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58 Background: It is estimated that more than 40% of patients with mCRPC have functional loss of phosphatase and tensin homolog (PTEN) tumor suppressor gene, which is associated with unfavorable prognosis and reduced response to androgen receptor-targeting therapy. We describe patient characteristics and survival outcomes by PTEN LOF status among patients with mCRPC in real-world clinical practice. Methods: We conducted a retrospective cohort study using data from the nationwide Flatiron Health-Foundation Medicine mCRPC Clinico-Genomic database (FH-FMI CGDB), a de-identified database linking data derived from electronic health records with genomic data derived from FMI comprehensive genomic profiling (CGP) tests. The study included patients ≥18 years old, with a primary diagnosis of mCRPC between 1/1/2013 and 6/30/2019 who underwent FMI CGP testing and who had a valid PTEN LOF status. Patients were included if their PTEN report date and mCRPC diagnosis date occurred before death or censoring. PTEN LOF status was identified via FMI’s CGP testing. Kaplan-Meier (KM) methods assessed overall survival (OS) by PTEN LOF status from the date of mCRPC diagnosis (later of metastasis and castration resistance) until death or end of study follow-up. A stratified Cox regression model was used to estimate the hazard of death. The Cox model was adjusted for age, race and sequence of metastasis/CRPC diagnoses, and was stratified by the year of mCRPC diagnosis. Adjustments to account for left-truncation and survivorship bias were made in the KM analysis and the Cox regression model. Results: Among the 458 patients who met the eligibility criteria, 174 (38%) had PTEN LOF. The majority of the study sample (76%) was diagnosed with castration-resistance after metastasis. The PTEN LOF group had a higher percentage of white patients (80% vs. 68%; p= 0.01) compared to the PTEN non-LOF group. The mean age of the study sample was 68 years, and there was no difference in mean age at diagnosis by PTEN LOF status ( p= 0.17). Based on the KM estimates adjusted for left-truncation, the median OS was 14.3 months (95% confidence interval [CI]: 11.1-19.7) in the PTEN LOF group compared to 18.3 months (95%CI: 15.5-21.5) in the PTEN non-LOF group (log-rank p= 0.049). In the multivariable Cox model, the PTEN LOF group had numerically 30% higher risk of death compared to the PTEN non-LOF group (hazard ratio = 1.30; 95% CI: 0.99-1.71; p= 0.057). Conclusions: Among real-world patients with mCRPC in the CGDB, PTEN LOF could be associated with poorer survival outcomes, potentially highlighting the unmet need among these patients. Additional studies with larger cohorts are needed to better evaluate the survival outcomes of patients with PTEN LOF. Therapeutic agents acting on the PTEN/PI3K/AKT/mTOR pathway are being tested in clinical trials, and could potentially improve outcomes in this subgroup of patients with mCRPC.

SummaryApoptosis is a conserved cellular pathway that results in the activation of cysteine-aspartyl proteases, or caspases. To dissect the nonredundant roles of the executioner caspase-3, -6, and -7 in orchestrating apoptosis, we have developed an orthogonal protease to selectively activate each isoform in human cells. Our approach uses a split-tobacco etch virus (TEV) protease under small-molecule control, which we call the SNIPer, with caspase alleles containing genetically encoded TEV cleavage sites. These studies reveal that all three caspases are transiently activated but only activation of caspase-3 or -7 is sufficient to induce apoptosis. Proteomic analysis shown here and from others reveals that 20 of the 33 subunits of the 26S proteasome can be cut by caspases, and we demonstrate synergy between proteasome inhibition and dose-dependent caspase activation. We propose a model of proteolytic reciprocal negative regulation with mechanistic implications for the combined clinical use of proteasome inhibitors and proapoptotic drugs.PaperClip

Motivation:Granzyme B (GrB) and caspases cleave specific protein substrates to induce apoptosis in virally infected and neoplastic cells. While substrates for both types of proteases have been determined experimentally, there are many more yet to be discovered in humans and other metazoans. Here, we present a bioinformatics method based on support vector machine (SVM) learning that identifies sequence and structural features important for protease recognition of substrate peptides and then uses these features to predict novel substrates. Our approach can act as a convenient hypothesis generator, guiding future experiments by high-confidence identification of peptide-protein partners. Results:The method is benchmarked on the known substrates of both protease types, including our literature-curated GrB substrate set (GrBah). On these benchmark sets, the method outperforms a number of other methods that consider sequence only, predicting at a 0.87 true positive rate (TPR) and a 0.13 false positive rate (FPR) for caspase substrates, and a 0.79 TPR and a 0.21 FPR for GrB substrates. The method is then applied to ∼25 000 proteins in the human proteome to generate a ranked list of predicted substrates of each protease type. Two of these predictions, AIF-1 and SMN1, were selected for further experimental analysis, and each was validated as a GrB substrate. Availability: All predictions for both protease types are publically available at http://salilab.org/peptide. A web server is at the same site that allows a user to train new SVM models to make predictions for any protein that recognizes specific oligopeptide ligands. Contact: craik@cgl.ucsf.edu; sali@salilab.org Supplementary information: Supplementary data are available at Bioinformatics online

In the presence of a suitable oxidizing agent, the Ni(II) complex of glycyl–glycyl–histidine (GGH) mediates efficient and specific oxidative protein cross-linking. The fusion of GGH to the N terminus of a protein allows for the cross-linking reagent to be delivered in a site-specific fashion, making this system extremely useful for analyzing protein–protein contacts in complicated mixtures of biomolecules. Tyrosine residues have been postulated to be the primary amino acid target of this reaction, and using the dimeric serine protease inhibitor ecotin, we previously demonstrated that engineering a tyrosine at the protein interface of a dimer dramatically increased cross-linking efficiency. Cross-linking increased four-fold for GGH-ecotin D137Y in comparison to wild-type GGH-ecotin, presumably through bityrosine formation at the dimer interface. Here we report the first complete structural analysis of the cross-linked GGH-ecotin D137Y dimer. Using a combination of mass spectrometric and chemical derivatization methods, a sole novel cross-link between the N-terminal glycine residues and the engineered tyrosine at position 137 has been characterized. The dimer cross-link is localized to a single site without other protein modifications, but different reaction pathways produce structurally related products. We propose a mechanism that involves covalent bond formation between the protein backbone and a dopaquinone moiety derived from a specific tyrosine residue. This finding establishes that it is not necessary to have two tyrosine residues within close proximity in the protein interface to obtain high protein cross-linking yields, and suggests that the cross-linking reagent may be of more general utility than previously thought.

SummaryThe mechanism of target cell lysis in cytotoxic lymphocyte-mediated death is not well understood, and the role of granzymes in this process is unclear. Chemical functional probes were thus prepared for the major granzymes A and B to deconvolute their role in natural killer cell-mediated lysis of target cells. These biotinylated and substrate specificity-based diphenyl phosphonates allowed facile evaluation of selectivity through activity-based profiling in cell lysates and intact cells. Both inhibitors were found to be extremely selective in vitro and in cells. Use of these inhibitors in cell-based assays revealed granzyme A to be a minor effector and granzyme B to be a major effector of target cell lysis by natural killer cells. These studies indicate that the proapoptotic granzyme B functions also as a pronecrotic effector of target cell death.

Granzyme M is a trypsin-fold serine protease that is specifically found in the granules of natural killer cells. This enzyme has been implicated recently in the induction of target cell death by cytotoxic lymphocytes, but unlike granzymes A and B, the molecular mechanism of action of granzyme M is unknown. We have characterized the extended substrate specificity of human granzyme M by using purified recombinant enzyme, several positional scanning libraries of coumarin substrates, and a panel of individual p-nitroanilide and coumarin substrates. In contrast to previous studies conducted using thiobenzyl ester substrates (Smyth, M. J., O'Connor, M. D., Trapani, J. A., Kershaw, M. H., and Brinkworth, R. I. (1996) J. Immunol. 156, 4174–4181), a strong preference for leucine at P1 over methionine was demonstrated. The extended substrate specificity was determined to be lysine = norleucine at P4, broad at P3, proline > alanine at P2, and leucine > norleucine > methionine at P1. The enzyme activity was found to be highly dependent on the length and sequence of substrates, indicative of a regulatory function for human granzyme M. Finally, the interaction between granzyme M and the serpins α1-antichymotrypsin, α1 -proteinase inhibitor, and proteinase inhibitor 9 was characterized by using a candidate-based approach to identify potential endogenous inhibitors. Proteinase inhibitor 9 was effectively hydrolyzed and inactivated by human granzyme M, raising the possibility that this orphan granzyme bypasses proteinase inhibitor 9 inhibition of granzyme B.

105 Background: GDC-0919, a small molecule inhibitor of indoleamine-2,3-dioxygenase 1 (IDO1), reduces tryptophan catabolism and kynurenine production within the tumor microenvironment that may promote normal effector T cell activity and an immunogenic state. IDO1 inhibition may complement targeting of PD-L1 with atezolizumab. Methods: A Phase Ib, open-label, study assessed safety, pharmacokinetics (PK), pharmacodynamics (PD), and anti-tumor activity (RECIST v1.1) of GDC-0919 and atezolizumab in pts with locally advanced or metastatic solid tumors. Pts were given escalating doses of GDC-0919 (50-1000 mg orally twice daily, for 21 days) and atezolizumab (1200 mg IV, every 3 weeks) using a standard 3+3 design. Results: As of 14Dec2016, 52 pts were treated in 6 cohorts of GDC-0919 plus atezolizumab. The median number of prior systemic therapies was 3 (range 1-9); 2 pts received prior immunotherapy. Pts received a median of 4 cycles of GDC-0919 and atezolizumab (range 1-17). No MTD was identified. Across all dose levels, 1 DLT was observed (Grade [G] 3 sepsis syndrome at GDC-0919 200 mg); no G4/5 AEs were attributed to study treatment. G3+ AEs, regardless of causality were reported in 34 (65%) pts. Related G3 AEs were reported in 7 (13%) pts, included nausea, rash, sepsis syndrome, fatigue, and pneumonitis. Two pts (4%) had AEs leading to treatment discontinuation, related in 1/2 (G3 pneumonitis). Combination PK was consistent with single agent observations and supports BID dosing of GDC-0919. Peripheral PD showed dose-dependent decreases in plasma kynurenine, consistent with systemic modulation of IDO1. Preliminary efficacy data from 45 pts with ≥ 1 on-treatment tumor assessments included 4 patients (9%) with partial response and 11 (24%) pts with stable disease. Conclusions: The combination of GDC-0919 and atezolizumab was generally well-tolerated and demonstrated peripheral IDO1 modulation and preliminary efficacy in a heterogeneous patient population during dose escalation. The study is currently enrolling pts with select tumor types in expansion cohorts to assess tumor PD and combination efficacy. Clinical trial information: NCT02471846.

Mass spectrometry-based proteomics is a powerful tool for identifying hundreds to thousands of posttranslational modifications in complex mixtures. However, it remains enormously challenging to simultaneously assess the intrinsic catalytic efficiencies ( k cat / K M ) of these modifications in the context of their natural interactors. Such fundamental enzymological constants are key to determining substrate specificity and for establishing the timing and importance of cellular signaling. Here, we report the use of selected reaction monitoring (SRM) for tracking proteolysis induced by human apoptotic caspases-3, -7, -8, and -9 in lysates and living cells. By following the appearance of the cleaved peptides in lysate as a function of time, we were able to determine hundreds of catalytic efficiencies in parallel. Remarkably, we find the rates of substrate hydrolysis for individual caspases vary greater than 500-fold indicating a sequential process. Moreover, the rank-order of substrate cutting is similar in apoptotic cells, suggesting that cellular structures do not dramatically alter substrate accessibility. Comparisons of extrinsic (TRAIL) and intrinsic (staurosporine) inducers of apoptosis revealed similar substrate profiles, suggesting the final proteolytic demolitions proceed by similarly ordered plans. Certain biological processes were rapidly targeted by the caspases, including multiple components of the endocyotic pathway and miRNA processing machinery. We believe this massively parallel and quantitative label-free approach to obtaining basic enzymological constants will facilitate the study of proteolysis and other posttranslational modifications in complex mixtures.

SummaryThe nearly 600 proteases in the human genome regulate a diversity of biological processes, including programmed cell death. Comprehensive characterization of protease signaling in complex biological samples is limited by available proteomic methods. We have developed a general approach for global identification of proteolytic cleavage sites using an engineered enzyme to selectively biotinylate free protein N termini for positive enrichment of corresponding N-terminal peptides. Using this method to study apoptosis, we have sequenced 333 caspase-like cleavage sites distributed among 292 protein substrates. These sites are generally not predicted by in vitro caspase substrate specificity but can be used to predict other physiological caspase cleavage sites. Structural bioinformatic studies show that caspase cleavage sites often appear in surface-accessible loops and even occasionally in helical regions. Strikingly, we also find that a disproportionate number of caspase substrates physically interact, suggesting that these dimeric proteases target protein complexes and networks to elicit apoptosis.

The cell death–inducing serine protease granzyme A (GzmA) has a unique disulfide-linked quaternary structure. The structure of human GzmA bound to a tripeptide CMK inhibitor, determined at a resolution of 2.4 A, reveals that the oligomeric state contributes to substrate selection by limiting access to the active site for potential macromolecular substrates and inhibitors. Unlike other serine proteases, tetrapeptide substrate preferences do not correlate well with natural substrate cleavage sequences. This suggests that the context of the cleavage sequence within a macromolecular substrate imposes another level of selection not observed with the peptide substrates. Modeling of inhibitors bound to the GzmA active site shows that the dimer also contributes to substrate specificity in a unique manner by extending the active-site cleft. The crystal structure, along with substrate library profiling and mutagenesis, has allowed us to identify and rationally manipulate key components involved in GzmA substrate specificity.

Resistance to human immunodeficiency virus type 1 protease (HIV PR) inhibitors results primarily from the selection of multiple mutations in the protease region. Because many of these mutations are selected for the ability to decrease inhibitor binding in the active site, they also affect substrate binding and potentially substrate specificity. This work investigates the substrate specificity of a panel of clinically derived protease inhibitor-resistant HIV PR variants. To compare protease specificity, we have used positional-scanning, synthetic combinatorial peptide libraries as well as a select number of individual substrates. The subsite preferences of wild-type HIV PR determined by using the substrate libraries are consistent with prior reports, validating the use of these libraries to compare specificity among a panel of HIV PR variants. Five out of seven protease variants demonstrated subtle differences in specificity that may have significant impacts on their abilities to function in viral maturation. Of these, four variants demonstrated up to fourfold changes in the preference for valine relative to alanine at position P2 when tested on individual peptide substrates. This change correlated with a common mutation in the viral NC/p1 cleavage site. These mutations may represent a mechanism by which severely compromised, drug-resistant viral strains can increase fitness levels. Understanding the altered substrate specificity of drug-resistant HIV PR should be valuable in the design of future generations of protease inhibitors as well as in elucidating the molecular basis of regulation of proteolysis in HIV.

A method is presented for the preparation and use of fluorogenic peptide substrates that allows for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of proteases. The substrates contain the fluorogenic leaving group 7-amino-4-carbamoylmethylcoumarin (ACC). Substrates incorporating the ACC leaving group show kinetic profiles comparable to those with the traditionally used 7-amino-4-methylcoumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient production of single substrates and substrate libraries by using 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase synthesis techniques. The approximately 3-fold-increased quantum yield of ACC over AMC permits reduction in enzyme and substrate concentrations. As a consequence, a greater number of substrates can be tolerated in a single assay, thus enabling an increase in the diversity space of the library. Soluble positional protease substrate libraries of 137,180 and 6,859 members, possessing amino acid diversity at the P4-P3-P2-P1 and P4-P3-P2 positions, respectively, were constructed. Employing this screening method, we profiled the substrate specificities of a diverse array of proteases, including the serine proteases thrombin, plasmin, factor Xa, urokinase-type plasminogen activator, tissue plasminogen activator, granzyme B, trypsin, chymotrypsin, human neutrophil elastase, and the cysteine proteases papain and cruzain. The resulting profiles create a pharmacophoric portrayal of the proteases to aid in the design of selective substrates and potent inhibitors.