Stabilization of amyloidogenic immunoglobulin light chains by small molecules.

In Ig light-chain (LC) amyloidosis (AL), the unique antibody LC protein that is secreted by monoclonal plasma cells in each patient misfolds and/or aggregates, a process leading to organ degeneration. As a step toward developing treatments for AL patients with substantial cardiac involvement who have difficulty tolerating existing chemotherapy regimens, we introduce small-molecule kinetic stabilizers of the native dimeric structure of full-length LCs, which can slow or stop the amyloidogenicity cascade at its origin. A protease-coupled fluorescence polarization-based high-throughput screen was employed to identify small molecules that kinetically stabilize LCs. NMR and X-ray crystallographic data demonstrate that at least one structural family of hits bind at the LC-LC dimerization interface within full-length LCs, utilizing variable-domain residues that are highly conserved in most AL patients. Stopping the amyloidogenesis cascade at the beginning is a proven strategy to ameliorate postmitotic tissue degeneration.

Mathematical Relationships of Individual Stereocenter er Values to dr Values.

A mathematical relationship is derived for relating the enantiomeric ratios (er values) of two individual stereocenters within a single chiral molecule to the diastereomeric ratio (dr). Whereas the er (or enantiomeric excess, ee) of chiral molecules is readily determined by chiral chromatography and dr values can be determined by chromatography or NMR, modern methods for the optical determination of er values at individual functional groups do not normally determine the er and dr of the entire molecule. We find there is only a special circumstance when knowledge of the er of two individual stereocenters can be used to predict the er of the enantiomers in each diastereomeric set, along with the dr of the stereoisomers. Under circumstances where this relationship fails, one will require a dr assay in addition to two individual er assays to fully characterize the stereochemical parameters of a reaction. Thus, with these circumstances in mind, we give mathematical relationships for determining complete stereoisomer speciation having the knowledge of individual stereocenter er values and a dr value.

Model Building Using Linear Free Energy Relationship Parameters-Eliminating Calibration Curves for Optical Analysis of Enantiomeric Excess.

Linear free energy relationship (LFER) parameters are routinely used to parametrize physicochemical effects while investigating reaction mechanisms. In this Communication, we describe an alternate application for LFERs: training sets for model building in an analytical application. In this study, the sterics, quantified by Charton parameters (Δv), of nine secondary chiral alcohol analytes were correlated to the circular dichroism output from a chiral alcohol optical sensor. To test the validity of the model, the correlative linear model was applied to determine the enantiomeric excess of samples of two alcohols without a priori knowledge of a calibration curve. The error in this method was comparable to those of previous experimental methods (<5%).

Expanded Porphyrin-Anion Supramolecular Assemblies: Environmentally Responsive Sensors for Organic Solvents and Anions.

Porphyrins have been used frequently to construct supramolecular assemblies. In contrast, noncovalent ensembles derived from expanded porphyrins, larger congeners of naturally occurring tetrapyrrole macrocycles, are all but unknown. Here we report a series of expanded porphyrin-anion supramolecular assemblies. These systems display unique environmentally responsive behavior. Addition of polar organic solvents or common anions to the ensembles leads to either a visible color change, a change in the fluorescence emission features, or differences in solubility. The actual response, which could be followed easily by the naked eye, was found to depend on the specifics of the assembly, as well as the choice of analyte. Using the ensembles of this study, it proved possible to differentiate between common solvents, such as diethyl ether, THF, ethyl acetate, acetone, alcohol, acetonitrile, DMF, and DMSO, identify complex solvent systems, as well as distinguish between the fluoride, chloride, bromide, nitrate, and sulfate anions.

Rapid optical methods for enantiomeric excess analysis: from enantioselective indicator displacement assays to exciton-coupled circular dichroism.

CONSPECTUS: The advent of high-throughput screening (HTS) for chiral catalysts has encouraged the development of fast methods for determining enantiomeric excess (ee). Traditionally, chromatographic methods such as chiral HPLC have been used for ee determination in HTS. These methods, however, are not optimal because of high duty cycle. Their long analysis time results in a bottleneck in the HTS process. A more ideal method for HTS that requires less analysis time such as chiroptical methods are thus of interest. In this Account, we summarize our efforts to develop host-guest systems for ee determination. The first part includes our enantioselective indicator displacement assays (eIDAs), and the second part focuses on our circular dichroism based host-guest systems. Our first eIDA utilizes chiral boronic acid receptors, along with prescreened indicators, to determine ee for chiral α-hydroxyacids and vicinal diols with ±7% average error (AE). To further the practicality for this system, a HTS protocol was developed. Our second eIDA uses diamino chiral ligands and Cu(II) as the receptor for the ee determination of α-amino acids. The system reported ±12% AE, and a HTS protocol was developed for this system. Our first CD based host-guest system uses metal complexes composed of Cu(I) or Pd(II) with enantiopure 2,2'-diphenylphosphino-1,1'-binaphthyl (BINAP) as host to determine the ee of chiral vicinal diamines (±4% AE), primary amines (±17% AE), and cyclohexanones (±7% AE). Primary amines and cyclohexanones were derivatized to form chiral imines or chiral hydrazones to allow coordination with the metal complex. Upon coordination of chiral analytes, the metal-to-ligand (BINAP) charge transfer band was modulated, thus allowing the discrimination of chiral analytes. As an effort to improve the accuracy for chiral primary amine ee determination, a system with a host composed of o-formylphenyl boronic acid (FPBA) and enantiopure 1,1'-bi-2-naphthol (BINOL) was used to reduce the AE to ±5.8%. In the presence of amines, the FPBA-BINOL host forms an imine-coordinated boronic ester, thus affecting the CD signal of the boron complex. Another chiral primary amine ee determination system was developed with Fe(II) and 3-hydroxy-2-pyridinecarbaldehyde. The chiral imines, formed by the pyridinecarbaldehyde and chiral amines, would coordinate to the Fe(II) ion yielding exciton-coupled circular dichroism (ECCD) active metal complexes. This system was able to determine the ee of chiral amines with ±5% AE. Furthermore, this imine-Fe(II) complex system also successfully determined the ee of α-chiral aldehydes with ±5% AE. Other ECCD based hosts were subsequently developed; one with bisquinolylpyridylamine and Cu(II) for chiral carboxylates and amino acids and another multicomponent system with pyridine chromophores for chiral secondary alcohol ee determination. Both of the systems were able to determine ee of the chiral analytes with ±3% AE. Overall, our group has developed ee determining host-guest systems that target various functionalities. To date, we are able to determine the ee of vicinal diols, α-hydroxyacids, vicinal diamines, cyclohexanones, amines, α-chiral aldehydes, carboxylates, amino acids, and secondary alcohols with ±7% or lower average error. Future development will involve improving the average error and employing the current systems to analyze real-life samples resulting from parallel syntheses.

A Synergistic Combinatorial and Chiroptical Study of Peptide Catalysts for Asymmetric Baeyer-Villiger Oxidation.

We report an approach to the asymmetric Baeyer-Villiger oxidation utilizing bioinformatics-inspired combinatorial screening for catalyst discovery. Scaled-up validation of our on-bead efforts with a circular dichroism-based assay of alcohols derived from the products of solution-phase reactions established the absolute configuration of lactone products; this assay proved equivalent to HPLC in its ability to evaluate catalyst performance, but was far superior in its speed of analysis. Further solution-phase screening of a focused library suggested a mode of asymmetric induction that draws distinct parallels with the mechanism of Baeyer-Villiger monooxygenases.

Standardization of the management of pediatric urolithiasis in the emergency department.

INTRODUCTION: The incidence of urolithiasis in the pediatric population is rising and medical expulsive therapy (MET) using alpha-adrenergic antagonists has been found to be effective in aiding in the passage of ureteral stones in children. A prior review of patients presenting to our quaternary children's hospital with urolithiasis found only 54 % were prescribed MET and these patients had increased rates of spontaneous stone passage. Thus, an ED urolithiasis management protocol was created to standardize evaluation and care of children with suspected urolithiasis. OBJECTIVE: To compare management of children with urolithiasis presenting to the ED before and after urolithiasis management protocol implementation. METHODS: This is a retrospective review of patients with urolithiasis who presented to our children's ED from 2011 to 2022. The primary outcome was rate of MET prescribing before and after pathway implementation in July 2017, thus the pre-implementation group comprises patients who presented to the ED from July 2011 to July 2017, and the post-protocol group includes those who presented from August 2017 to April 2022. Secondary outcomes included CT utilization in the ED, surgical intervention rate, proportion with spontaneous stone passage, and frequency of urology consultation. Two-sample t-test and Fisher's exact test were used to compare the outcomes of interest before and after protocol implementation. RESULTS: Of 337 patients who presented to the ED after protocol implementation, 120 met inclusion criteria. When comparing outcomes before and after implementation of the protocol, there was significantly decreased use of CT scans (79 % vs 50 %, p < 0.0001) and increased prescribing of MET (54 % vs 82 %, p < 0.0001). There was a significant decrease in opioids prescribed (44 % vs 26.7 %, p = 0.0040), and an increased rate of spontaneous stone passage (34 % vs 46 %, p = 0.0483). Lastly, there was a significant reduction in the rate of surgery for stone management after the implementation of protocol (35 % vs 17 %, p = 0.0020) DISCUSSION: As the ED is the primary site of presentation for many children with urolithiasis, standardization of evaluation and management provides an opportunity to improve outcomes in this population. We found that implementation of an ED urolithiasis management protocol was associated with decreased use of CT scans, decreased opioid prescribing, increased spontaneous stone passage, and decreased rate of surgical management for children with ureteral stones. CONCLUSIONS: This study demonstrates the positive impact of standardizing care for children presenting to the ED with urolithiasis.

Prevalence of and factors associated with burnout in pediatric urology fellows.

INTRODUCTION: The 2016 AUA census found 39% of practicing urologists experienced burnout. Burnout is a syndrome of emotional exhaustion, depersonalization, and decreased feelings of personal accomplishment due to workplace stress. Despite the demands of training, little is known about the prevalence of burnout in pediatric urology fellows. OBJECTIVE: To determine the prevalence of burnout in pediatric urology fellows and factors associated with higher levels of burnout. STUDY DESIGN: The Maslach Burnout Inventory (MBI) and an anonymous survey of personal and training characteristics were distributed electronically to pediatric urology fellows in April 2023. The MBI is a standardized and validated 22-item questionnaire used to quantify burnout and is comprised of three subscales: Emotional Exhaustion (EE), Depersonalization (DP), and Personal Accomplishment (PA). As per prior literature, higher scores in EE (>27) or DP (>10) were defined as high burnout. Demographic and training characteristics were compared between fellows with high and low to moderate levels of burnout with t tests and Fischer's exact tests. RESULTS: The survey response rate was 85% (29/34); 48% clinical fellows, 52% research fellows. Mean age of respondents was 31.4 years (SD 2.6), 59% female, 70% married, and 37% a parent. Mean scores for EE, DP, and PA were 15 (SD 10.2), 4 (SD 4.0), and 39 (SD 10.3), respectively, with no significant difference between year in fellowship. Nineteen percent of fellows met criteria for high burnout and 41% met criteria for moderate to high burnout based on EE or DP scores. Factors significantly associated with high burnout included number nights of call per month and number of projects required to complete in fellowship. Strategies fellows used most to combat burnout included quality time with family and friends, sleep, exercise, and watching TV/movies. DISCUSSION: Nearly 20% of pediatric urology fellows scored for high levels of burnout and over 40% scored for moderate to high levels of burnout. There appears to be an association with increased call and project workload requirements with increased levels of burnout, and efforts to combat burnout could specifically address these factors. Fellows with more children at home had lower levels of burnout, and many trainees described quality time with family and friends as their preferred strategy to prevent burnout. CONCLUSION: This survey-based study identifies risk factors for burnout in pediatric urology fellows. Fellows can use the information presented to consider personalized strategies to prevent burnout through training and into their careers.

Patient and Provider Education Safely Reduces Opioid Prescribing After Pediatric Urologic Surgery.

OBJECTIVE: To examine current opioid prescribing and determine what clinical factors were associated with use of opioids after urologic surgery after a previous study from our institution found that education regarding opioid prescribing practices significantly decreased post-operative opioid prescriptions from 61% to 34% (p<0.0001). METHODS: From 2017 to 2023, a questionnaire querying what medications were used for postoperative pain was administered to patients/families at a postoperative visit. Survey results and demographic factors were obtained via retrospective chart review. Fisher's exact and t-tests compared patients who did and did not use opioids. RESULTS: 1,630 patients' families completed a survey, with mean age 5.3 years, 95% male. Over the study period 550 patients (34%) were prescribed opioids and 474/1,630 (29%) used opioids post-operatively. Patients who used opioids were significantly older (7 vs 4 years, p<0.0001). Endoscopic surgery (p=0.0005), buried penis/torsion/chordee repair (p<0.0001), meatoplasty/skin bridge (p<0.0001), and alternating acetaminophen and ibuprofen (p<0.0001) were associated with decreased opioid use. Families of patients who used opioids had higher rates of calling clinic (6% vs 2%, p=0.0011) and visiting the Emergency Department (ED) with pain concerns (3% vs 0.7%, p=0.002). In 2017, 63% of patients were prescribed opioids after surgery compared with 6% in 2023 (p<0.0001). CONCLUSIONS: Most pediatric urologic surgeries can be performed without outpatient post-operative opioids. After education, we decreased opioid prescribing to only 5% of patients. The patients who were prescribed opioids had higher rates of ED visits or calling the clinic nurses with pain concerns.

Divergent Proteome Reactivity Influences Arm-Selective Activation of the Unfolded Protein Response by Pharmacological Endoplasmic Reticulum Proteostasis Regulators.

Pharmacological activation of the activating transcription factor 6 (ATF6) arm of the unfolded protein response (UPR) has proven useful for ameliorating proteostasis deficiencies in cellular and mouse models of numerous etiologically diverse diseases. Previous high-throughput screening efforts identified the small molecule AA147 as a potent and selective ATF6 activating compound that operates through a mechanism involving metabolic activation of its 2-amino-p-cresol substructure affording a quinone methide, which then covalently modifies a subset of endoplasmic reticulum (ER) protein disulfide isomerases (PDIs). Another compound identified in this screen, AA132, also contains a 2-amino-p-cresol moiety; however, this compound showed less transcriptional selectivity, instead globally activating all three arms of the UPR. Here, we show that AA132 activates global UPR signaling through a mechanism analogous to that of AA147, involving metabolic activation and covalent modification of proteins including multiple PDIs. Chemoproteomic-enabled analyses show that AA132 covalently modifies PDIs to a greater extent than AA147. However, the extent of PDI labeling by AA147 approaches a plateau more rapidly than PDI labeling by AA132. These observations together suggest that AA132 can access a larger pool of proteins for covalent modification, possibly because its activated form is less susceptible to quenching than activated AA147. In other words, the lower reactivity of activated AA132 allows it to persist longer and modify more PDIs in the cellular environment. Collectively, these results suggest that AA132 globally activates the UPR through increased engagement of ER PDIs. Consistent with this, reducing the cellular concentration of AA132 decreases PDI modifications and enables selective ATF6 activation. Our results highlight the relationship between metabolically activatable-electrophile stability, ER proteome reactivity, and the transcriptional response observed with the enaminone chemotype of ER proteostasis regulators, enabling continued development of next-generation ATF6 activating compounds.

Divergent Proteome Reactivity Influences Arm-Selective Activation of Pharmacological Endoplasmic Reticulum Proteostasis Regulators.

Pharmacological activation of the activating transcription factor 6 (ATF6) arm of the Unfolded Protein Response (UPR) has proven useful for ameliorating proteostasis deficiencies in a variety of etiologically diverse diseases. Previous high-throughput screening efforts identified the small molecule AA147 as a potent and selective ATF6 activating compound that operates through a mechanism involving metabolic activation of its 2-amino- p -cresol substructure affording a quinone methide, which then covalently modifies a subset of ER protein disulfide isomerases (PDIs). Intriguingly, another compound identified in this screen, AA132, also contains a 2-amino- p -cresol moiety; however, this compound showed less transcriptional selectivity, instead globally activating all three arms of the UPR. Here, we show that AA132 activates global UPR signaling through a mechanism analogous to that of AA147, involving metabolic activation and covalent PDI modification. Chemoproteomic-enabled analyses show that AA132 covalently modifies PDIs to a greater extent than AA147. Paradoxically, activated AA132 reacts slower with PDIs, indicating it is less reactive than activated AA147. This suggests that the higher labeling of PDIs observed with activated AA132 can be attributed to its lower reactivity, which allows this activated compound to persist longer in the cellular environment prior to quenching by endogenous nucleophiles. Collectively, these results suggest that AA132 globally activates the UPR through increased engagement of ER PDIs. Consistent with this, reducing the cellular concentration of AA132 decreases PDI modifications and allows for selective ATF6 activation. Our results highlight the relationship between metabolically activatable-electrophile stability, ER proteome reactivity, and the transcriptional response observed with the enaminone chemotype of ER proteostasis regulators, enabling continued development of next-generation ATF6 activating compounds.

From Substituent Effects to Applications: Enhancing the Optical Response of a Four-Component Assembly for Reporting EE Values.

High-throughput screening for asymmetric catalysts has stimulated an interest in optically-based enantiomeric-excess (ee) sensors, primarily for their improved time and cost efficiency when compared to the standard HPLC analysis. We present herein substituent-effect studies on a recently reported Zn(II) multicomponent assembly that is used for chiral, secondary alcohol ee detemination. The systematic altering of assemblies formed from select substituted pyridyl ligands pointed to the conclusion that steric effects dominate the mode of interaction at the pyridyl 3- and 6- positions. From these results we identified a new Zn(II)-centered multicomponent assembly with a higher dynamic range than previously reported. Calibration curves of the CD signals resulting from the new assembly led to an ee assay with a 1.7% error. To further the utility of the new assembly, a correlation was developed between alcohol substituent size to the respective enantiopure CD value.