The RNA Polymerase α Subunit Recognizes the DNA Shape of the Upstream Promoter Element.

We demonstrate here that the α subunit C-terminal domain of Escherichia coli RNA polymerase (αCTD) recognizes the upstream promoter (UP) DNA element via its characteristic minor groove shape and electrostatic potential. In two compositionally distinct crystallized assemblies, a pair of αCTD subunits bind in tandem to the UP element consensus A-tract that is 6 bp in length (A6-tract), each with their arginine 265 guanidinium group inserted into the minor groove. The A6-tract minor groove is significantly narrowed in these crystal structures, as well as in computationally predicted structures of free and bound DNA duplexes derived by Monte Carlo and molecular dynamics simulations, respectively. The negative electrostatic potential of free A6-tract DNA is substantially enhanced compared to that of generic DNA. Shortening the A-tract by 1 bp is shown to "knock out" binding of the second αCTD through widening of the minor groove. Furthermore, in computationally derived structures with arginine 265 mutated to alanine in either αCTD, either with or without the "knockout" DNA mutation, contact with the DNA is perturbed, highlighting the importance of arginine 265 in achieving αCTD-DNA binding. These results demonstrate that the importance of the DNA shape in sequence-dependent recognition of DNA by RNA polymerase is comparable to that of certain transcription factors.

Meaningful Change Thresholds for the Psoriasis Symptoms and Signs Diary: A Secondary Analysis of a Randomized Clinical Trial.

Importance: Change from baseline score on the validated Psoriasis Symptoms and Signs Diary (PSSD) is a widely used, patient-reported end point in clinical trials for psoriasis. Meaningful score change thresholds anchored to patient-reported assessments have not been established in a clinical trial setting. Objective: To evaluate meaningful within-patient score change thresholds for the PSSD using data from the phase 3 Program to Evaluate the Efficacy and Safety of Deucravacitinib, a Selective TYK2 Inhibitor (POETYK), PSO-1 clinical trial, which compared the efficacy and safety of deucravacitinib vs placebo and apremilast among adults with moderate to severe plaque psoriasis. Design, Setting, and Participants: In this predefined analysis using data from the POETYK PSO-1 multicenter, randomized, double-blind, placebo-controlled phase 3 clinical trial, conducted from August 7, 2018, to September 2, 2020, 666 adults with moderate to severe plaque psoriasis completed the PSSD daily throughout the trial. Meaningful change thresholds were derived by anchoring mean PSSD score change from baseline to week 16 to category improvements on the Patient Global Impression of Change (PGI-C) and the Patient Global Impression of Severity (PGI-S). Interventions: Deucravacitinib, 6 mg, once daily; placebo; or apremilast, 30 mg, twice daily. Main Outcome and Measures: The main outcome was score change from baseline to week 16 on the PSSD, anchored to the PGI-C and PGI-S. Results: The trial included 666 patients (mean [SD] age, 46.1 [13.4] years; 453 men [68.0%]). Three thresholds were identified using an analysis set of 609 patients. Score improvement of at least 15 points from baseline reflected meaningful within-patient change anchored to the PGI-C. Score improvements of 25 points were supported by both the PGI-C and the PGI-S, while a 30-point score change identified patients with greater improvements in their psoriasis symptoms and signs. Conclusions and Relevance: This analysis suggests that PSSD score improvements of 15, 25, or 30 points represent increasing improvements in disease burden that are meaningful to patients with psoriasis.

Deucravacitinib in Moderate to Severe Psoriasis: Clinical and Quality-of-Life Outcomes in a Phase 2 Trial.

INTRODUCTION: Deucravacitinib is an oral, selective tyrosine kinase 2 inhibitor that demonstrated therapeutic benefit in a Phase 2 clinical trial of adults with moderate to severe plaque psoriasis. This analysis was designed to evaluate the effect of deucravacitinib on additional clinical and quality-of-life (QoL) outcomes and assess the relationship between these outcomes in adults with psoriasis. METHODS: Post-hoc analysis of a 12-week Phase 2 trial was conducted for the three most efficacious dosage groups (3 mg twice daily, 6 mg twice daily, 12 mg once daily) and placebo. Investigator assessments for efficacy included Psoriasis Area and Severity Index (PASI), body surface area (BSA) involvement, and static Physician's Global Assessment; QoL was assessed using the Dermatology Life Quality Index (DLQI). Treatment responses and their associations were evaluated over time. RESULTS: Deucravacitinib elicited improvement versus placebo as early as Week 4 for most efficacy measures (including changes in absolute PASI and BSA), with efficacy trends observed from Week 2 to Week 12. Improvements in QoL, assessed by achievement of a DLQI overall score of 0/1 (no effect at all on patient's life), followed a pattern similar to deucravacitinib-related clinical outcomes over 12 weeks. Overall, patients with greater improvements in psoriasis-related clinical signs and symptoms also reported greater improvement in QoL. However, complete skin clearance was not required for achieving DLQI 0/1. CONCLUSION: Deucravacitinib treatment produced early response and similar trends in improvements across multiple efficacy assessments and QoL in moderate to severe plaque psoriasis. Deucravacitinib has the potential to become a promising new oral therapy for this condition. TRIAL REGISTRATION: ClinicalTrials.gov identifier; NCT02931838.

Psoriasis is a skin disease that affects up to 2% of the population. In psoriasis, red, scaly lesions develop on the skin driven by an aberrant immune response. Psoriasis impacts not only physical and mental health but also quality of life (QoL). Deucravacitinib is being investigated as a treatment for psoriasis. We performed a Phase 2 dose-ranging, placebo-controlled, 12-week study of deucravacitinib in adults with moderate to severe psoriasis. Patients in the USA, Australia, Canada, Germany, Japan, Latvia, Mexico, and Poland participated. The study showed that oral treatment with deucravacitinib was effective using a disease severity score (percentage of patients with ≥ 75% reduction from baseline in Psoriasis Area and Severity Index score) at Week 12­placebo 7% and deucravacitinib 67%­75% for the three highest dosages­and was generally well tolerated. We further analyzed the association between efficacy and a QoL measure, the Dermatology Life Quality Index (DLQI), in patients who received placebo or the most effective dosages of deucravacitinib (≥ 3 mg twice daily). Deucravacitinib was effective at the three dosage levels tested. Skin improvement occurred early during treatment and was mirrored by improvements in DLQI score during the 12 weeks of treatment. Although some patients did not have complete clearance of their psoriasis, a large percentage of those patients still achieved considerable improvement in QoL as measured by achieving a DLQI score of 0/1 (i.e., no effect at all on the patient's QoL).

Daclatasvir and sofosbuvir treatment of decompensated liver disease or post-liver transplant hepatitis C virus recurrence in patients with advanced liver disease/cirrhosis in a real-world cohort.

We report the findings of an early access program providing treatment for chronic hepatitis C virus infection (any genotype) with daclatasvir and sofosbuvir with/without ribavirin to patients with Child-Pugh class C cirrhosis or prior liver transplant recipients with recurrent hepatitis C virus infection and advanced fibrosis/cirrhosis. Patients had <12-month life expectancies per the local investigator. Patients received daclatasvir 60 mg and sofosbuvir 400 mg once daily, with/without ribavirin, for 24 weeks. Sustained virologic response (SVR) at posttreatment week 12 (SVR12) was measured. Assessments adhered to local standards. One patient (prior Child-Pugh class C who improved to class B) enrolled by exemption was included in the overall data but not the class C cohort efficacy/safety data. Of the 77 treated patients, including 62 liver transplant recipients (genotype 1, n = 43, 69%; genotype 3, n = 16, 26%) and 14 patients with Child-Pugh class C cirrhosis (genotype 1, n = 4, 29%; genotype 3, n = 10, 71%), 63 (82%) completed treatment. SVR12 rates by modified intention-to-treat analysis (excluding nonvirologic failures lost to follow-up and withdrawal [consent/no reason]) in the overall, liver transplant, and Child-Pugh class C cohorts were 84% (n = 64/76), 90% (n = 56/62), and 62% (n = 8/13), respectively. Rates increased to 96% (n = 64/67), 97% (n = 56/58), and 89% (n = 8/9), respectively, in patients with available virologic data (including early discontinuations); 22/23 patients with genotype 3 (96%) achieved SVR12. Single cases of virologic nonresponse and relapse (both in liver transplant recipients with genotype 1) and viral breakthrough (Child-Pugh class C; genotype 3) occurred. Six patients died, 10 had adverse events leading to discontinuation, and 30 experienced serious adverse events. Conclusion: Daclatasvir plus sofosbuvir, with/without ribavirin, provided high SVR12 rates and was generally well tolerated in patients with life-threatening disease and high unmet needs. (Hepatology Communications 2018;2:354-363).

Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: recognition of pyrimidine-purine and purine-purine steps.

The catabolite activator protein (CAP) bends DNA in the CAP-DNA complex, typically introducing a sharp DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site, 5'-A1A2A3T4G5T6G7A8T9C10T11 -3' ("primary kink"). In previous work, we showed that CAP recognizes the nucleotide immediately 5' to the primary-kink site, T6, through an "indirect-readout" mechanism involving sequence effects on energetics of primary-kink formation. Here, to understand further this example of indirect readout, we have determined crystal structures of CAP-DNA complexes containing each possible nucleotide at position 6. The structures show that CAP can introduce a DNA kink at the primary-kink site with any nucleotide at position 6. The DNA kink is sharp with the consensus pyrimidine-purine step T6G7 and the non-consensus pyrimidine-purine step C6G7 (roll angles of approximately 42 degrees, twist angles of approximately 16 degrees ), but is much less sharp with the non-consensus purine-purine steps A6G7 and G6G7 (roll angles of approximately 20 degrees, twist angles of approximately 17 degrees). We infer that CAP discriminates between consensus and non-consensus pyrimidine-purine steps at positions 6-7 solely based on differences in the energetics of DNA deformation, but that CAP discriminates between the consensus pyrimidine-purine step and non-consensus purine-purine steps at positions 6-7 both based on differences in the energetics of DNA deformation and based on qualitative differences in DNA deformation. The structures further show that CAP can achieve a similar, approximately 46 degrees per DNA half-site, overall DNA bend through a sharp DNA kink, a less sharp DNA kink, or a smooth DNA bend. Analysis of these and other crystal structures of CAP-DNA complexes indicates that there is a large, approximately 28 degrees per DNA half-site, out-of-plane component of CAP-induced DNA bending in structures not constrained by end-to-end DNA lattice interactions and that lattice contacts involving CAP tend to involve residues in or near biologically functional surfaces.

No evident association between efavirenz use and suicidality was identified from a disproportionality analysis using the FAERS database.

OBJECTIVE: To assess the potential association of selected antiretrovirals (ARVs), including efavirenz, with suicidality. DESIGN: Retrospective analysis of the Food and Drug Administration Adverse Event Reporting System (FAERS), by performing a Multi-Item Gamma Poisson Shrinker (MGPS) disproportionality analysis. METHODS: MGPS disproportionality analysis, a technique to identify associations between drugs and adverse events, was performed using cumulative data from the FAERS database collected up to August 2012. This method yields an Empirical Bayesian Geometric Mean score and corresponding 90% confidence interval (EB05, EB95). EB05 scores ≥ 2 were pre-defined as a signal for a potential drug-event association. The FAERS database includes spontaneous adverse-event reports from consumers and healthcare professionals. All FAERS reports of suicidality (including suicidal ideation, suicide attempt and completed suicide or a composite of these) in patients taking efavirenz (as single agent or in fixed-dose combination), atazanavir, darunavir, etravirine, nevirapine and raltegravir were identified. A number of parallel analyses were performed to assess the validity of the methodology: fluoxetine and sertraline, antidepressants with a known association with suicidality, and raltegravir, an ARV with rhabdomyolysis and myopathy listed as "uncommon" events in the US-prescribing information. RESULTS: A total of 29,856 adverse event reports were identified among patients receiving efavirenz, atazanavir, darunavir, etravirine, nevirapine and raltegravir, of which 457 were reports of suicidality events. EB05 scores observed for the composite suicidality term for efavirenz (EB05=0.796), and other ARVs (EB05=0.279-0.368), were below the pre-defined threshold. Fluoxetine and sertraline gave EB05 scores for suicidality >2. Raltegravir gave EB05 scores >2 for myopathy and rhabdomyolysis. CONCLUSIONS: The pre-determined threshold for signals for suicidality, including suicidal ideation, suicide attempt, completed suicide and a composite suicidality endpoint, was not exceeded for efavirenz and other ARVs in this analysis. Efavirenz has been associated with suicidality in clinical trials. Further studies that adjust for confounding factors are needed to better understand any potential association with ARVs and suicidality.

Signatures of protein-DNA recognition in free DNA binding sites.

One obstacle to achieving complete understanding of the principles underlying sequence-dependent recognition of DNA is the paucity of structural data for DNA recognition sequences in their free (unbound) state. Here, we carried out crystallization screening of 50 DNA duplexes containing cognate protein binding sites and obtained new crystal structures of free DNA binding sites for three distinct modes of DNA recognition: anti-parallel beta strands (MetR), helix-turn-helix motif + hinge helices (PurR), and zinc fingers (Zif268). Structural changes between free and protein-bound DNA are manifested differently in each case. The new DNA structures reveal that distinctive sequence-dependent DNA geometry dominates recognition by MetR, protein-induced bending of DNA dictates recognition by PurR, and deformability of DNA along the A-B continuum is important in recognition by Zif268. Together, our findings show that crystal structures of free DNA binding sites provide new information about the nature of protein-DNA interactions and thus lend insights towards a structural code for DNA recognition.