Delineating Protein-Protein Curvilinear Dissociation Pathways and Energetics with Naïve Multiple-Walker Umbrella Sampling Simulations.

The protein-protein interaction energetics can be obtained by calculating the potential of mean force (PMF) from umbrella sampling (US) simulations, in which samplings are often enhanced along a predefined vector as the reaction coordinate. However, any slight change in the vector may significantly vary the calculated PMF, and therefore the energetics using a random choice of vector may mislead. A non-predefined curve path-based sampling enhancement approach is a natural alternative, but was relatively less explored for protein-protein systems. In this work, dissociation of the barnase-barstar complex is simulated by implementing non-predefined curvilinear pathways in US simulations. A simple variational principle is applied to determine the lower bound PMF, which could be used to derive the standard free energy of binding. Two major dissociation pathways, which include interactions with the RNA-binding loop and the Val 36 to Gly 40 loop, are observed. Further, the proposed approach was used to discriminate the decoys from protein-protein docking studies. © 2019 Wiley Periodicals, Inc.

Discovery and evaluation of novel FAAH inhibitors in neuropathic pain model.

Conceptual design and modification of urea moiety in chemotype PF-3845/04457845, the bench marking irreversible inhibitor of fatty acid amide hydrolase (FAAH), led to discovery of a novel nicotinamide-based lead 12a having reversible mechanism of action. Focused SAR around the pyridine heterocycle (Ar) in 12a (Tables 1 and 2) resulted into four shortlisted compounds, (-)-12a, (-)-12i, (-)-12l-m. The required (-)-enantiomers were obtained via diastereomeric resolution of a novel chiral dissymmetric intermediate 15. Based on comparative profile of FAAH potency, metabolic stability in liver microsome, liability of inhibiting major hCYP450 isoforms, rat PK, and brain penetration ability, two SAR optimized compounds, (-)-12l and (-)-12m, were selected for efficacy study in rat model of chemotherapy-induced peripheral neuropathy (CIPN). Both the compounds exhibited dose related antihyperalgesic effects, when treated with 3-30 mg/kg po for 7 days. The effects at 30 mg/kg are comparable to that of PF-04457845 (10 mg/kg) and Tramadol (40 mg/kg).

The Imipridone ONC213 Targets α-Ketoglutarate Dehydrogenase to Induce Mitochondrial Stress and Suppress Oxidative Phosphorylation in Acute Myeloid Leukemia.

Eradication of acute myeloid leukemia (AML) is therapeutically challenging; many patients succumb to AML despite initially responding to conventional treatments. Here, we showed that the imipridone ONC213 elicits potent antileukemia activity in a subset of AML cell lines and primary patient samples, particularly in leukemia stem cells, while producing negligible toxicity in normal hematopoietic cells. ONC213 suppressed mitochondrial respiration and elevated α-ketoglutarate by suppressing α-ketoglutarate dehydrogenase (αKGDH) activity. Deletion of OGDH, which encodes αKGDH, suppressed AML fitness and impaired oxidative phosphorylation, highlighting the key role for αKGDH inhibition in ONC213-induced death. ONC213 treatment induced a unique mitochondrial stress response and suppressed de novo protein synthesis in AML cells. Additionally, ONC213 reduced the translation of MCL1, which contributed to ONC213-induced apoptosis. Importantly, a patient-derived xenograft from a relapsed AML patient was sensitive to ONC213 in vivo. Collectively, these findings support further development of ONC213 for treating AML. SIGNIFICANCE: In AML cells, ONC213 suppresses αKGDH, which induces a unique mitochondrial stress response, and reduces MCL1 to decrease oxidative phosphorylation and elicit potent antileukemia activity. See related commentary by Boët and Sarry, p. 950.

Comparison of a high-throughput high-content intracellular Leishmania donovani assay with an axenic amastigote assay.

Visceral leishmaniasis is a neglected tropical disease with significant health impact. The current treatments are poor, and there is an urgent need to develop new drugs. Primary screening assays used for drug discovery campaigns have typically used free-living forms of the Leishmania parasite to allow for high-throughput screening. Such screens do not necessarily reflect the physiological situation, as the disease-causing stage of the parasite resides inside human host cells. Assessing the drug sensitivity of intracellular parasites on scale has recently become feasible with the advent of high-content screening methods. We describe here a 384-well microscopy-based intramacrophage Leishmania donovani assay and compare it to an axenic amastigote system. A panel of eight reference compounds was tested in both systems, as well as a human counterscreen cell line, and our findings show that for most clinically used compounds both axenic and intramacrophage assays report very similar results. A set of 15,659 diverse compounds was also screened using both systems. This resulted in the identification of seven new antileishmanial compounds and revealed a high false-positive rate for the axenic assay. We conclude that the intramacrophage assay is more suited as a primary hit-discovery platform than the current form of axenic assay, and we discuss how modifications to the axenic assay may render it more suitable for hit-discovery.

A Curvilinear-Path Umbrella Sampling Approach to Characterizing the Interactions Between Rapamycin and Three FKBP12 Variants.

Rapamycin is an immunosuppressant macrolide that exhibits anti-proliferative properties through inhibiting the mTOR kinase. In fact, the drug first associates with the FKBP12 enzyme before interacting with the FRB domain of its target. Despite the availability of structural and thermodynamic information on the interaction of FKBP12 with rapamycin, the energetic and mechanistic understanding of this process is still incomplete. We recently reported a multiple-walker umbrella sampling simulation approach to characterizing the protein-protein interaction energetics along curvilinear paths. In the present paper, we extend our investigations to a protein-small molecule duo, the FKBP12•rapamycin complex. We estimate the binding free energies of rapamycin with wild-type FKBP12 and two mutants in which a hydrogen bond has been removed, D37V and Y82F. Furthermore, the underlying mechanistic details are analyzed. The calculated standard free energies of binding agree well with the experimental data, and the roles of the hydrogen bonds are shown to be quite different for each of these two mutated residues. On one hand, removing the carboxylate group of D37 strongly destabilizes the association; on the other hand, the hydroxyl group of Y82 is nearly unnecessary for the stability of the complex because some nonconventional, cryptic, indirect interaction mechanisms seem to be at work.

Facile Synthesis of Pure and Cr-Doped WO3 Thin Films for the Detection of Xylene at Room Temperature.

This paper focused on the preparation of pure and Cr-doped tungsten trioxide (WO3) thin films using the spray pyrolysis method. Different techniques were adopted to analyze these films' structural and morphological properties. The X-ray detection analysis showed that the average crystallite size of the WO3-nanostructured thin films increased as the Cr doping concentration increased. The atomic force microscopy results showed that the root-mean-square roughness of the films increased with Cr doping concentration up to 3 wt % and then decreased. The increased roughness is favorable for gas-sensing applications. Surface morphology and elemental analysis of the films were studied by field emission scanning electron microscopy with energy-dispersive X-ray spectroscopy measurements. The 3 wt % Cr-WO3 has a large nanoflake-like structure with high surface roughness and porous morphology. Gas-sensing characteristics of undoped and Cr-doped WO3 thin films were investigated with various gases at room temperature. The results showed that 3 wt % Cr-doped WO3 film performed the maximum response toward 50 ppm of xylene with excellent selectivity at room temperature. We believe that increased lattice defects, surface morphology, and roughness due to Cr doping in the WO3 crystal matrix might be responsible for increased xylene sensitivity.

The PPE18 of Mycobacterium tuberculosis interacts with TLR2 and activates IL-10 induction in macrophage.

The pathophysiological functions of proline-glutamic acid (PE)/proline-proline-glutamic acid (PPE) family of proteins of Mycobacterium tuberculosis are not well understood. In this study, we demonstrate that one of the PPE proteins, PPE18 can stimulate macrophages to secrete IL-10, known to favor a Th2 type response. The recombinant PPE18 was found to specifically interact with the TLR2 leading to an early and sustained activation of p38 MAPK, which is critical for IL-10 induction. In silico docking analyses and mutation experiments indicate that PPE18 specifically interacts with the leucine rich repeat 11 approximately 15 domain of TLR2 and the site of interaction is different from that of a synthetic lipopeptide Pam(3)CSK(4) known to activate predominantly ERK 1/2. When PMA-differentiated THP-1 macrophages were infected with a mutant Mycobacterium tuberculosis strain lacking the PPE18, produced poorer levels of IL-10 as compared with those infected with the wild-type strain. In contrast, an M. smegmatis strain overexpressing the PPE18 induced higher levels of IL-10 in infected macrophages. Our data indicate that the PPE18 protein may trigger an anti-inflammatory response by inducing IL-10 production.

Chemical Genetics Screen Identifies COPB2 Tool Compounds That Alters ER Stress Response and Induces RTK Dysregulation in Lung Cancer Cells.

Activating mutations in the epidermal growth factor receptor (EGFR) are common driver mutations in non-small cell lung cancer (NSCLC). First, second and third generation EGFR tyrosine kinase inhibitors (TKIs) are effective at inhibiting mutant EGFR NSCLC, however, acquired resistance is a major issue, leading to disease relapse. Here, we characterize a small molecule, EMI66, an analog of a small molecule which we previously identified to inhibit mutant EGFR signalling via a novel mechanism of action. We show that EMI66 attenuates receptor tyrosine kinase (RTK) expression and signalling and alters the electrophoretic mobility of Coatomer Protein Complex Beta 2 (COPB2) protein in mutant EGFR NSCLC cells. Moreover, we demonstrate that EMI66 can alter the subcellular localization of EGFR and COPB2 within the early secretory pathway. Furthermore, we find that COPB2 knockdown reduces the growth of mutant EGFR lung cancer cells, alters the post-translational processing of RTKs, and alters the endoplasmic reticulum (ER) stress response pathway. Lastly, we show that EMI66 treatment also alters the ER stress response pathway and inhibits the growth of mutant EGFR lung cancer cells and organoids. Our results demonstrate that targeting of COPB2 with EMI66 presents a viable approach to attenuate mutant EGFR signalling and growth in NSCLC.

Mapping conformational transitions in cyclic AMP receptor protein: crystal structure and normal-mode analysis of Mycobacterium tuberculosis apo-cAMP receptor protein.

Cyclic AMP (cAMP) receptor protein, which acts as the sensor of cAMP levels in cells, is a well-studied transcription factor that is best known for allosteric changes effected by the binding of cAMP. Although genetic and biochemical data on the protein are available from several sources, structural information about the cAMP-free protein has been lacking. Therefore, the precise atomic events that take place upon binding of cAMP, leading to conformational changes in the protein and its activation to bind DNA, have been elusive. In this work we solved the cAMP-free crystal structure of the Mycobacterium tuberculosis homolog of cAMP receptor protein at 2.9 A resolution, and carried out normal-mode analysis to map conformational transitions among its various conformational states. In our structure, the cAMP-binding domain holds onto the DNA-binding domain via strong hydrophobic interactions, thereby freezing the latter in a conformation that is not competent to bind DNA. The two domains release each other in the presence of cAMP, making the DNA-binding domain more flexible and allowing it to bind its cognate DNA via an induced-fit mechanism. The structure of the cAMP-free protein and results of the normal-mode analysis therefore highlight an elegant mechanism of the allosteric changes effected by the binding of cAMP.

Utilization of virtual reality for endotracheal intubation training.

Tracheal intubation is performed for urgent airway control in injured patients. Current methods of training include working on cadavers and manikins, which lack the realism of a living human being. Work in this field has been limited due to the complex nature of simulating in real-time, the interactive forces and deformations which occur during an actual patient intubation. This study addressed the issue of intubation training in an attempt to bridge the gap between actual and virtual patient scenarios. The haptic device along with the real-time performance of the simulator give it both visual and physical realism. The three-dimensional viewing and interaction available through virtual reality make it possible for physicians, pre-hospital personnel and students to practice many endotracheal intubations without ever touching a patient. The ability for a medical professional to practice a procedure multiple times prior to performing it on a patient will both enhance the skill of the individual while reducing the risk to the patient.

A virtual environment for esophageal intubation training.

Esophageal intubations are performed for urgent airway control in injured patients. Current methods of training include working on cadavers and mannequins, which lack the realism of a living human being. Work in this field has been limited due to the complex nature of simulating in real-time the interactive forces and deformations which occur during an actual patient intubation. This study addressed the issue of intubation training in an attempt to bridge the gap between actual and virtual patient scenarios. The two haptic devices along with the real-time performance of the simulator give it both visual and physical realism. The three dimensional viewing and interaction available through virtual reality make it possible for physicians, pre-hospital personnel and students to practice many esophageal intubations without ever touching a patient. The ability for a medical professional to practice a procedure multiple times prior to performing it on a patient will both enhance the skill of the individual while reducing the risk to the patient.

A static-cidal assay for Trypanosoma brucei to aid hit prioritisation for progression into drug discovery programmes.

Human African Trypanosomiasis is a vector-borne disease of sub-Saharan Africa that causes significant morbidity and mortality. Current therapies have many drawbacks, and there is an urgent need for new, better medicines. Ideally such new treatments should be fast-acting cidal agents that cure the disease in as few doses as possible. Screening assays used for hit-discovery campaigns often do not distinguish cytocidal from cytostatic compounds and further detailed follow-up experiments are required. Such studies usually do not have the throughput required to test the large numbers of hits produced in a primary high-throughput screen. Here, we present a 384-well assay that is compatible with high-throughput screening and provides an initial indication of the cidal nature of a compound. The assay produces growth curves at ten compound concentrations by assessing trypanosome counts at 4, 24 and 48 hours after compound addition. A reduction in trypanosome counts over time is used as a marker for cidal activity. The lowest concentration at which cell killing is seen is a quantitative measure for the cidal activity of the compound. We show that the assay can identify compounds that have trypanostatic activity rather than cidal activity, and importantly, that results from primary high-throughput assays can overestimate the potency of compounds significantly. This is due to biphasic growth inhibition, which remains hidden at low starting cell densities and is revealed in our static-cidal assay. The assay presented here provides an important tool to follow-up hits from high-throughput screening campaigns and avoid progression of compounds that have poor prospects due to lack of cidal activity or overestimated potency.