Kayode, Olumide

Proteases are ubiquitous enzymes that catalyze the hydrolysis of peptide bonds within protein substrates; they have served as key model enzymes for studying the molecular basis for catalytic power and specificity. Protease substrate specificity is most often defined in terms of linear sequence motifs that flank the cleavage site; however, the natural substrates of proteases are proteins with 3‐dimensional shapes and complex conformational dynamics that are not well represented by 1‐dimensional sequence alone. These structural and dynamical properties can impact recognition and binding of substrates by proteases, as well as the efficiency of catalysis itself. In this study, we explore the importance of substrate structure and dynamics for proteolysis using as our model the cleavage of the Kunitz‐BPTI family of canonical serine protease inhibitors by mesotrypsin. Bovine pancreatic trypsin inhibitor (BPTI), an archetypal serine protease inhibitor of the Kunitz family, has a high affinity substrate‐like interaction with trypsin, yet is cleaved many orders of magnitude slower than other peptide substrates. Mesotrypsin, a trypsin variant, has been shown to hydrolyze Kunitz family inhibitors at accelerated rates; this is especially true of human Kunitz domain inhibitors. Amyloid precursor protein inhibitor (APPI) and amyloid precursor like protein‐2 Kunitz domain (APLP2‐KD), two human Kunitz domain family members, are hydrolyzed by mesotrypsin several hundred times faster than BPTI. Here, we present a new crystal structure of a cleavage intermediate of APLP2‐KD bound to mesotrypsin, refined to 1.4 Å resolution. This newly solved structure reveals a dramatic substrate conformational change that we hypothesize is required during Kunitz domain cleavage. Using this structure along with published APPI and BPTI complexes, we have modeled acyl‐enzyme intermediates of mesotrypsin and we have carried out molecular dynamic simulations to explore the conformational transformation that allows the progression of the hydrolysis reaction. We identify dynamic features of the inhibitor scaffolds, located far from the enzyme binding site, that correlate with observed inhibitor cleavage rates and plausibly explain their differential propensity to undergo both conformational transformation and cleavage. Our findings implicate global scaffold flexibility as a determinant of proteolytic rate, as rigid substrates possessing stabilizing features that render them highly resistant to conformational change are proteolyzed more slowly than more flexible substrates of similar structure. Support or Funding Information NIH R01 CA154387 Mayo Graduate school

Abstract Purpose: To determine the dose limiting toxicities (DLT), maximum tolerated dose (MTD), and recommended phase II dose (RP2D) of veliparib in combination with weekly topotecan in patients with solid tumors. Correlative studies were included to assess the impact of topotecan and veliparib on poly(ADP-ribose) levels in peripheral blood mononuclear cells, serum pharmacokinetics of both agents, and potential association of germline repair gene mutations with outcome. Experimental Design: Eligible patients had metastatic nonhematologic malignancies with measurable disease. Using a 3 + 3 design, patients were treated with veliparib orally twice daily on days 1–3, 8–10, and 15–17 and topotecan intravenously on days 2, 9, and 16 every 28 days. Tumor responses were assessed by RECIST. Results: Of 58 patients enrolled, 51 were evaluable for the primary endpoint. The MTD and RP2D was veliparib 300 mg twice daily on days 1–3, 8–10, and 15–17 along with topotecan 3 mg/m2 on days 2, 9, and 16 of a 28-day cycle. DLTs were grade 4 neutropenia lasting >5 days. The median number of cycles was 2 (1–26). The objective response rate was 10%, with 1 complete and 4 partial responses. Twenty-two patients (42%) had stable disease ranging from 4 to 26 cycles. Patients with germline BRCA1, BRCA2, or RAD51D mutations remained on study longer than those without homologous recombination repair (HRR) gene mutations (median 4 vs. 2 cycles). Conclusions: Weekly topotecan in combination with veliparib has a manageable safety profile and appears to warrant further investigation.

Engineered protein therapeutics offer advantages, including strong target affinity, selectivity and low toxicity, but like natural proteins can be susceptible to proteolytic degradation, thereby limiting their effectiveness. A compelling therapeutic target is mesotrypsin, a protease up-regulated with tumour progression, associated with poor prognosis, and implicated in tumour growth and progression of many cancers. However, with its unique capability for cleavage and inactivation of proteinaceous inhibitors, mesotrypsin presents a formidable challenge to the development of biological inhibitors. We used a powerful yeast display platform for directed evolution, employing a novel multi-modal library screening strategy, to engineer the human amyloid precursor protein Kunitz protease inhibitor domain (APPI) simultaneously for increased proteolytic stability, stronger binding affinity and improved selectivity for mesotrypsin inhibition. We identified a triple mutant APPIM17G/I18F/F34V, with a mesotrypsin inhibition constant (Ki) of 89 pM, as the strongest mesotrypsin inhibitor yet reported; this variant displays 1459-fold improved affinity, up to 350 000-fold greater specificity and 83-fold improved proteolytic stability compared with wild-type APPI. We demonstrated that APPIM17G/I18F/F34V acts as a functional inhibitor in cell-based models of mesotrypsin-dependent prostate cancer cellular invasiveness. Additionally, by solving the crystal structure of the APPIM17G/I18F/F34V–mesotrypsin complex, we obtained new insights into the structural and mechanistic basis for improved binding and proteolytic resistance. Our study identifies a promising mesotrypsin inhibitor as a starting point for development of anticancer protein therapeutics and establishes proof-of-principle for a novel library screening approach that will be widely applicable for simultaneously evolving proteolytic stability in tandem with desired functionality for diverse protein scaffolds.

Abstract Background: ABT-888 is an inhibitor of poly(ADP-ribose) polymerase1 (PARP1) and PARP2, two highly conserved enzymes implicated in DNA repair, maintenance of genomic stability, and regulation of transcription. Topotecan (TPT) is a water soluble camptothecin derivative with antitumor activity against ovarian tumors. The combination of ABT-888 with TPT is under investigation in phase I trials because PARP inhibition sensitizes tumors cells to TPT in vitro and in vivo. In those trials, the pharmacokinetics of TPT and ABT-888 are being studied to determine if ABT-888 levels are sufficient to modulate PARP activity and to investigate the potential interaction between TPT and ABT-888. Methods: Advanced cancer patients with ECOG performance scores 0-2, adequate hematological, renal and hepatic function were treated with ABT-888 on days 1-3, 8-10, and 15-17 every 28 days. Topotecan was administered on days 2, 9 and 16 every 28 days. DLTs were defined as grade 4 neutropenia, grade 4 thrombocytopenia, grade 4 anemia or ≥ grade 3 non-hematologic toxicities despite maximal supportive care. ABT-888 and Toptoecan pharmacokinetics were characterized in all patients. Analysis of urinary recovery is ongoing. Results: In this ongoing Phase I trial, 13 female patients with ovarian (n=12) or breast (n=1) cancer have been enrolled to date and pharmacokinetic data is available for 12 of those patients. ABT-888/TPT dose levels are: Level 1- 10 mg/ 2 mg/m2, Level 2- 20 mg/2 mg/m2, Level 3- 10 mg/3 mg/m2, and Level 4- 20 mg/3 mg/m2. The combination of ABT-888 with topotecan has been tolerable, with no dose limiting toxicity, and dose escalation continues at dose level 5. Topotecan t1/2 and clearance values were 3.0 ± 0.5 hrs and 9.50 ± 1.51 L/hr/m2 when administered alone, and were not affected by administration with ABT-888. When administered alone, ABT-888 t1/2, Cmax, AUC0-∞ values after the 10 mg dose were 8.2 ± 2.4 hrs, 432 ± 217 nM, and 2568 ± 1300 nM*hr, respectively. ABT-888 t1/2, Cmax, AUC values after the 20 mg dose were 5.7 ± 3.1 hrs, 600 ± 165 nM, and 3870± 934 respectively. When administered with TPT, ABT-888 t1/2, Cmax, AUC values after the 10 mg dose were 7.2 ± 2.1 hrs, 412 ± 283 nM, and 2705± 1089 nM*hr are: nM*hr respectively. ABT-888 t1/2, Cmax and AUC values after the 20 mg dose combined with TPT were 5.8 ± 2.2 hrs, 664 ± 97.6 nM, and 4283± 936 nM*hr respectively. Conclusions: Plasma clearance of TPT and ABT-888 does not appear to be altered when they are administered together. The effect of co-administration of these agents on renal clearance is presently under investigation and those results will be presented. This work was supported by NIH grants R25 GM75148-04, U01CA069912-16S and MM01-RR00585. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1301. doi:10.1158/1538-7445.AM2011-1301