA to demonstrate the binding of the generated D-peptides to PHF6* and full-length Tau fibrils.

Associated Anomalies and Outcome in Patients with Prenatal Diagnosis of Aortic Arch Anomalies as Aberrant Right Subclavian Artery, Right Aortic Arch and Double Aortic Arch.

We aimed to evaluate retrospectively associated anomalies and outcome in prenatal aortic arch anomalies (AAAs). We included ninety patients with aberrant right subclavian artery (ARSA), right aortic arch (RAA) with mirror image branching (RAA-mirror) or aberrant left subclavian artery (RAA-ALSA) and double aortic arch (DAA) between 2011 and 2020. In total, 19/90 (21.1%) had chromosomal anomalies, the highest rate being within the ARSA subgroup (17/46, 37%). All (13/13) of the RAA-mirror subgroup, 10/27 (37.0%) of RAA-ALSA, 13/46 (28.3%) of ARSA and 0/4 within the DAA subgroup had additional intracardiac anomaly. The rate of extracardiac anomalies was 30.7% in RAA-mirror, 28.3% in ARSA, 25.0% in DAA and 22.2% in the RAA-ALSA subgroup. A total of 42/90 (46.7%) had isolated AAAs: three (7.1%) with chromosomal anomalies, all trisomy 21 (3/26, 11.5%) within the ARSA subgroup. Out of 90, 19 (21.1%) were lost to follow-up (FU). Two (2.2%) intrauterine deaths occurred, and six (6.7%) with chromosomal anomalies terminated their pregnancy. In total, 63 (70.0%) were liveborn, 3/63 (4.8%) with severe comorbidity had compassionate care and 3/60 (5.0%) were lost to FU. The survival rate in the intention-to-treat cohort was 53/57 (93%). Forty-one (77.4%) presented with vascular ring/sling, two (4.9%) with RAA-ALSA developed symptoms and one (2.4%) needed an operation. We conclude that intervention due to vascular ring is rarely necessary. NIPT could be useful in isolated ARSA cases without higher a priori risk for trisomy 21 and after exclusion of other anomalies.

L-Glycosidase-Cleavable Natural Glycans Facilitate the Chemical Synthesis of Correctly Folded Disulfide-Bonded D-Proteins.

D-peptide ligands can be screened for therapeutic potency and enzymatic stability using synthetic mirror-image proteins (D-proteins), but efficient acquisition of these D-proteins can be hampered by the need to accomplish their in vitro folding, which often requires the formation of correctly linked disulfide bonds. Here, we report the finding that temporary installation of natural O-linked-ß-N-acetyl-D-glucosamine (O-GlcNAc) groups onto selected D-serine or D-threonine residues of the synthetic disulfide-bonded D-proteins can facilitate their folding in vitro, and that the natural glycosyl groups can be completely removed from the folded D-proteins to afford the desired chirally inverted D-protein targets using naturally occurring O-GlcNAcase. This approach enabled the efficient chemical syntheses of several important but difficult-to-fold D-proteins incorporating disulfide bonds including the mirror-image tumor necrosis factor alpha (D-TNFα) homotrimer and the mirror-image receptor-binding domain of the Omicron spike protein (D-RBD). Our work establishes the use of O-GlcNAc to facilitate D-protein synthesis and folding and proves that D-proteins bearing O-GlcNAc can be good substrates for naturally occurring O-GlcNAcase.

Utility of the Angle between the Right Aortic Arch and First Branch for Detecting Double Aortic Arch via Fetal Echocardiography.

INTRODUCTION: The presence of a double aortic arch (DAA) is manifested by compressive symptoms, requiring surgery. DAA cases are classified as either complete or incomplete type. DAA and a right aortic arch with mirror image branching (mRAA) have a similar configuration to the first branch artery. The first branch of the mRAA is the left brachiocephalic artery, which appears to be the same as that of an incomplete DAA due to blood flow interruption. The present retrospective study aimed to evaluate the differences between DAA and mRAA by fetal echocardiography. METHODS: This single retrospective cohort study included all patients diagnosed with complete DAA, incomplete DAA, or mRAA at our facility between 2010 and 2022. The patients were diagnosed with complete DAA, incomplete DAA, or mRAA after birth and remaining fetal echocardiograms. The patients were divided into the DAA (complete DAA: n = 4, incomplete DAA: n = 3) and mRAA (n = 4) groups. The following three outcomes were compared: (1) angle between the right aortic arch and first branch (RF angle), (2) ratio of height to width of the region bounded by the aortic arch, first branch of the aortic arch, and descending aorta, and (3) maximum tracheal diameter on a three-vessel trachea view. RESULTS: The incomplete DAA cases were difficult to diagnose via fetal echocardiography. On fetal echocardiography, the RF angle was significantly steeper in the DAA group than in the mRAA group (median 57° [36°-69°] vs. 75° [62°-94°]; p < 0.05). The DAA and RAA groups showed no significant differences in the ratio of height to width of the region bounded by the aortic arch, first branch of the aortic arch, and descending aorta (median 0.57 [0.17-0.68] vs. 0.73 [0.56-1.0]) and maximum tracheal diameter (median 2.5 [1.4-3.3] vs. 3.2 [2.8-3.5] mm). The cut-off value for the presence of DAA was an RF angle <71°. CONCLUSION: The DAA group (complete and incomplete DAA) had a significantly steeper RF angle than the mRAA group. Therefore, RF angle measurement could improve the fetal diagnosis and postnatal prognosis of DAA.

Case report: Mirror-image hypermetropic anisometropia in nontwin brothers.

Mirror-image is a term to describe a physical characteristic of some identical twins that appear asymmetrically on opposite sides as if they are looking in a mirror. Mirror-image anisometropia in monozygotic twins was previously reported in the ophthalmic literature. In this article, we describe a case of mirror-image hypermetropic anisometropia in siblings aged 8 and 5 years old. Nontwin brothers, 8 and 5 years old, with mirror-image hypermetropic anisometropia, were referred to the pediatric ophthalmology clinic. Corrected distant visual acuity was 20/20 (ocula dextra [OD]), 20/60 (ocula sinistra [OS]) in the older brother, and 20/50 (OD), 20/20 (OS) in the younger brother. Cycloplegic refraction was + 3.5 - 1.25 × 180 (OD), +7.75 - 1.5 × 30 (OS), and + 7.0 - 0.75 × 20 (OD), +2.0 - 1.0 × 170 (OS) in the older and younger brother, respectively. The axial length difference between the two eyes was 1.47 mm in the older and 2.01 mm in the younger brother. Hypermetropic anisometropia that may lead to anisometropic amblyopia may happen in nontwin brothers. This emphasizes the importance of complete ophthalmologic examination in the siblings of all patients with anisometropia.

Measuring "Where": A Comparative Analysis of Methods Measuring Spatial Perception.

The literature offers various methods for measuring sound localization. In this study, we aimed to compare these methods to determine their effectiveness in addressing different research questions by examining the effect sizes obtained from each measure. Data from 150 participants who identified the location of a sound source were analyzed to explore the effects of speaker angle, stimuli, HPD type, and condition (with/without HPD) on sound localization, using six methods for analysis: mean absolute deviation (MAD), root-mean-squared error (RMSE), very large errors (VLE), percentage of errors larger than the average error observed in a group of participants (pMean), percentage of errors larger than half the distance between two consecutive loudspeakers (pHalf), and mirror image reversal errors (MIRE). Results indicated that the MIRE measure was the most sensitive to the effects of speaker angle and HPD type, while the VLE measure was most sensitive to the effect of stimuli type. The condition variable provided the largest effect sizes, with no difference observed between measures. The data suggest that when effect sizes are substantial, all methods are adequate. However, for cases where the effect size is expected to be small, methods that yield larger effect sizes should be considered, considering their alignment with the research question.

Design and evaluation of substrate-product analog inhibitors for racemases and epimerases utilizing a 1,1-proton transfer mechanism.

Racemases and epimerases catalyze the inversion of stereochemistry at asymmetric carbon atoms to generate stereoisomers that often play important roles in normal and pathological physiology. Consequently, there is interest in developing inhibitors of these enzymes for drug discovery. A strategy for the rational design of substrate-product analog (SPA) inhibitors of racemases and epimerases utilizing a direct 1,1-proton transfer mechanism is elaborated. This strategy assumes that two groups on the asymmetric carbon atom remain fixed at active-site binding determinants, while the hydrogen and third, motile group move during catalysis, with the latter potentially traveling between an R- and S-pocket at the active site. SPAs incorporate structural features of the substrate and product, often with geminal disubstitution on the asymmetric carbon atom to simultaneously present the motile group to both the R- and S-pockets. For racemases operating on substrates bearing three polar groups (glutamate, aspartate, and serine racemases) or with compact, hydrophobic binding pockets (proline racemase), substituent motion is limited and the design strategy furnishes inhibitors with poor or modest binding affinities. The approach is most successful when substrates have a large, motile hydrophobic group that binds at a plastic and/or capacious hydrophobic site. Potent inhibitors were developed for mandelate racemase, isoleucine epimerase, and α-methylacyl-CoA racemase using the SPA inhibitor design strategy, exhibiting binding affinities ranging from substrate-like to exceeding that of the substrate by 100-fold. This rational approach for designing inhibitors of racemases and epimerases having the appropriate active-site architectures is a useful strategy for furnishing compounds for drug development.