The ORC/Cdc6/MCM2-7 complex facilitates MCM2-7 dimerization during prereplicative complex formation.

The replicative mini-chromosome-maintenance 2-7 (MCM2-7) helicase is loaded in Saccharomyces cerevisiae and other eukaryotes as a head-to-head double-hexamer around origin DNA. At first, ORC/Cdc6 recruits with the help of Cdt1 a single MCM2-7 hexamer to form an 'initial' ORC/Cdc6/Cdt1/MCM2-7 complex. Then, on ATP hydrolysis and Cdt1 release, the 'initial' complex is transformed into an ORC/Cdc6/MCM2-7 (OCM) complex. However, it remains unclear how the OCM is subsequently converted into a MCM2-7 double-hexamer. Through analysis of MCM2-7 hexamer-interface mutants we discovered a complex competent for MCM2-7 dimerization. We demonstrate that these MCM2-7 mutants arrest during prereplicative complex (pre-RC) assembly after OCM formation, but before MCM2-7 double-hexamer assembly. Remarkably, only the OCM complex, but not the 'initial' ORC/Cdc6/Cdt1/MCM2-7 complex, is competent for MCM2-7 dimerization. The MCM2-7 dimer, in contrast to the MCM2-7 double-hexamer, interacts with ORC/Cdc6 and is salt-sensitive, classifying the arrested complex as a helicase-loading intermediate. Accordingly, we found that overexpression of the mutants cause cell-cycle arrest and dominant lethality. Our work identifies the OCM complex as competent for MCM2-7 dimerization, reveals MCM2-7 dimerization as a limiting step during pre-RC formation and defines critical mechanisms that explain how origins are licensed

Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA

In eukaryotes, the Cdt1-bound replicative helicase core MCM2-7 is loaded onto DNA by the ORC-Cdc6 ATPase to form a prereplicative complex (pre-RC) with an MCM2-7 double hexamer encircling DNA. Using purified components in the presence of ATP-γS, we have captured in vitro an intermediate in pre-RC assembly that contains a complex between the ORC-Cdc6 and Cdt1-MCM2-7 heteroheptamers called the OCCM. Cryo-EM studies of this 14-subunit complex reveal that the two separate heptameric complexes are engaged extensively, with the ORC-Cdc6 N-terminal AAA+ domains latching onto the C-terminal AAA+ motor domains of the MCM2-7 hexamer. The conformation of ORC-Cdc6 undergoes a concerted change into a right-handed spiral with helical symmetry that is identical to that of the DNA double helix. The resulting ORC-Cdc6 helicase loader shows a notable structural similarity to the replication factor C clamp loader, suggesting a conserved mechanism of action

Efficacy and Safety of Ixekizumab in the Treatment of Radiographic Axial Spondyloarthritis:Sixteen-Week Results From a Phase III Randomized, Double-Blind, Placebo-Controlled Trial in Patients With Prior Inadequate Response to or Intolerance of Tumor Necrosis Factor Inhibitors

Objective: To investigate the efficacy and safety of ixekizumab in patients with active radiographic axial spondyloarthritis (SpA) and prior inadequate response to or intolerance of 1 or 2 tumor necrosis factor inhibitors (TNFi). Methods: In this phase III randomized, double-blind, placebo-controlled trial, adult patients with an inadequate response to or intolerance of 1 or 2 TNFi and an established diagnosis of axial SpA (according to the Assessment of SpondyloArthritis international Society [ASAS] criteria for radiographic axial SpA, with radiographic sacroiliitis defined according to the modified New York criteria and ≥1 feature of SpA) were recruited and randomized 1:1:1 to receive placebo or 80-mg subcutaneous ixekizumab every 2 weeks (IXEQ2W) or 4 weeks (IXEQ4W), with an 80-mg or 160-mg starting dose. The primary end point was 40% improvement in disease activity according to the ASAS criteria (ASAS40) at week 16. Secondary outcomes and safety were also assessed. Results: A total of 316 patients were randomized to receive placebo (n = 104), IXEQ2W (n = 98), or IXEQ4W (n = 114). At week 16, significantly higher proportions of IXEQ2W patients (n = 30 [30.6%]; P = 0.003) or IXEQ4W patients (n = 29 [25.4%]; P = 0.017) had achieved an ASAS40 response versus the placebo group (n = 13 [12.5%]), with statistically significant differences reported as early as week 1 with ixekizumab treatment. Statistically significant improvements in disease activity, function, quality of life, and spinal magnetic resonance imaging–evident inflammation were observed after 16 weeks of ixekizumab treatment versus placebo. Treatment-emergent adverse events (AEs) with ixekizumab treatment were more frequent than with placebo. Serious AEs were similar across treatment arms. One death was reported (IXEQ2W group). Conclusion: Ixekizumab treatment for 16 weeks in patients with active radiographic axial SpA and previous inadequate response to or intolerance of 1 or 2 TNFi yields rapid and significant improvements in the signs and symptoms of radiographic axial SpA versus placebo

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to <90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], >300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery