Citations

High structural and sequence similarity within protein families can pose significant challenges to the development of selective inhibitors, especially toward proteolytic enzymes. Such enzymes usually belong to large families of closely similar proteases and may also hydrolyze, with different rates, protein- or peptide-based inhibitors. To address this challenge, we employed a combinatorial yeast surface display library approach complemented with a novel pre-equilibrium, competitive screening strategy for facile assessment of the effects of multiple mutations on inhibitor association rates and binding specificity. As a proof of principle for this combined approach, we utilized this strategy to alter inhibitor/protease association rates and to tailor the selectivity of the amyloid β-protein precursor Kunitz protease inhibitor domain (APPI) for inhibition of the oncogenic protease mesotrypsin, in the presence of three competing serine proteases, anionic trypsin, cationic trypsin and kallikrein-6. We generated a variant, designated APPIP13W/M17G/I18F/F34V, with up to 30-fold greater specificity relative to the parental APPIM17G/I18F/F34V protein, and 6500- to 230 000-fold improved specificity relative to the wild-type APPI protein in the presence of the other proteases tested. A series of molecular docking simulations suggested a mechanism of interaction that supported the biochemical results. These simulations predicted that the selectivity and specificity are affected by the interaction of the mutated APPI residues with nonconserved enzyme residues located in or near the binding site. Our strategy will facilitate a better understanding of the binding landscape of multispecific proteins and will pave the way for design of new drugs and diagnostic tools targeting proteases and other proteins.

93 Background: Tumor mutational load (TML) has been proposed as a biomarker of responsiveness to novel immune checkpoint inhibitors (ICIs) due its association with increased neoantigen formation and tumor immunogenicity. Gastrointestinal cancers (GICs) are generally insensitive to ICIs; therefore the aim of this study was to evaluate the TML, clinically relevant genomic alterations (CRGAs), and PDL-1 status of GIC patients (pts). Methods: We analyzed samples of pts with GICs using a multiplatform profiling tool (Caris Life Sciences, Phoenix, AZ). TML and CRGAs were calculated based on next generation sequencing and mutational load was stratified as high (≥17 mut/Mb), intermediate (8-16 mut/Mb), and low (≤ 7 mut/Mb). PD-L1 status was determined by IHC. Descriptive statistics and simple linear regression were used for analysis. Results: A total of 85 pts with GICs were analyzed. The median pt age was 65. 53% were males and 47% were females. Tumors were colorectal 51%, pancreatic 13%, esophageal 13%, gastric 9%, hepatobiliary 11%, anal canal 4%, and small intestine 1%. 80 cases (94%) had an adequate sample for profiling and TML was available in 59 cases (74%). Cases were 5% TML-high, 56% TML-intermediate, and 39% TML-low. The median TML was 9 mut/Mb and median CRGAs was 3. The most commonly detected CRGAs were TP53 (56%), APC (44%), KRAS (40%), ATM (18%) and PTEN (6%). A positive but very weak correlation was found between TML and number of CRGAs (R2 = 0.3). PD-L1 status was available in 74 cases (93%) with 86% being PD-L1 negative and 14% PD-L1 positive. PD-L1 expression was most commonly seen in esophageal (50%) and anal canal (30%) cancers. Among 57 tumors tested for both biomarkers, no correlation was found between a high TML and PD-L1 expression. Conclusions: TML varies among GICs, and higher TML scores were not associated with PD-L1 expression, but there was a weak correlation between TML and the number of CRGAs. Further analysis by stratification of GIC type and prognostic analysis to assess the correlation between TMB and response to ICIs is warranted.

ABSTRACTBackground: Dysregulation of the specialized lipid metabolism involved in myelin synthesis and maintenance by oligodendrocytes has been associated with the unique neuropathology of MSA. We hypothesized that apolipoprotein E, which is associated with neurodegeneration, may also play a role in the pathogenesis of MSA.Objective: This study evaluated genetic associations of Apolipoprotein E alleles with risk of MSA and α‐synuclein pathology, and also examined whether apolipoprotein E isoforms differentially affect α‐synuclein uptake in a oligodendrocyte cell.Methods: One hundred sixty‐eight pathologically confirmed MSA patients, 89 clinically diagnosed MSA patients, and 1,277 control subjects were genotyped for Apolipoprotein E. Human oligodendrocyte cell lines were incubated with α‐synuclein and recombinant human apolipoprotein E, with internalized α‐synuclein imaged by confocal microscopy and cells analyzed by flow cytometry.Results: No significant association with risk of MSA or was observed for either Apolipoprotein E ɛ2 or ɛ4. α‐Synuclein burden was also not associated with Apolipoprotein E alleles in the pathologically confirmed patients. Interestingly, in our cell assays, apolipoprotein E ɛ4 significantly reduced α‐synuclein uptake in the oligodendrocytic cell line.Conclusions: Despite differential effects of apolipoprotein E isoforms on α‐synuclein uptake in a human oligodendrocytic cell, we did not observe a significant association at the Apolipoprotein E locus with risk of MSA or α‐synuclein pathology. © 2018 International Parkinson and Movement Disorder Society

SNF'ing out antitumor immunity Immune checkpoint inhibitors induce durable tumor regressions in some, but not all, cancer patients. Understanding the mechanisms that determine tumor sensitivity to these drugs could potentially expand the number of patients who benefit (see the Perspective by Ghorani and Quezada). Pan et al. discovered that tumor cells in which a specific SWI/SNF chromatin remodeling complex had been experimentally inactivated were more sensitive to T cell–mediated killing. The cells were more responsive to interferon-γ, leading to increased secretion of cytokines that promote antitumor immunity. Miao et al. examined the genomic features of tumors from patients with metastatic renal cell carcinoma who had been treated with immune checkpoint inhibitors. Tumors harboring inactivating mutations in PBRM1 , which encodes a subunit of the same SWI/SNF complex, were more likely to respond to the drugs. Science , this issue p. 770 , p. 801 ; see also p. 745

We evaluated a method of baroreflex testing involving sequential intravenous bolus injections of nitroprusside followed by phenylephrine and phenylephrine followed by nitroprusside in 18 healthy men and women, and we drew inferences regarding human sympathetic and vagal baroreflex mechanisms. We recorded the electrocardiogram, photoplethysmographic finger arterial pressure, and peroneal nerve muscle sympathetic activity. We then contrasted least squares linear regression slopes derived from the depressor (nitroprusside) and pressor (phenylephrine) phases with 1) slopes derived from spontaneous fluctuations of systolic arterial pressures and R-R intervals, and 2) baroreflex gain derived from cross-spectral analyses of systolic pressures and R-R intervals. We calculated sympathetic baroreflex gain from integrated muscle sympathetic nerve activity and diastolic pressures. We found that vagal baroreflex slopes are less when arterial pressures are falling than when they are rising and that this hysteresis exists over pressure ranges both below and above baseline levels. Although pharmacological and spontaneous vagal baroreflex responses correlate closely, pharmacological baroreflex slopes tend to be lower than those derived from spontaneous fluctuations. Sympathetic baroreflex slopes are similar when arterial pressure is falling and rising; however, small pressure elevations above baseline silence sympathetic motoneurons. Vagal, but not sympathetic baroreflex gains vary inversely with subjects’ ages and their baseline arterial pressures. There is no correlation between sympathetic and vagal baroreflex gains. We recommend repeated sequential nitroprusside followed by phenylephrine doses as a simple, efficientmeans to provoke and characterize human vagal and sympathetic baroreflex responses.

BackgroundPatients with regionally advanced melanoma are at high risk of distant failure and unlikely to be cured by surgery alone. Neoadjuvant therapy may provide benefit in these patients.ObjectivesTo evaluate our experience with neoadjuvant systemic therapy in high‐risk stage III patients.MethodsRetrospective review of patients with advanced stage III disease who received neoadjuvant therapy between August 2009 and August 2016 at Mayo Clinic Rochester.ResultsTwenty‐three cases met our inclusion criteria, 16 with resectable disease and 7 with unresectable disease. No patients with resectable disease and one patient with borderline resectable disease progressed regionally, prohibiting surgical resection. Five of seven patients with unresectable disease were down‐staged to a resectable state. Six of twenty‐three (26%) had a CR and five are alive at last follow‐up. Fifteen of twenty three patients (65%) progressed with a median progression free survival of 11 months; however, the 5 year overall survival estimate was 84%.ConclusionsNeoadjuvant systemic therapy is a reasonable approach for patients with advanced but resectable/borderline resectable disease and the risk of losing regional control is low. Our data also suggest some patients with unresectable disease will be converted to resectable and a complete clinical response to treatment can be obtained in approximately one quater of patients.