Bacteriophage antidefense genes that neutralize TIR and STING immune responses.

Programmed cell suicide of infected bacteria, known as abortive infection (Abi), serves as an immune defense strategy to prevent the propagation of bacteriophage viruses. Many Abi systems utilize bespoke cyclic nucleotide immune messengers generated upon infection to mobilize cognate death effectors. Here, we identify a family of bacteriophage nucleotidyltransferases (NTases) that synthesize competitor cyclic dinucleotide (CDN) ligands and inhibit TIR NADase effectors activated via a linked STING CDN sensor domain (TIR-STING). Through a functional screen of NTase-adjacent phage genes, we uncover candidate inhibitors of cell suicide induced by heterologous expression of tonically active TIR-STING. Among these, we demonstrate that a virus MazG-like nucleotide pyrophosphohydrolase, Atd1, depletes the starvation alarmone (p)ppGpp, revealing a potential role for the alarmone-activated host toxin MazF as an executioner of TIR-driven Abi. Phage NTases and counterdefenses like Atd1 preserve host viability to ensure virus propagation and represent tools to modulate TIR and STING immune responses.

Chemical scaffold recycling: Structure-guided conversion of an HIV integrase inhibitor into a potent influenza virus RNA-dependent RNA polymerase inhibitor designed to minimize resistance potential.

Influenza is one of the leading causes of disease-related mortalities worldwide. Several strategies have been implemented during the past decades to hinder the replication cycle of influenza viruses, all of which have resulted in the emergence of resistant virus strains. The most recent example is baloxavir marboxil, where a single mutation in the active site of the target endonuclease domain of the RNA-dependent-RNA polymerase renders the recent FDA approved compound ∼1000-fold less effective. Raltegravir is a first-in-class HIV inhibitor that shows modest activity to the endonuclease. Here, we have used structure-guided approaches to create rationally designed derivative molecules that efficiently engage the endonuclease active site. The design strategy was driven by our previously published structures of endonuclease-substrate complexes, which allowed us to target functionally conserved residues and reduce the likelihood of resistance mutations. We succeeded in developing low nanomolar equipotent inhibitors of both wild-type and baloxavir-resistant endonuclease. We also developed macrocyclic versions of these inhibitors that engage the active site in the same manner as their 'open' counterparts but with reduced affinity. Structural analyses provide clear avenues for how to increase the affinity of these cyclic compounds.

Targeting KDM4 for treating PAX3-FOXO1-driven alveolar rhabdomyosarcoma.

Chimeric transcription factors drive lineage-specific oncogenesis but are notoriously difficult to target. Alveolar rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue sarcoma transformed by the pathognomonic Paired Box 3-Forkhead Box O1 (PAX3-FOXO1) fusion protein, which governs a core regulatory circuitry transcription factor network. Here, we show that the histone lysine demethylase 4B (KDM4B) is a therapeutic vulnerability for PAX3-FOXO1+ RMS. Genetic and pharmacologic inhibition of KDM4B substantially delayed tumor growth. Suppression of KDM4 proteins inhibited the expression of core oncogenic transcription factors and caused epigenetic alterations of PAX3-FOXO1-governed superenhancers. Combining KDM4 inhibition with cytotoxic chemotherapy led to tumor regression in preclinical PAX3-FOXO1+ RMS subcutaneous xenograft models. In summary, we identified a targetable mechanism required for maintenance of the PAX3-FOXO1-related transcription factor network, which may translate to a therapeutic approach for fusion-positive RMS.

Site-specific antibody-drug conjugates with variable drug-to-antibody-ratios for AML therapy.

Random conjugations of chemotherapeutics to monoclonal antibodies result in heterogeneous antibody-drug conjugates (ADCs) with suboptimal pharmacological properties. We recently developed a new technology for facile generation of homogeneous ADCs by harnessing human CD38 catalytic domain and its dinucleotide-derived covalent inhibitor, termed ADP-ribosyl cyclase-enabled ADCs (ARC-ADCs). Herein we advance this technology by designing and synthesizing ARC-ADCs with customizable drug-to-antibody ratios (DARs). Through varying numbers and locations of CD38 fused to an antibody targeting human C-type lectin-like molecule-1 (hCLL-1), ARC-ADCs featuring DARs of 2 and 4 were rapidly generated via a single step with cytotoxic monomethyl auristatin F (MMAF) as payloads. In contrast to anti-hCLL-1 ARC-ADC carrying 2 drug molecules, anti-hCLL-1 ARC-ADC with a DAR of 4 shows highly potent activity in killing hCLL-1-positive acute myeloid leukemia (AML) cells both in vitro and in vivo. This work provides novel ADC candidates for combating AML and supports ARC-ADC as a general and versatile approach for producing site-specific ADCs with defined DARs.

Measuring how two proteins affect each other's net charge in a crowded environment.

Theory predicts that the net charge (Z) of a protein can be altered by the net charge of a neighboring protein as the two approach one another below the Debye length. This type of charge regulation suggests that a protein's charge and perhaps function might be affected by neighboring proteins without direct binding. Charge regulation during protein crowding has never been directly measured due to analytical challenges. Here, we show that lysine specific protein crosslinkers (NHS ester-Staudinger pairs) can be used to mimic crowding by linking two non-interacting proteins at a maximal distance of ~7.9 Å. The net charge of the regioisomeric dimers and preceding monomers can then be determined with lysine-acyl "protein charge ladders" and capillary electrophoresis. As a proof of concept, we covalently linked myoglobin (Zmonomer  = -0.43 ± 0.01) and α-lactalbumin (Zmonomer  = -4.63 ± 0.05). Amide hydrogen/deuterium exchange and circular dichroism spectroscopy demonstrated that crosslinking did not significantly alter the structure of either protein or result in direct binding (thus mimicking crowding). Ultimately, capillary electrophoretic analysis of the dimeric charge ladder detected a change in charge of ΔZ = -0.04 ± 0.09 upon crowding by this pair (Zdimer  = -5.10 ± 0.07). These small values of ΔZ are not necessarily general to protein crowding (qualitatively or quantitatively) but will vary per protein size, charge, and solvent conditions.

17-DMAG dually inhibits Hsp90 and histone lysine demethylases in alveolar rhabdomyosarcoma.

Histone lysine demethylases (KDMs) play critical roles in oncogenesis and therefore may be effective targets for anticancer therapy. Using a time-resolved fluorescence resonance energy transfer demethylation screen assay, in combination with multiple orthogonal validation approaches, we identified geldanamycin and its analog 17-DMAG as KDM inhibitors. In addition, we found that these Hsp90 inhibitors increase degradation of the alveolar rhabdomyosarcoma (aRMS) driver oncoprotein PAX3-FOXO1 and induce the repressive epigenetic mark H3K9me3 and H3K36me3 at genomic loci of PAX3-FOXO1 targets. We found that as monotherapy 17-DMAG significantly inhibits expression of PAX3-FOXO1 target genes and multiple oncogenic pathways, induces a muscle differentiation signature, delays tumor growth and extends survival in aRMS xenograft mouse models. The combination of 17-DMAG with conventional chemotherapy significantly enhances therapeutic efficacy, indicating that targeting KDM in combination with chemotherapy may serve as a therapeutic approach to PAX3-FOXO1-positive aRMS.

High-Throughput Microplate-Based Fluorescence Assays for Studying Stochastic Aggregation of Superoxide Dismutase-1.

Investigating in vitro kinetics of superoxide dismutase-1 (SOD1) aggregation with high-throughput microplate-based assays provides valuable information regarding SOD1 pathogenesis in amyotrophic lateral sclerosis (ALS) and opens venues for the development of effective therapies. In this chapter, we first explain the step-by-step purification and demetallation of wild-type (WT) and ALS-variant SOD1 proteins from Saccharomyces cerevisiae (baker's yeast). We then describe the methodology for a microplate-based fluorescence assay that is used to study real-time kinetics of metal-free (apo)-SOD1 aggregation. This technique is highly sensitive, semiautomated, requires minimum modifications to protein, and produces a plethora of data in a short period of time. We also describe a new approach for extracting clinically relevant information from SOD1 aggregation data using Kaplan-Meier estimators.

Glycerolipid Headgroups Control Rate and Mechanism of Superoxide Dismutase-1 Aggregation and Accelerate Fibrillization of Slowly Aggregating Amyotrophic Lateral Sclerosis Mutants.

Interactions between superoxide dismutase-1 (SOD1) and lipid membranes might be directly involved in the toxicity and intercellular propagation of aggregated SOD1 in amyotrophic lateral sclerosis (ALS), but the chemical details of lipid-SOD1 interactions and their effects on SOD1 aggregation remain unclear. This paper determined the rate and mechanism of nucleation of fibrillar apo-SOD1 catalyzed by liposomal surfaces with identical hydrophobic chains (RCH2(O2C18H33)2), but headgroups of different net charge and hydrophobicity (i.e., R(CH2)N+(CH3)3, RPO4-(CH2)2N+(CH3)3, and RPO4-). Under semiquiescent conditions (within a 96 well microplate, without a gyrating bead), the aggregation of apo-SOD1 into thioflavin-T-positive (ThT(+)) amyloid fibrils did not occur over 120 h in the absence of liposomal surfaces. Anionic liposomes triggered aggregation of apo-SOD1 into ThT(+) amyloid fibrils; cationic liposomes catalyzed fibrillization but at slower rates and across a narrower lipid concentration; zwitterionic liposomes produced nonfibrillar (amorphous) aggregates. The inability of zwitterionic liposomes to catalyze fibrillization and the dependence of fibrillization rate on anionic lipid concentration suggests that membranes catalyze SOD1 fibrillization by a primary nucleation mechanism. Membrane-catalyzed fibrillization was also examined for eight ALS variants of apo-SOD1, including G37R, G93R, D90A, and E100G apo-SOD1 that nucleate slower than or equal to WT SOD1 in lipid-free, nonquiescent amyloid assays. All ALS variants (with one exception) nucleated faster than WT SOD1 in the presence of anionic liposomes, wherein the greatest acceleratory effects were observed among variants with lower net negative surface charge (G37R, G93R, D90A, E100G). The exception was H46R apo-SOD1, which did not form ThT(+) species.

Direct Measurement of Charge Regulation in Metalloprotein Electron Transfer.

Determining whether a protein regulates its net electrostatic charge during electron transfer (ET) will deepen our mechanistic understanding of how polypeptides tune rates and free energies of ET (e.g., by affecting reorganization energy, and/or redox potential). Charge regulation during ET has never been measured for proteins because few tools exist to measure the net charge of a folded protein in solution at different oxidation states. Herein, we used a niche analytical tool (protein charge ladders analyzed with capillary electrophoresis) to determine that the net charges of myoglobin, cytochrome c, and azurin change by 0.62±0.06, 1.19±0.02, and 0.51±0.04 units upon single ET. Computational analysis predicts that these fluctuations in charge arise from changes in the pKa  values of multiple non-coordinating residues (predominantly histidine) that involve between 0.42-0.90 eV. These results suggest that ionizable residues can tune the reactivity of redox centers by regulating the net charge of the entire protein-cofactor-solvent complex.

Lysine acylation in superoxide dismutase-1 electrostatically inhibits formation of fibrils with prion-like seeding.

The acylation of lysine residues in superoxide dismutase-1 (SOD1) has been previously shown to decrease its rate of nucleation and elongation into amyloid-like fibrils linked to amyotrophic lateral sclerosis. The chemical mechanism underlying this effect is unclear, i.e. hydrophobic/steric effects versus electrostatic effects. Moreover, the degree to which the acylation might alter the prion-like seeding of SOD1 in vivo has not been addressed. Here, we acylated a fraction of lysine residues in SOD1 with groups of variable hydrophobicity, charge, and conformational entropy. The effect of each acyl group on the rate of SOD1 fibril nucleation and elongation were quantified in vitro with thioflavin-T (ThT) fluorescence, and we performed 594 iterate aggregation assays to obtain statistically significant rates. The effect of the lysine acylation on the prion-like seeding of SOD1 was assayed in spinal cord extracts of transgenic mice expressing a G85R SOD1-yellow fluorescent protein construct. Acyl groups with >2 carboxylic acids diminished self-assembly into ThT-positive fibrils and instead promoted the self-assembly of ThT-negative fibrils and amorphous complexes. The addition of ThT-negative, acylated SOD1 fibrils to organotypic spinal cord failed to produce the SOD1 inclusion pathology that typically results from the addition of ThT-positive SOD1 fibrils. These results suggest that chemically increasing the net negative surface charge of SOD1 via acylation can block the prion-like propagation of oligomeric SOD1 in spinal cord.

Kaplan-Meier Meets Chemical Kinetics: Intrinsic Rate of SOD1 Amyloidogenesis Decreased by Subset of ALS Mutations and Cannot Fully Explain Age of Disease Onset.

Over 150 mutations in SOD1 (superoxide dismutase-1) cause amyotrophic lateral sclerosis (ALS), presumably by accelerating SOD1 amyloidogenesis. Like many nucleation processes, SOD1 fibrillization is stochastic (in vitro), which inhibits the determination of aggregation rates (and obscures whether rates correlate with patient phenotypes). Here, we diverged from classical chemical kinetics and used Kaplan-Meier estimators to quantify the probability of apo-SOD1 fibrillization (in vitro) from ∼103 replicate amyloid assays of wild-type (WT) SOD1 and nine ALS variants. The probability of apo-SOD1 fibrillization (expressed as a Hazard ratio) is increased by certain ALS-linked SOD1 mutations but is decreased or remains unchanged by other mutations. Despite this diversity, Hazard ratios of fibrillization correlated linearly with (and for three mutants, approximately equaled) Hazard ratios of patient survival (R2 = 0.67; Pearson's r = 0.82). No correlation exists between Hazard ratios of fibrillization and age of initial onset of ALS (R2 = 0.09). Thus, Hazard ratios of fibrillization might explain rates of disease progression but not onset. Classical kinetic metrics of fibrillization, i.e., mean lag time and propagation rate, did not correlate as strongly with phenotype (and ALS mutations did not uniformly accelerate mean rate of nucleation or propagation). A strong correlation was found, however, between mean ThT fluorescence at lag time and patient survival (R2 = 0.93); oligomers of SOD1 with weaker fluorescence correlated with shorter survival. This study suggests that SOD1 mutations trigger ALS by altering a property of SOD1 or its oligomers other than the intrinsic rate of amyloid nucleation (e.g., oligomer stability; rates of intercellular propagation; affinity for membrane surfaces; and maturation rate).

How Do Gyrating Beads Accelerate Amyloid Fibrillization?

The chemical and physical mechanisms by which gyrating beads accelerate amyloid fibrillization in microtiter plate assays are unclear. Identifying these mechanisms will help optimize high-throughput screening assays for molecules and mutations that modulate aggregation and might explain why different research groups report different rates of aggregation for identical proteins. This article investigates how the rate of superoxide dismutase-1 (SOD1) fibrillization is affected by 12 different beads with a wide range of hydrophobicity, mass, stiffness, and topology but identical diameter. All assays were performed on D90A apo-SOD1, which is a stable and wild-type-like variant of SOD1. The most significant and uniform correlation between any material property of each bead and that bead's effect on SOD1 fibrillization rate was with regard to bead mass. A linear correlation existed between bead mass and rate of fibril elongation (R2 = 0.7): heavier beads produced faster rates and shorter fibrils. Nucleation rates (lag time) also correlated with bead mass, but only for non-polymeric beads (i.e., glass, ceramic, metallic). The effect of bead mass on fibrillization correlated (R2 = 0.96) with variations in buoyant forces and contact forces (between bead and microplate well), and was not an artifact of residual momentum during intermittent gyration. Hydrophobic effects were observed, but only for polymeric beads: lag times correlated negatively with contact angle of water and degree of protein adhesion (surface adhesion and hydrophobic effects were negligible for non-polymeric beads). These results demonstrate that contact forces (alone) explain kinetic variation among non-polymeric beads, whereas surface hydrophobicity and contact forces explain kinetic variation among polymeric beads. This study also establishes conditions for high-throughput amyloid assays of SOD1 that enable the control over fibril morphologies and produce eightfold faster lag times and fourfold less stochasticity than in previous studies.

Gibbs Energy of Superoxide Dismutase Heterodimerization Accounts for Variable Survival in Amyotrophic Lateral Sclerosis.

The exchange of subunits between homodimeric mutant Cu, Zn superoxide dismutase (SOD1) and wild-type (WT) SOD1 is suspected to be a crucial step in the onset and progression of amyotrophic lateral sclerosis (ALS). The rate, mechanism, and ΔG of heterodimerization (ΔGHet) all remain undetermined, due to analytical challenges in measuring heterodimerization. This study used capillary zone electrophoresis to measure rates of heterodimerization and ΔGHet for seven ALS-variant apo-SOD1 proteins that are clinically diverse, producing mean survival times between 2 and 12 years (postdiagnosis). The ΔGHet of each ALS variant SOD1 correlated with patient survival time after diagnosis (R(2) = 0.98), with more favorable ΔGHet correlating with shorter survival by 4.8 years per kJ. Rates of heterodimerization did not correlate with survival time or age of disease onset. Metalation diminished the rate of subunit exchange by up to ∼38-fold but only altered ΔGHet by <1 kJ mol(-1). Medicinal targeting of heterodimer thermodynamics represents a plausible strategy for prolonging life in SOD1-linked ALS.

Stochastic Formation of Fibrillar and Amorphous Superoxide Dismutase Oligomers Linked to Amyotrophic Lateral Sclerosis.

Recent reports suggest that the nucleation and propagation of oligomeric superoxide dismutase-1 (SOD1) is effectively stochastic in vivo and in vitro. This perplexing kinetic variability-observed for other proteins and frequently attributed to experimental error-plagues attempts to discern how SOD1 mutations and post-translational modifications linked to amyotrophic lateral sclerosis (ALS) affect SOD1 aggregation. This study used microplate fluorescence spectroscopy and dynamic light scattering to measure rates of fibrillar and amorphous SOD1 aggregation at high iteration (ntotal = 1.2 × 10(3)). Rates of oligomerization were intrinsically irreproducible and populated continuous probability distributions. Modifying reaction conditions to mimic random and systematic experimental error could not account for kinetic outliers in standard assays, suggesting that stochasticity is not an experimental artifact, rather an intrinsic property of SOD1 oligomerization (presumably caused by competing pathways of oligomerization). Moreover, mean rates of fibrillar and amorphous nucleation were not uniformly increased by mutations that cause ALS; however, mutations did increase kinetic noise (variation) associated with nucleation and propagation. The stochastic aggregation of SOD1 provides a plausible statistical framework to rationalize how a pathogenic mutation can increase the probability of oligomer nucleation within a single cell, without increasing the mean rate of nucleation across an entire population of cells.

Arresting amyloid with coulomb's law: acetylation of ALS-linked SOD1 by aspirin impedes aggregation.

Although the magnitude of a protein's net charge (Z) can control its rate of self-assembly into amyloid, and its interactions with cellular membranes, the net charge of a protein is not viewed as a druggable parameter. This article demonstrates that aspirin (the quintessential acylating pharmacon) can inhibit the amyloidogenesis of superoxide dismutase (SOD1) by increasing the intrinsic net negative charge of the polypeptide, i.e., by acetylation (neutralization) of multiple lysines. The protective effects of acetylation were diminished (but not abolished) in 100 mM NaCl and were statistically significant: a total of 432 thioflavin-T amyloid assays were performed for all studied proteins. The acetylation of as few as three lysines by aspirin in A4V apo-SOD1-a variant that causes familial amyotrophic lateral sclerosis (ALS)-delayed amyloid nucleation by 38% and slowed amyloid propagation by twofold. Lysines in wild-type- and ALS-variant apo-SOD1 could also be peracetylated with aspirin after fibrillization, resulting in supercharged fibrils, with increases in formal net charge of ∼2 million units. Peracetylated SOD1 amyloid defibrillized at temperatures below unacetylated fibrils, and below the melting temperature of native Cu2,Zn2-SOD1 (e.g., fibril Tm = 84.49°C for acetylated D90A apo-SOD1 fibrils). Targeting the net charge of native or misfolded proteins with small molecules-analogous to how an enzyme's Km or Vmax are medicinally targeted-holds promise as a strategy in the design of therapies for diseases linked to protein self-assembly.

Metal-ion-specific screening of charge effects in protein amide H/D exchange and the Hofmeister series.

In this study, protein charge ladders and mass spectrometry were used to quantify how metal cations in the Hofmeister series (Na(+), K(+), Li(+), Mg(2+), and Ca(2+)) permute the effects of lysine acetylation on the rate of amide H/D exchange in a representative protein (myoglobin, Mb). The successive acetylation of up to 18 Lys-ε-NH3(+) groups in Mb caused a linear decrease in its global rate of amide H/D exchange (as measured by mass spectrometry), despite also decreasing the thermostability of Mb by >10 °C. The ability of a metal cation to screen kinetic electrostatic effects during H/D exchange-and to abolish the protective effect of acetylation against H/D exchange-was found to depend on the position of the cation in the Hofmeister series. Na(+) and K(+) cations did not fully equalize the rates of H/D exchange among each "rung" of the charge ladder, whereas Mg(2+) and Ca(2+) did equalize rates without eliminating the hydrophobic core of the protein (i.e., without unfolding Mb); Li(+) exhibited intermediate effects. The ability of Mg(2+) and Ca(2+) to completely screen electrostatic effects associated with the H/D exchange of charge isomers of Mb suggests that Mg(2+) or Ca(2+) (but not Na(+) or K(+)) can be used to quantify the magnitude by which electrostatic charge contributes to the observed rates of amide H/D exchange in proteins.

Protein charge ladders reveal that the net charge of ALS-linked superoxide dismutase can be different in sign and magnitude from predicted values.

This article utilized "protein charge ladders"-chemical derivatives of proteins with similar structure, but systematically altered net charge-to quantify how missense mutations that cause amyotrophic lateral sclerosis (ALS) affect the net negative charge (Z) of superoxide dismutase-1 (SOD1) as a function of subcellular pH and Zn(2+) stoichiometry. Capillary electrophoresis revealed that the net charge of ALS-variant SOD1 can be different in sign and in magnitude-by up to 7.4 units per dimer at lysosomal pH-than values predicted from standard pKa values of amino acids and formal oxidation states of metal ions. At pH 7.4, the G85R, D90A, and G93R substitutions diminished the net negative charge of dimeric SOD1 by up to +2.29 units more than predicted; E100K lowered net charge by less than predicted. The binding of a single Zn(2+) to mutant SOD1 lowered its net charge by an additional +2.33 ± 0.01 to +3.18 ± 0.02 units, however, each protein regulated net charge when binding a second, third, or fourth Zn(2+) (ΔZ < 0.44 ± 0.07 per additional Zn(2+) ). Both metalated and apo-SOD1 regulated net charge across subcellular pH, without inverting from negative to positive at the theoretical pI. Differential scanning calorimetry, hydrogen-deuterium exchange, and inductively coupled plasma mass spectrometry confirmed that the structure, stability, and metal content of mutant proteins were not significantly affected by lysine acetylation. Measured values of net charge should be used when correlating the biophysical properties of a specific ALS-variant SOD1 protein with its observed aggregation propensity or clinical phenotype.

Colorimetric and longitudinal analysis of leukocoria in recreational photographs of children with retinoblastoma.

Retinoblastoma is the most common primary intraocular tumor in children. The first sign that is often reported by parents is the appearance of recurrent leukocoria (i.e., "white eye") in recreational photographs. A quantitative definition or scale of leukocoria--as it appears during recreational photography--has not been established, and the amount of clinical information contained in a leukocoric image (collected by a parent) remains unknown. Moreover, the hypothesis that photographic leukocoria can be a sign of early stage retinoblastoma has not been tested for even a single patient. This study used commercially available software (Adobe Photoshop®) and standard color space conversion algorithms (operable in Microsoft Excel®) to quantify leukocoria in actual "baby pictures" of 9 children with retinoblastoma (that were collected by parents during recreational activities i.e., in nonclinical settings). One particular patient with bilateral retinoblastoma ("Patient Zero") was photographed >7, 000 times by his parents (who are authors of this study) over three years: from birth, through diagnosis, treatment, and remission. This large set of photographs allowed us to determine the longitudinal and lateral frequency of leukocoria throughout the patient's life. This study establishes: (i) that leukocoria can emerge at a low frequency in early-stage retinoblastoma and increase in frequency during disease progression, but decrease upon disease regression, (ii) that Hue, Saturation and Value (i.e., HSV color space) are suitable metrics for quantifying the intensity of retinoblastoma-linked leukocoria; (iii) that different sets of intraocular retinoblastoma tumors can produce distinct leukocoric reflections; and (iv) the Saturation-Value plane of HSV color space represents a convenient scale for quantifying and classifying pupillary reflections as they appear during recreational photography.