Somdutta Sen

Progressive degradation of serum samples limits proteomic biomarker discovery.

Preanalytical variables play a key role in discovery of biomarkers. Although the effect of several preanalytical variables on the mass spectral profiles has been studied extensively, little is known about long-term storage of serum samples. This is important because samples used in case-control or epidemiological studies are usually stored for a long time before analysis. Here we evaluated long-term storage effects on mass spectral peak patterns of serum peptides extracted using weak cation exchange magnetic beads. For this, 20 serum samples stored at -80 degrees C were divided equally into two groups based on their storage time. We found that intensities of 26 mass spectral peaks significantly varied between these two groups. Intensities of these peaks significantly correlated with storage time. Genetic algorithm-based models generated using these 26 peaks could classify 63 additional samples into these two groups with 100% and 96% accuracy, respectively. We also show that storing samples for 10 months at -80 and -20 degrees C results in the appearance/disappearance or intensity variation of peaks, some of which were previously reported as disease biomarkers.

Plasma peptidome profiling of acute hepatitis E patients by MALDI-TOF/TOF

BackgroundHepatitis E is endemic to resource-poor regions, where it manifests as sporadic cases and large waterborne outbreaks. The disease severity ranges from acute self-limited hepatitis with low mortality to fulminant hepatic failure with high mortality. It is believed that the host response plays an important role in determining the progression and outcome of this disease. We profiled the plasma peptidome from hepatitis E patients to discover suitable biomarkers and understand disease pathogenesis.ResultsThe peptidome (< 10 kDa) fraction of plasma was enriched and analyzed by mass spectrometry. A comparative analysis of the peptide pattern of hepatitis E patients versus healthy controls was performed using ClinPro Tools. We generated a peptide profile that could be used for selective identification of hepatitis E cases. We have identified five potential biomarker peaks with m/z values of 9288.6, 7763.6, 4961.5, 1060.572 and 2365.139 that can be used to reliably differentiate between hepatitis E patients and controls with areas under the receiver operating characteristic curve (AUROC) values of 1.00, 0.954, 0.989, 0.960 and 0.829 respectively. A number of proteins involved in innate immunity were identified to be differentially present in the plasma of patients compared to healthy controls.ConclusionsBesides the utility of this approach for biomarker discovery, identification of changes in endogenous peptides in hepatitis E patient plasma has increased our understanding of disease pathogenesis. We have identified peptides in plasma that can reliably distinguish hepatitis E patients from healthy controls. Results from this and an earlier proteomics study are discussed.

Discovery of Novel and Orally Bioavailable Inhibitors of PI3 Kinase Based on Indazole Substituted Morpholino-Triazines

A new class of potent PI3Kα inhibitors is identified based on aryl substituted morpholino-triazine scaffold. The identified compounds showed not only a high level of enzymatic and cellular potency in nanomolar range but also high oral bioavailability. The three lead molecules (based on their in vitro potency) when evaluated further for in vitro metabolic stability as well as pharmacokinetic profile led to the identification of 26, as a candidate for further development. The IC50 and EC50 value of 26 is 60 and 500 nM, respectively, for PI3Kα enzyme inhibitory activity and ovarian cancer (A2780) cell line. The identified lead also showed a high level of microsomal stability and minimal inhibition activity for CYP3A4, CYP2C19, and CYP2D6 at 10 μM concentrations. The lead compound 26, demonstrated excellent oral bioavailability with an AUC of 5.2 μM at a dose of 3 mpk in mice and found to be well tolerated in mice when dosed at 30 mpk BID for 5 days.

In vitro segregation of marker genes in anther cultures of rice

SummarySegregation of genes controlling expression of anthocyanin pigmentation in rice (Oryza sativa L. subsp.indica) leaf blade and leaf sheath was examined in the microspore-derived plants. The segregation pattern of marker genes was found to fit closely the expected gametic segregation ratios among microspore-derived green as well as albino plants. Microspore-derived in vitro regenerated plants expressed genetic traits similar to seedlings. The results indicate that the germ cell culture technique can be of significance while monitoring gene action, i.e. anthocyanin synthesis at monoploid phase of plant development.

Somatic cell genetic studies inBrassica species. II. Production of androgenetic haploid plants inBrassica nigra (L.)Koch

SummaryCallus growth and its subsequent regeneration into complete plantlets was achieved from in vitro cultured anthers ofBrassica nigra (L.)Koch. Callus was induced on a modified N6 medium containing trace elements, organics of B5 medium and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Morphogenesis of callus in the form of shoots on MS medium containing indole-3-acetic acid (IAA) and N6-benzyladenine (BA) 0.5 mg/l each and embryoids on MS medium containing 0.5–1.0 mg/l IAA and 3.0–5.0 mg/l BA could be accomplished. Chromosomal analysis revealed presence of 41% haploids (n=8) amongst the regenerated plants.

Synthesis and evaluation of pyrrolotriazine based molecules as PI3 kinase inhibitors

Over activation of the PI3K/Akt/mTOR pathway is found in most cancer tumor types. Controlled regulation of this pathway using PI3K inhibitors can provide therapeutic significance in cancer treatment. Herein, we report the synthesis and evaluation of pyrrolotriazine based novel small molecules as pan-PI3K inhibitors. The SAR studies based on in vitro potency along with microsomal metabolic stability screening, identified 18 as a preclinical lead found to be suitable for in vivo evaluation. The identified lead was also found to be a selective inhibitor of PI3K isoforms and mTOR when screened across a panel of 23 homologous kinases.

Abstract 4515: SPR965: an oral PI3K/ mTOR C1/C2 inhibitor for the treatment of solid tumors

Background The discovery and development of inhibitors for the PI3K/AKT/mTOR signaling pathway is an attractive area of research due to its association with several oncogenic malignancies. This signaling pathway controls cellular growth as well as survival via regulation of widely divergent physiological processes, i.e. cell cycle progression, differentiation, transcription, translation and apoptosis. Constitutive activation of the PI3 kinase alpha and/or the downstream protein mTOR has been implicated in the progression of a large variety of solid tumors. Literature reports suggest the importance of developing combined and specific inhibitor of both PI3K and mTOR kinases. Described herein is the discovery of a novel small molecule, SPR965, a potent, and orally bioavailable inhibitor for class 1 PI3 Kinase and mTOR kinases with the potential for the clinical treatment of various solid tumors, specifically prostate, ovarian and colon cancers. The complete preclinical profile of SPR965 is presented in detail. Methods Drug candidates were evaluated in an in vitro enzyme assays to determine their inhibitory activity PI3 and mTOR C1/ C2 kinases. Promising candidates were then evaluated in proliferation assays using various human cancer cell lines (PC3 - prostate, SKOV3 - ovarian, HCT-116 - colon and A2780 - ovarian). The most promising leads were then evaluated against a panel of 456 kinases to determine the level of selectivity for our primary targets - PI3K and mTOR C1/C2. Compounds meeting the desired threshold of potency and selectivity were evaluated for their in vivo pharmacokinetic profile (iv/ po) in rodents followed by studies in mouse xenograft models (SKOV3 and HCT-116). Results SPR965 is a potent inhibitor of PI3K alpha and mTOR with an IC50 of 24 and 25 nM respectively. SPR965 is also a highly selective inhibitor of PI3 and mTOR C1/C2 kinases when evaluated in a screen against 456 kinases. Further studies demonstrated that SPR965 is a potent inhibitor of proliferation in a multiple cell lines and in several xenograft models. The proliferation inhibition activity (EC50) in A2780 is 17 nM, PC3 is 30 nM, SKOV3 is 74 nM, and HCT-116 is 163 nM. SPR965 is one of the most efficacious PI3/mTOR kinase inhibitor yet reported, with ED50 = 0.5 mg/Kg as determined in a SKOV3 xenograft mouse model and ED50 = 0.6 mg/ Kg in HCT-116 xenograft mouse model. This dose level is markedly lower than those reported for other reported inhibitors of this pathway. Pharmacokinetic studies in Sprague Dawley rats and in NUDE mice indicated that the oral bioavailability of SPR965was ∼100% and 75% respectively. Conclusion SPR965 is one of the most efficacious inhibitor of the PI3 and mTOR kinases when compared to the reported ED50s of other reported compounds. It also is one of the most selective and highly bioavailable inhibitor of the PI3 and mTOR kinases. Further studies are underway to support first-in-human trials with SPR965 where we hope to demonstrate its unique therapeutic benefits. Citation Format: Reena Arora, Bakul K. Dutta, Ravinder Goel, Frank P. Hollinger, Bilash Kulia, Dinesh Mahajan, Amal R. Mahapatra, Milind Sagar, Somdutta Sen, Amit Sharma, Sundeep Dugar. SPR965: an oral PI3K/ mTOR C1/C2 inhibitor for the treatment of solid tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4515. doi:10.1158/1538-7445.AM2014-4515

Abstract 2653: Preclinical activity of dual PI3K/mTOR inhibitor SPR965 in multiple myeloma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Introduction: Relapsed and refractory multiple myeloma (MM) remains a significant clinical challenge and drugs with new mechanisms of action to overcome resistance are needed. Constitutive activation of the PI3K/Akt/mTOR pathway in MM promotes tumorigenesis through propagation of the cell cycle, protein synthesis, and inhibition of apoptosis. Anti-MM effects of rapalogs are limited due to feedback activation of the PI3K/Akt signaling pathway. Agents capable of targeting PI3K and mTORC1 or both mTORC1/C2 are able to partially overcome such resistance mechanisms. However, the mTORC2 mediated increase in pAkt (Ser473) after PI3K/mTORC1 inhibition and the activation of PI3K after mTORC1/C2 inhibition could still contribute to resistance to such agents. We sought to investigate the effects of SPR965 (synthesized and provided by Sphaera Pharma, Singapore under an MTA), an orally bioavailable novel small molecule with inhibition of class 1 PI3 kinase and mTORC1/C2 kinases on MM cell lines and patient cells. Results: SPR965 induced cytotoxicity and inhibited proliferation in all MM cell lines examined with IC50 values between 25-500nM. To understand if SPR965 induced apoptotic cell death, annexin/PI staining followed by flow cytometric assays were performed, which showed a clear increase in cells undergoing apoptosis. Western blots showed increase in caspase-3, -9, and PARP cleavage confirming apoptotic induction by SPR965. Importantly, SPR965 caused potent increase in apoptosis in cytogenetically distinct MM patient cells. Next, we examined the mechanism of action of SPR965. SPR965 caused potent mTORC1 inhibition evidenced by decreased p4E-BP1, p-p70S6K, and pS6 at doses as low as 25nM. SPR965 was able to inhibit PI3K activity as shown by decreased pPDK1 and pBTK at slightly higher concentrations of 75nM. Phosphorylation of Akt S473, a TORC2 substrate, increased at initial low concentrations of SPR965, but was attenuated at concentrations of 100 nM and above, demonstrating dose-dependent inhibition of mTORC2. Such effects were also observed when we performed western blots on MM patient derived primary plasma cells. Increased levels of p27 (KIP1) were observed suggestive of G1 growth arrest, which was confirmed on cell cycle analysis. Since SPR965 caused an increase in pAkt (both T308 and S473) at doses up to 75nM, we examined if SPR965 was able to synergize with an Akt inhibitor MK2206 at doses lower than 75nM. Our results showed potent synergy suggesting that the pAkt up regulation contributes to partial resistance, which is inhibited by SPR965 at doses of 100nM and higher, doses that are still clinically achievable. Conclusion: Our findings demonstrate SPR965 induces apoptosis of MM cells through the inhibition of PI3K and mTORC1/C2 activity. Further studies are underway to better characterize the mechanism of action of SPR965, all of which will be informative for the drug to be used as a single agent or in combination with other agents in relapsed MM. Citation Format: Jeremy T. Larsen, Vijay Ramakrishnan, Jessica Haug, Teresa Kimlinger, Somdutta Sen, Dinesh Mahajan, Sundeep Dugar, S. Vincent Rajkumar, Shaji K. Kumar. Preclinical activity of dual PI3K/mTOR inhibitor SPR965 in multiple myeloma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2653. doi:10.1158/1538-7445.AM2015-2653