Inhibition of the SARS-CoV-2 main protease (MPpro) is a major focus of drug discovery efforts against COVID-19. Here we report a hit expansion of non-covalent inhibitors of MPpro. Starting from a recently discovered scaffold (The COVID Moonshot Consortium. Open Science Discovery of Oral Non-Covalent SARS-CoV-2 Main Protease Inhibitor Therapeutics. bioRxiv 2020.10.29.339317) represented by an isoquinoline series, we searched a database of over a billion compounds using a cheminformatics molecular fingerprinting approach. We identified and tested 48 compounds in enzyme inhibition assays, of which 21 exhibited inhibitory activity above 50% at 20 mu M. Among these, four compounds with IC50 values around 1 mu M were found. Interestingly, despite the large search space, the isoquinolone motif was conserved in each of these four strongest binders. Roomtemperature X-ray structures of co-crystallized protein-inhibitor complexes were determined up to 1.9 angstrom resolution for two of these compounds as well as one of the stronger inhibitors in the original isoquinoline series, revealing essential interactions with the binding site and water molecules. Molecular dynamics simulations and quantum chemical calculations further elucidate the binding interactions as well as electrostatic effects on ligand binding. The results help explain the strength of this new non-covalent scaffold for MPpro inhibition and inform lead optimization efforts for this series, while demonstrating the effectiveness of a high-throughput computational approach to expanding a pharmacophore library.

Neuroendocrine Carcinomas (NECs) are a distinct family of poorly differentiated morphology, sharing molecular, clinical and outcome characteristics. No standard-of-care exists after the failure of first-line platinum-based chemotherapy (CT) regimens. Nivo±ipi has been reported to provide survival benefit versus standard CT in metastatic solid cancers. GCO-001 NIPINEC is a multicenter, non-comparative, randomized (1:1) phase 2 trial with a two-step design (early stopping for futility after 50% of accrual). Main inclusion criteria were histologically proven NEC (large- and small-cell for gastroenteropancreatic (GEP) NECs, and large-cell for lung NECs), in 2nd or 3rd line refractory to platinum-based CT, and PS 0-2. Patients received nivo 3 mg/kg q2w ± ipi 1 mg/kg q6w, for 2 years or until progression or unacceptable toxicity. The primary endpoint was objective response rate (ORR) at 8 weeks assessed by investigators (power of 90%, alpha risk at 5% two-sided). Other endpoints included progression-free survival (PFS), overall survival (OS) and safety. NCT number: NCT03591731. From Dec 2018 to Mar 2021, 185 patients (93 GEP and 92 lung) were enrolled in 50 centers. Median age was 64.4 years (range 26.4–87.1), 71% males, 70% smokers, 91% with ECOG PS 0 or 1, 24% with asymptomatic brain metastases. 170 (92%) patients were evaluable for the primary endpoint. ORR at 8 weeks was 7.2% (95%CI [2.7-15.1]) with nivo and 14.9% (95%CI [8.2-24.2]) with nivo+ipi. Median PFS was 1.8 months (95%CI [1.7-2.0]) with nivo and 1.9 months (95%CI [1.6-2.1]) with nivo+ipi. Median OS was 7.2 months (95%CI [3.7-14.1]) with nivo and 5.8 months (95%CI [3.3-7.6]) with nivo+ipi. 3 patients (3.5%) and 7 patients (8.9%) experienced treatment discontinuation because of toxicity with nivo and nivo+ipi respectively. 2 treatment-related deaths were observed with nivo (meningoencephalitis and pneumonitis). Most frequent grade ≥ 3 AEs were asthenia (7.3%), anaemia (6.3%) and alkaline phosphatase increased (5.8%). Nivo+Ipi only reached the primary endpoint in 2nd/3rd line NECs pts with acceptable toxicity.

Aortic dissections are uncommon but potentially life-threatening emergencies requiring a high index of clinical suspicion for emergency medicine providers. The most reliable diagnostic test for an acute aortic dissection is CT angiography, although this test cannot be performed at the bedside. Transthoracic echocardiography (TTE) has yielded a lower sensitivity, mainly due to its inability to diagnose Type B dissections while being nearly 100% sensitive for Type A dissections using dilation of the aortic root as the criteria. We developed an ultrasound protocol combining TTE with an evaluation of the abdominal aorta. The goal of this study was to determine the sensitivity of this point-of-care ultrasound protocol in the evaluation of patients with aortic dissection. Single center, retrospective review of all patients evaluated in the ED after our protocol had been established from January 1, 2010 through March 31, 2017 who had a diagnosis of aortic dissection confirmed by CT angiography. According to our protocol, we used 3 signs from TTE to suggest AD: the presence of either a pericardial effusion or intimal flap, or an aortic outflow track size of greater than 3.5 cm during diastole (measured from inner wall to inner wall within 2cm of the aortic annulus). In the abdominal aorta, the presence of an undulating intimal flap suggested AD. The presence of any of these findings was considered a positive study for dissection. We excluded patients with known dissections, transfers from OSH, and 12 newly diagnosed AD without ultrasounds. The Fisher Exact test was utilized for data analysis. 442 ultrasounds were performed for suspected AD. 28 patients were identified during the study period. 12 had a Stanford type A dissection. 16 had a Stanford type B. 27 of the 28 patients had at least one of the aforementioned findings. The only patient not diagnosed with bedside ultrasound had a Stanford type B dissection limited to the descending thoracic aorta. The most common positive finding was an intimal flap, identified in 23 out of 28 patients, including 15 type B patients. These criteria showed a sensitivity of 96.4% (95% CI 81.03% - 99.91%) and a specificity of 90.8% (95% CI 87.62% - 93.42%) for aortic dissection (100% for type A & 93.75% for type B). Our protocol, provided an overall NPV of 99.73% (95% CI 98.21% - 99.96%) for both type A and B dissections. (Fisher Exact = 0, p < .001; x2 [1] = 155.06, p < .001). By combining TTE with abdominal aortic ultrasound in patients with suspected aortic dissection, we were able to diagnose 96.4% of patients who presented to our institution with an aortic dissection (100% of type A dissections). Furthermore, the presence of an intimal flap was also 100% specific for aortic dissection. Future prospective studies can further evaluate test characteristics of this combined ultrasound protocol.

Methyl-coenzyme M reductase, the rate-limiting enzyme in methanogenesis and anaerobic methane oxidation, is responsible for the biological production of more than 1 billion tons of methane per year. The mechanism of methane synthesis is thought to involve either methyl-nickel(III) or methyl radical/Ni(II)-thiolate intermediates. We employed transient kinetic, spectroscopic, and computational approaches to study the reaction between the active Ni(I) enzyme and substrates. Consistent with the methyl radical-based mechanism, there was no evidence for a methyl-Ni(III) species; furthermore, magnetic circular dichroism spectroscopy identified the Ni(II)-thiolate intermediate. Temperature-dependent transient kinetics also closely matched density functional theory predictions of the methyl radical mechanism. Identifying the key intermediate in methanogenesis provides fundamental insights to develop better catalysts for producing and activating an important fuel and potent greenhouse gas.

The reduction of N2 to NH3 by Mo-dependent nitrogenase at its active-site metal cluster FeMo-cofactor utilizes reductive elimination of Fe-bound hydrides with obligatory loss of H2 to activate the enzyme for binding/reduction of N2. Earlier work showed that wild-type nitrogenase and a nitrogenase with amino acid substitutions in the MoFe protein near FeMo-cofactor can catalytically reduce CO2 by two or eight electrons/protons to carbon monoxide (CO) and methane (CH4) at low rates. Here, it is demonstrated that nitrogenase preferentially reduces CO2 by two electrons/protons to formate (HCOO(-)) at rates >10 times higher than rates of CO2 reduction to CO and CH4. Quantum mechanical calculations on the doubly reduced FeMo-cofactor with a Fe-bound hydride and S-bound proton (E2(2H) state) favor a direct reaction of CO2 with the hydride ("direct hydride transfer" reaction pathway), with facile hydride transfer to CO2 yielding formate. In contrast, a significant barrier is observed for reaction of Fe-bound CO2 with the hydride ("associative" reaction pathway), which leads to CO and CH4. Remarkably, in the direct hydride transfer pathway, the Fe-H behaves as a hydridic hydrogen, whereas in the associative pathway it acts as a protic hydrogen. MoFe proteins with amino acid substitutions near FeMo-cofactor (α-70(Val→Ala), α-195(His→Gln)) are found to significantly alter the distribution of products between formate and CO/CH4.

The surface chemistry of metal oxide particles is governed by the charge that develops at the interface with aqueous solution. Mineral transformation, biogeochemical reactions, remediation, and sorption dynamics are profoundly affected in response. Here we report implementation of replica-exchange constant-pH molecular dynamics simulations that use classical molecular dynamics for exploring configurational space and Metropolis Monte Carlo walking through protonation space with a simulated annealing escape route from metastable configurations. By examining the archetypal metal oxide, goethite (α-FeOOH), we find that electrostatic potential gradients spontaneously arise between intersecting low-index crystal faces and across explicitly treated oxide nanoparticles at a magnitude exceeding the Johnson-Nyquist voltage fluctuation. Fluctuations in adsorbed proton density continuously repolarize the surface potential bias between edge-sharing crystal faces, at a rate slower than the reported electron-polaron hopping rate in goethite interiors. This suggests that these spontaneous surface potential fluctuations will control the net movement of charge carriers in the lattice.

Mo-dependent nitrogenase catalyzes the biological reduction of N2 to two NH3 molecules at FeMo-cofactor buried deep inside the MoFe protein. Access of substrates, such as N2, to the active site is likely restricted by the surrounding protein, requiring substrate channels that lead from the surface to the active site. Earlier studies on crystallographic structures of the MoFe protein have suggested three putative substrate channels. Here, we have utilized submicrosecond atomistic molecular dynamics simulations to allow the nitrogenase MoFe protein to explore its conformational space in an aqueous solution at physiological ionic strength, revealing a putative substrate channel. The viability of this observed channel was tested by examining the free energy of passage of N2 from the surface through the channel to FeMo-cofactor, resulting in the discovery of a very low energy barrier. These studies point to a viable substrate channel in nitrogenase that appears during thermal motions of the protein in an aqueous environment and that approaches a face of FeMo-cofactor earlier implicated in substrate binding.

Control of the reactivity of the nickel center of the [NiFe] hydrogenase and other metalloproteins commonly involves outer coordination sphere ligands that act to modify the geometry and physical properties of the active site metal centers. We carried out a combined set of classical molecular dynamics and quantum/classical mechanics calculations to provide quantitative estimates of how dynamic fluctuations of the active site within the protein matrix modulate the electronic structure at the catalytic center. Specifically we focused on the dynamics of the inner and outer coordination spheres of the cysteinate-bound Ni-Fe cluster in the catalytically active Ni-C state. There are correlated movements of the cysteinate ligands and the surrounding hydrogen-bonding network, which modulate the electron affinity at the active site and the proton affinity of a terminal cysteinate. On the basis of these findings, we hypothesize a coupling between protein dynamics and electron and proton transfer reactions critical to dihydrogen production.

The staggered cross decaheme configuration of electron transfer cofactors in the outer-membrane cytochrome MtrF serves as a prototype for conformationally gated multiheme electron transport. Derived from the bacterium Shewanella oneidensis, the staggered cross configuration reveals intersecting c-type octaheme and tetraheme "wires" containing thermodynamic "hills" and "valleys" (Proc. Natl. Acad. Sci. U. S. A. 2014, 11, 611-616), suggesting that the protein structure may include a dynamical mechanism for conductance and pathway switching depending on enzymatic functional need. Here, we applied classical molecular and statistical mechanics calculations of large-amplitude protein dynamics in MtrF, to address its potential to modulate pathway conductance, including assessment of the effect of the total charge state. Explicit solvent molecular dynamics simulations of fully oxidized and fully reduced MtrF showed that the slowest mode of collective decaheme motion is 9096 similar between the oxidized and reduced states and consists primarily of interheme separation with minor rotational contributions. The frequency of this motion is 1.7 x 10(7) s(-1) both for fully oxidized and fully reduced MtrF, slower than the downhill electron transfer rates between stacked heme pairs at the octaheme termini and faster than the electron transfer rates between parallel hemes in the tetraheme chain. This implies that MtrF uses slow conformational fluctuations to modulate electron flow along the octaheme pathway, apparently for the purpose of increasing the residence time of electrons on lowest potential hemes 4 and 9. This apparent gating mechanism should increase the success rate of electron transfer from MtrF to low potential environmental acceptors via these two solvent-exposed hemes.

Abstract This paper discusses the key challenges associated with the design of a Dry Tree Semi (DTS) global configuration with robust performance and easy execution, as well as the integration of the topside facilities with hull at quayside. The authors will address the issues associated with a safe wellbay design in order to satisfy all functional requirements of simultaneous drilling and production operations, and compensation of riser stroke. More importantly, the authors will present technically robust and cost-effective solutions to overcome these challenges through extensive engineering assessment, computer simulation, and model test verification. The authors focus on improving functional and operational safety of the DTS system while minimizing project execution risks. Through innovative design, engineering analysis, and model test verification of the DTS system, important and valuable results will be presented in this paper, which include overcoming various conflicting issues associated with wellbay arrangement and functional performance under simultaneous drilling and production operations; compensating for the most challenging issue of riser performance on a DTS utilizing long stroke riser tensioner technology; and deriving a hull-mooring-riser global configuration that not only carries robust performance during operation, but also offers the benefits during project execution.

Calmodulin (CaM) contains two structurally homologous domains that cooperatively bind to a range of different target proteins, such that upon binding the opposing N- and C-domains wrap around the CaM-binding sequence. The N-domain has a binding affinity (Kd = 24 µM) that is approximately 3-orders of magnitude weaker than the C-domain (Kd = 11 nM) to the plasma membrane Ca-ATPase (PMCA). These large differences in binding affinities facilitate ordered binding, which is necessary for the productive activation of many target proteins. To better understand design principles that facilitate molecular recognition, we have used directed evolution combined with yeast surface display to identify mutations that enhance binding between the N-domain of CaM and the PMCA, permitting the identification of combinatorial families of mutate N-domain proteins with nanomolar binding affinities (similar to that of the C-domain). All observed mutations occur at noninterfacial sites that are naturally variable in Nature, suggesting that hypervariable sites between different species may fine tune binding affinities. Mutations in the N-domain that selectively destabilize the unbound state commonly result in enhanced binding affinities with other CaM-binding sequences (i.e., skeletal myosin light chain kinase and ryanodine receptor). In contrast, mutations that structurally couple with residues in the binding interface result in selective high-affinity binding to the PMCA; these mutations commonly result in decreased binding to other target proteins. In total, these results indicate the value of using directed evolution approaches to identify underlying principles that determine binding affinities. This research was supported by the Defense Threat Reduction Agency (DTRA).

Calmodulin (CaM) is a highly flexible calcium-binding protein that mediates signal transduction through an ability to differentially bind to highly variable binding sequences in target proteins. To identify how binding affects CaM motions, and its relationship to conformational entropy and target peptide sequence, we have employed fully atomistic, explicit solvent molecular dynamics simulations of unbound CaM and CaM bound to five different target peptides. The calculated CaM conformational binding entropies correlate with experimentally derived conformational entropies with a correlation coefficient R2 of 0.95. Selected side-chain interactions with target peptides restrain interhelical loop motions, acting to tune the conformational entropy of the bound complex via widely distributed CaM motions. In the complex with the most conformational entropy retention (CaM in complex with the neuronal nitric oxide synthase binding sequence), Lys-148 at the C-terminus of CaM forms transient salt bridges alternating between Glu side chains in the N-domain, the central linker, and the binding target. Additional analyses of CaM structures, fluctuations, and CaM-target interactions illuminate the interplay between electrostatic, side chain, and backbone properties in the ability of CaM to recognize and discriminate against targets by tuning its conformational entropy, and suggest a need to consider conformational dynamics in optimizing binding affinities.

Classical molecular force-field parameters describing the structure and motion of metal clusters in [NiFe] hydrogenase enzymes can be used to compare the dynamics and thermodynamics of [NiFe] under different oxidation, protonation, and ligation circumstances. Using density functional theory (DFT) calculations of small model clusters representative of the active site and the proximal, medial, and distal Fe/S metal centers and their attached protein side chains, we have calculated classical force-field parameters for [NiFe] in reduced and oxidized states, including internal coordinates, force constants, and atom-centered charges. Derived force constants revealed that cysteinate ligands bound to the metal ions are more flexible in the Ni-B active site, which has a bridging hydroxide ligand, than in the Ni-C active site, which has a bridging hydride. Ten nanosecond all-atom, explicit-solvent MD simulations of [NiFe] hydrogenase in oxidized and reduced catalytic states established the stability of the derived force-field parameters in terms of Cα and metal cluster fluctuations. Average active site structures from the protein MD simulations are consistent with [NiFe] structures from the Protein Data Bank, suggesting that the derived force-field parameters are transferrable to other hydrogenases beyond the structure used for testing. A comparison of experimental H2-production rates demonstrated a relationship between cysteinate side chain rotation and activity, justifying the use of a fully dynamic model of [NiFe] metal cluster motion.

The change in calmodulin's conformational entropy upon binding to target peptides favorably influences target binding thermodynamics. Experiments by Wand and co-workers (Nature 19, 2007, 325–329) demonstrated that calmodulin conformational entropy calculated from NMR order parameters correlates linearly with the overall binding entropy from isothermal titration calorimetry and is a significant contributor to binding affinity, a hypothesis that can be directly tested using computational molecular dynamics. We calculated 100 nanosecond trajectories for calcium-saturated calmodulin and five of the six calmodulin-target complexes from the Wand study for which structures are available (CaMKK, CaMK1, smMLCK, eNOS and nNOS) using fully atomistic, explicit solvent, constant temperature and pressure (300 K, 1 atm) molecular dynamics with the AMBER03 force field and the TIP3P solvent model. These simulations enabled us to compare the low- and high-frequency CaM motions associated with target binding and the conformational entropy changes associated with the process using the quasiharmonic approximation. The calculated entropies of CaM bound to the targets relative to unbound CaM correlate extremely well with the NMR-derived conformational entropies and ITC binding entropies (correlation coefficients R are 0.89), and trajectory analysis revealed that observed binding entropies are due to increased helix flexibility in calmodulin's N-domain and the motion of CaM residues with long sidechains, particularly methionines and glutamates, consistent with the induced-disorder description of peptide binding to flexible proteins (Molecular Pharmaceutics, 2009, 430–437).

Objective: Eosinophilic oesophagitis (EoO) is a clinicopathological condition defined by proton pump inhibitorrefractory oesophageal symptoms combined with oesophageal eosinophilia. The pharmacodynamic effect of mepolizumab (a humanised anti-interleukin-5 monoclonal antibody) in EoO was evaluated.Methods: Eleven adults with active EoO (> 20 peak eosinophil number/high power field (hpf) and dysphagia) were randomised to 750 mg of mepolizumab (n = 5) or placebo (n = 6) and received two intravenous infusions, 1 week apart. Those not in complete remission (< 5 peak eosinophil number/hpf) after 8 weeks received two further doses 4 weeks apart, 1500 mg of mepolizumab or placebo. The effect of mepolizumab was assessed clinically, endoscopically, histologically, and via blood and tissue biomarkers.Results: As assessed by immunofluorescence, a marked reduction of mean oesophageal eosinophilia (p = 0.03) was seen in the mepolizumab group (-54%) compared with the placebo group (-5%) 4 weeks after initiation of treatment. No further reduction of eosinophil numbers was observed in response to the two additional infusions in either group. Mepolizumab reduced tenascin C (p = 0.033) and transforming growth factor beta 1 (p = 0.05) expression in the oesophageal epithelial layer 13 weeks after initiation of treatment. Clinically, limited improvement of symptoms was seen, although a trend was seen between 4 and 13 weeks after initiation of mepolizumab treatment. Mepolizumab was well tolerated.Conclusions: Mepolizumab significantly reduced eosinophil numbers in oesophageal tissues in adult patients with active EoO, and changes in the expression of molecules associated with oesophageal remodelling were reversed. Minimal clinical improvement was achieved in a subgroup of patients with EoO. Mepolizumab had an acceptable safety profile, even at the high 1500 mg dose level.

With cancer-related fatalities being the second leading cause of death in the USA, understanding the activity of effective chemotherapeutic agents is critical to addressing prostate and other cancers. Celecoxib, an FDA-approved drug for the treatment of colon tumors, has been used successfully as a lead compound in the development of antiproliferative agents. The ability of celecoxib to inhibit the development and progression of tumors has been connected to a number of mechanisms of actions that are both dependent on and independent of its cyclooxygenase-2 activity. A structure-based approach has been employed to develop a model that underscores the structural significance of celecoxib as an antiproliferative agent. By evaluating the structure activity of this library of molecules, we were able to create a QSAR model for predicting the antiproliferative activity of structurally similar molecules. The development of the model will be presented in this paper.