Caulfield, Thomas R.

Aggregated α-synuclein (α-SYN) proteins, encoded by the SNCA gene, are hallmarks of Lewy body disease (LBD), affecting multiple brain regions. However, the specific mechanisms underlying α-SYN pathology in cortical neurons, crucial for LBD-associated dementia, remain unclear. Here, we recapitulated α-SYN pathologies in human induced pluripotent stem cells (iPSCs)–derived cortical organoids generated from patients with LBD with SNCA gene triplication. Single-cell RNA sequencing, combined with functional and molecular validation, identified synaptic and mitochondrial dysfunction in excitatory neurons exhibiting high expression of the SNCA gene, aligning with observations in the cortex of autopsy-confirmed LBD human brains. Furthermore, we screened 1280 Food and Drug Administration–approved drugs and identified four candidates (entacapone, tolcapone, phenazopyridine hydrochloride, and zalcitabine) that inhibited α-SYN seeding activity in real-time quaking-induced conversion assays with human brains, reduced α-SYN aggregation, and alleviated mitochondrial dysfunction in SNCA triplication organoids and excitatory neurons. Our findings establish human cortical LBD models and suggest potential therapeutic drugs targeting α-SYN aggregation for LBD.

Background: While ‘immuno-competence’ is a well-known term, it lacks an operational definition. To address this omission, this study explored whether the temporal and structured data of the complete blood cell count (CBC) can rapidly estimate immuno-competence. To this end, one or more ratios that included data on all monocytes, lymphocytes and neutrophils were investigated. Materials and methods: Longitudinal CBC data collected from 101 COVID-19 patients (291 observations) were analyzed. Dynamics were estimated with several approaches, which included non-structured (the classic CBC format) and structured data. Structured data were assessed as complex ratios that capture multicellular interactions among leukocytes. In comparing survivors with non-survivors, the hypothesis that immuno-competence may exhibit feedback-like (oscillatory or cyclic) responses was tested. Results: While non-structured data did not distinguish survivors from non-survivors, structured data revealed immunological and statistical differences between outcomes: while survivors exhibited oscillatory data patterns, non-survivors did not. In survivors, many variables (including IL-6, hemoglobin and several complex indicators) showed values above or below the levels observed on day 1 of the hospitalization period, displaying L-shaped data distributions (positive kurtosis). In contrast, non-survivors did not exhibit kurtosis. Three immunologically defined data subsets included only survivors. Because information was based on visual patterns generated in real time, this method can, potentially, provide information rapidly. Discussion: The hypothesis that immuno-competence expresses feedback-like loops when immunological data are structured was not rejected. This function seemed to be impaired in immuno-suppressed individuals. While this method rapidly informs, it is only a guide that, to be confirmed, requires additional tests. Despite this limitation, the fact that three protective (survival-associated) immunological data subsets were observed since day 1 supports many clinical decisions, including the early and personalized prognosis and identification of targets that immunomodulatory therapies could pursue. Because it extracts more information from the same data, structured data may replace the century-old format of the CBC.

Complete loss-of-function mutations in the PRKN gene are a major cause of early-onset Parkinson’s disease (PD). PRKN encodes the Parkin protein, an E3 ubiquitin ligase that works in conjunction with the ubiquitin kinase PINK1 in a distinct quality control pathway to tag damaged mitochondria for autophagic clearance, i.e., mitophagy. According to previous structural investigations, Parkin protein is typically kept in an inactive conformation via several intramolecular, auto-inhibitory interactions. Here, we performed molecular dynamics simulations (MDS) to provide insights into conformational changes occurring during the de-repression of Parkin and the gain of catalytic activity. We analyzed four different Parkin-activating mutations that are predicted to disrupt certain aspects of its auto-inhibition. All four variants showed greater conformational motions compared to wild-type protein, as well as differences in distances between domain interfaces and solvent-accessible surface area, which are thought to play critical roles as Parkin gains catalytic activity. Our findings reveal that the studied variants exert a notable influence on Parkin activation as they alter the opening of its closed inactive structure, a finding that is supported by recent structure- and cell-based studies. These findings not only helped further characterize the hyperactive variants but overall improved our understanding of Parkin’s catalytic activity and nominated targets within Parkin’s structure for potential therapeutic designs.

Proteases are ubiquitous enzymes that catalyze the hydrolysis of peptide bonds within protein substrates; they have served as key model enzymes for studying the molecular basis for catalytic power and specificity. Protease substrate specificity is most often defined in terms of linear sequence motifs that flank the cleavage site; however, the natural substrates of proteases are proteins with 3‐dimensional shapes and complex conformational dynamics that are not well represented by 1‐dimensional sequence alone. These structural and dynamical properties can impact recognition and binding of substrates by proteases, as well as the efficiency of catalysis itself. In this study, we explore the importance of substrate structure and dynamics for proteolysis using as our model the cleavage of the Kunitz‐BPTI family of canonical serine protease inhibitors by mesotrypsin. Bovine pancreatic trypsin inhibitor (BPTI), an archetypal serine protease inhibitor of the Kunitz family, has a high affinity substrate‐like interaction with trypsin, yet is cleaved many orders of magnitude slower than other peptide substrates. Mesotrypsin, a trypsin variant, has been shown to hydrolyze Kunitz family inhibitors at accelerated rates; this is especially true of human Kunitz domain inhibitors. Amyloid precursor protein inhibitor (APPI) and amyloid precursor like protein‐2 Kunitz domain (APLP2‐KD), two human Kunitz domain family members, are hydrolyzed by mesotrypsin several hundred times faster than BPTI. Here, we present a new crystal structure of a cleavage intermediate of APLP2‐KD bound to mesotrypsin, refined to 1.4 Å resolution. This newly solved structure reveals a dramatic substrate conformational change that we hypothesize is required during Kunitz domain cleavage. Using this structure along with published APPI and BPTI complexes, we have modeled acyl‐enzyme intermediates of mesotrypsin and we have carried out molecular dynamic simulations to explore the conformational transformation that allows the progression of the hydrolysis reaction. We identify dynamic features of the inhibitor scaffolds, located far from the enzyme binding site, that correlate with observed inhibitor cleavage rates and plausibly explain their differential propensity to undergo both conformational transformation and cleavage. Our findings implicate global scaffold flexibility as a determinant of proteolytic rate, as rigid substrates possessing stabilizing features that render them highly resistant to conformational change are proteolyzed more slowly than more flexible substrates of similar structure. Support or Funding Information NIH R01 CA154387 Mayo Graduate school

(1) Background: Mutations in NFκB1, a transcriptional regulator of immunomodulating proteins, are a known cause of inborn errors of immunity. Our proband is a 22-year-old male with a diagnosis of common variable immunodeficiency (CVID), cytopenias with massive splenomegaly, and nodular regenerative hyperplasia of the liver. Genetic studies identified a novel, single-point mutation variant in NFκB1, c. T638A p. V213E. (2) Methods: Next-generation panel sequencing of the patient uncovered a novel single-point mutation in the NFκB1 gene that was modeled using the I-TASSER homology-modeling software, and molecular dynamics were assessed using the YASARA2 software (version 20.14.24). (3) Results: This variant replaces valine with glutamic acid at position 213 in the NFκB1 sequence. Molecular modeling and molecular dynamic studies showed altered dynamics in and around the rel homology domain, ankyrin regions, and death domain of the protein. We postulate that these changes alter overall protein function. (4) Conclusions: This case suggests the pathogenicity of a novel variant using protein-modeling techniques and molecular dynamic simulations.

Abstract BACKGROUND Mutations of NFkB1 are a known cause of inborn errors of immunity resulting in immunodeficiency due to the role of NFkB1 as a transcriptional regulator of immunomodulating proteins. We present a patient with a diagnosis of common variable immunodeficiency (CVID), cytopenias with massive splenomegaly, and nodular regenerative hyperplasia of the liver. Our proband, a 22-year-old male, presents with a clinical picture of immunodeficiency, and has a maternal cousin with a similar gene mutation but differing phenotype. Next generation panel sequencing (NGPS) was utilized to discover a novel, single point mutation variant in NFkB1, V213E, that is present in our proband. METHODS The novel single-point mutation V213E in the NFkB gene was modeled in-silico using the I-TASSER homology modeling software. Molecular dynamics simulations of both the wild type and variant mutant form fitted to accommodate the novel mutation were conducted and analyzed to assess for possible pathogenicity of the novel mutation. RESULTS This variant replaces Valine with Glutamic Acid at position 213 in the transcription regulator’s sequence. We perform molecular modeling to analyze the proteomic impact and difference in molecular dynamics (MDS) which depicted suppressed dynamics in the ankyrin region and death domain of the protein complex. Analysis of the proband’s family showed no significant family history except for the maternal cousin with a mutation in NFkB1. CONCLUSION This case both highlights the heterogeneity in phenotype of NFkB1 pathogenic variants as shown in this patient, full penetrance of the variant mutation, and suggests the pathogenicity of a new variant through protein modeling techniques and molecular dynamic simulations.

We have previously shown computationally that Mycolactone (MLN), a toxin produced by Mycobacterium ulcerans, strongly binds to Munc18b and other proteins, presumably blocking degranulation and exocytosis of blood platelets and mast cells. We investigated the effect of MLN on endocytosis using similar approaches, and it bound strongly to the N-terminal of the clathrin protein and a novel SARS-CoV-2 fusion protein. Experimentally, we found 100% inhibition up to 60 nM and 84% average inhibition at 30 nM in SARS-CoV-2 live viral assays. MLN was also 10× more potent than remdesivir and molnupiravir. MLN’s toxicity against human alveolar cell line A549, immortalized human fetal renal cell line HEK293, and human hepatoma cell line Huh7.1 were 17.12%, 40.30%, and 36.25%, respectively. The cytotoxicity IC50 breakpoint ratio versus anti-SARS-CoV-2 activity was more than 65-fold. The IC50 values against the alpha, delta, and Omicron variants were all below 0.020 µM, and 134.6 nM of MLN had 100% inhibition in an entry and spread assays. MLN is eclectic in its actions through its binding to Sec61, AT2R, and the novel fusion protein, making it a good drug candidate for treating and preventing COVID-19 and other similarly transmitted enveloped viruses and pathogens.

Abstract Matrix metalloproteinases (MMPs) regulate the degradation of extracellular matrix (ECM) components in biological processes. MMP activity is controlled by natural tissue inhibitors of metalloproteinases (TIMPs) that non-selectively inhibit the function of multiple MMPs via interaction with the MMPs' Zn2+-containing catalytic pocket. Recent studies suggest that TIMPs engineered to confer MMP specificity could be exploited for therapeutic purposes, but obtaining specific TIMP-2 inhibitors has proved to be challenging. Here, in an effort to improve MMP specificity, we incorporated the metal-binding non-canonical amino acids (NCAAs), 3,4-dihydroxyphenylalanine (L-DOPA) and (8-hydroxyquinolin-3-yl)alanine (HqAla), into the MMP-inhibitory N-terminal domain of TIMP2 (N-TIMP2) at selected positions that interact with the catalytic Zn2+ ion (S2, S69, A70, L100) or with a structural Ca2+ ion (Y36). Evaluation of the inhibitory potency of the NCAA-containing variants towards MMP-2, MMP-9 and MMP-14 in vitro revealed that most showed a significant loss of inhibitory activity towards MMP-14, but not towards MMP-2 and MMP-9, resulting in increased specificity towards the latter proteases. Substitutions at S69 conferred the best improvement in selectivity for both L-DOPA and HqAla variants. Molecular modeling revealed how MMP-2 and MMP-9 are better able to accommodate the bulky NCAA substituents at the intermolecular interface with N-TIMP2. The models also showed that, rather than coordinating to Zn2+, the NCAA side chains formed stabilizing polar interactions at the intermolecular interface with MMP-2 and MMP-9. The findings illustrate how incorporation of NCAAs can be used to probe and exploit differential tolerance for substitution within closely related protein-protein complexes to achieve improved specificity.

Bcl-2 and Mcl-1 proteins play a role in multiple myeloma (MM) cell survival, for which targeted inhibitors are being developed. AT-101 is an oral drug, which disrupts Bcl-2 and Mcl-1 function, impedes mitochondrial bioenergetic processes and induces apoptosis in MM cells. When combined with lenalidomide and dexamethasone (Rd), AT-101 significantly reduced tumor burden in an in vivo xenograft model of MM. These data provided rationale for a phase I/II study to establish the effective dose of AT-101 in combination with Rd (ARd regimen) in relapsed/refractory MM. A total of 10 patients were enrolled, most with high-risk cytogenetics (80%) and prior stem cell transplant (70%). Three patients were lenalidomide-refractory, 2 were bortezomib-refractory and 3 were daratumumab-refractory. The ARd combination was well tolerated with most common grade 3/4 adverse events being cytopenia’s. The overall response rate was 40% and clinical benefit rate was 90%. The median progression free survival was 14.9 months (95% CI 7.1-NE). Patients responsive to ARd showed a decrease in Bcl-2:Bim or Mcl-1:Noxa protein complexes, increased CD8+ T and NK cells and depletion of T and B-regulatory cells. The ARd regimen demonstrated an acceptable safety profile and promising efficacy in patients with relapsed/refractory MM prompting further investigation in additional patients.

AbstractBackgroundAlzheimer’s disease (AD) is the most common form of age‐related dementia characterized by the presence of amyloid‐β (Aβ) and tau pathologies leading to neurodegeneration and cognitive impairment. Wnt/β‐catenin signaling modulates multiple pathophysiological processes including synaptic plasticity, neuronal survival, neuroinflammation, Aβ production and tau phosphorylation, but this signaling pathway is greatly inhibited in the AD brain. Notably, two SNPs and an alternative splice variant of Wnt co‐receptor LRP6, which result in down‐regulation of Wnt/β‐catenin signaling, are associated with increased risk of developing AD. In addition, apoE4, the strongest risk factor of late‐onset AD, interacts with and decreases cell surface abundance of LRP6 in astrocytes. Studies from our laboratory have demonstrated that the level of LRP6 is significantly downregulated in human AD brains, and that deficiency in LRP6‐mediated Wnt/β‐catenin signaling leads to synaptic abnormalities and amyloid pathology in amyloid model mice. Therefore, LRP6 is an attractive therapeutic target for restoring Wnt/β‐catenin signaling in the AD brain.MethodIn our previous studies, we have discovered niclosamide, an FDA‐approved anthelminthic drug, inhibits Wnt/β‐catenin signaling via promoting LRP6 degradation. Herein, we demonstrated that CI‐994, an analog of niclosamide, exhibits opposite effects on LRP6 level and Wnt/β‐catenin signaling. Following structural optimization, we have generated a series of novel potent Wnt modulators.ResultThe leading compound W2A‐16 displays a good pharmacokinetic profile with high oral bioavailability and brain penetration. Importantly, W2A‐16 significantly activates Wnt/β‐catenin signaling and inhibits tau phosphorylation in AD patient‐specific iPSC‐derived cerebral organoids. Moreover, W2A‐16 attenuates amyloid pathology and gliosis and improves cognitive function in 5xFAD amyloid model mice. Further, W2A‐16 suppresses tau phosphorylation and neuroinflammation, attenuates body weight loss, and improves the survival of PS19 tauopathy mice.ConclusionTogether, these findings demonstrate that W2A‐16 is a strong drug candidate for AD therapy.