A CD38-directed, single-chain T-cell engager targets leukemia stem cells through IFN-γ-induced CD38 expression.

ABSTRACT: Treatment resistance of leukemia stem cells (LSCs) and suppression of the autologous immune system represent major challenges to achieve a cure in acute myeloid leukemia (AML). Although AML blasts generally retain high levels of surface CD38 (CD38pos), LSCs are frequently enriched in the CD34posCD38neg blast fraction. Here, we report that interferon gamma (IFN-γ) reduces LSCs clonogenic activity and induces CD38 upregulation in both CD38pos and CD38neg LSC-enriched blasts. IFN-γ-induced CD38 upregulation depends on interferon regulatory factor 1 transcriptional activation of the CD38 promoter. To leverage this observation, we created a novel compact, single-chain CD38-CD3 T-cell engager (BN-CD38) designed to promote an effective immunological synapse between CD38pos AML cells and both CD8pos and CD4pos T cells. We demonstrate that BN-CD38 engages autologous CD4pos and CD8pos T cells and CD38pos AML blasts, leading to T-cell activation and expansion and to the elimination of leukemia cells in an autologous setting. Importantly, BN-CD38 engagement induces the release of high levels of IFN-γ, driving the expression of CD38 on CD34posCD38neg LSC-enriched blasts and their subsequent elimination. Critically, although BN-CD38 showed significant in vivo efficacy across multiple disseminated AML cell lines and patient-derived xenograft models, it did not affect normal hematopoietic stem cell clonogenicity and the development of multilineage human immune cells in CD34pos humanized mice. Taken together, this study provides important insights to target and eliminate AML LSCs.

Design and in vitro analysis of SIRT2 inhibitor targeting Parkinson's disease.

Inhibition of Sirtuin2 (SIRT2) protein rescues the α-synuclein toxicity in vitro and in vivo models of Parkinson's disease (PD). Thioacetyl group can structurally mimic the acetyl group and restrain the deacetylating p53 reaction by SIRT2. This work evaluated the biological activity of designed pentapeptides inhibitor containing N-thioacetyl-lysine against SIRT2. Pentapeptide by introducing thioacetyl-lysine as an inhibitor of SIRT2 was screened by molecular docking and synthesized by solid phase method. The inhibition of pure recombinant SIRT2 as well as SIRT2 in serum of PD patients by peptide was done by fluorescent activity assay. The inhibition of SIRT2 was assessed in PC12 cell line by measuring acetylated α-tubulin level. The peptide YKK(ε-thioAc)AM and HRK(ε-thioAc)AM were found to be SIRT2 inhibitors by molecular docking. However, YKK(ε-thioAc)AM was more specific towards SIRT2 than SIRT1 (Sirtuin1). It inhibited recombinant SIRT2 by IC50 value of 0.15 µM and KD values 9.92 × 10-8/M. It also inhibited serum SIRT2 of PD. It increased the acetylation of α-tubulin in PC12 neuroblastoma cells which is essential for maintaining the microtubular cell functions of brain. It can be concluded that novel peptide YKK(ε-thioAc)AM may be a platform for therapeutic agent for Parkinson's disease targeting SIRT2.

Requirement of GTP binding for TIF-90-regulated ribosomal RNA synthesis and oncogenic activities in human colon cancer cells.

Transcription initiation factor 90 (TIF-90), an alternatively spliced variant of TIF-IA, differs by a 90 base pair deletion of exon 6. TIF-90 has been shown to regulate ribosomal RNA (rRNA) synthesis by interacting with polymerase I (Pol I) during the initiation of ribosomal DNA (rDNA) transcription in the nucleolus. Recently, we showed that TIF-90-mediated rRNA synthesis can play an important role in driving tumorigenesis in human colon cancer cells. Here we show that TIF-90 binds GTP at threonine 310, and that GTP binding is required for TIF-90-enhanced rRNA synthesis. Overexpression of activated AKT induces TIF-90 T310, but not a GTP-binding site (TIF-90 T310N) mutant, to translocate into the nucleolus and increase rRNA synthesis. Complementing this result, treatment with mycophenolic acid (MPA), an inhibitor of GTP production, dissociates TIF-90 from Pol I and hence abolishes AKT-increased rRNA synthesis by way of TIF-90 activation. Thus, TIF-90 requires bound GTP to fulfill its function as an enhancer of rRNA synthesis. Both TIF variants are highly expressed in colon cancer cells, and depletion of TIF-IA expression in these cells results in significant sensitivity to MPA-inhibited rRNA synthesis and reduced cell proliferation. Finally, a combination of MPA and AZD8055 (an inhibitor of both AKT and mTOR) synergistically inhibits rRNA synthesis, in vivo tumor growth, and other oncogenic activities of primary human colon cancer cells, suggesting a potential avenue for the development of therapeutic treatments by targeting the regulation of rRNA synthesis by TIF proteins.

Development of peptide inhibitor as a therapeutic agent against head and neck squamous cell carcinoma (HNSCC) targeting p38alpha MAP kinase.

BACKGROUND: The p38alpha MAP kinase pathway is involved in inflammation, cell differentiation, growth, apoptosis and production of pro-inflammatory cytokines TNF-alpha and IL-1beta. The overproduction of these cytokines plays an important role in cancer. The aim of this work was to design a peptide inhibitor on the basis of structural information of the active site of p38alpha. METHODS: A tetrapeptide, VWCS as p38alpha inhibitor was designed on the basis of structural information of the ATP binding site by molecular modeling. The inhibition study of peptide with p38alpha was performed by ELISA, binding study by Surface Plasmon Resonance and anti-proliferative assays by MTT and flow cytometry. RESULTS: The percentage inhibition of designed VWCS against pure p38alpha protein and serum of HNSCC patients was 70.30 and 71.5%, respectively. The biochemical assay demonstrated the K(D) and IC50 of the selective peptide as 7.22 x 10(-9) M and 20.08 nM, respectively. The VWCS as inhibitor significantly reduced viability of oral cancer KB cell line with an IC50 value of 10 microM and induced apoptosis by activating Caspase 3 and 7. CONCLUSIONS: VWCS efficiently interacted at the ATP binding pocket of p38alpha with high potency and can be used as a potent inhibitor in case of HNSCC. GENERAL SIGNIFICANCE: VWCS can act as an anticancer agent as it potentially inhibits the cell growth and induces apoptosis in oral cancer cell-line in a dose as well as time dependent manner. Hence, p38alpha MAP kinase inhibitor can be a potential therapeutic agent for human oral cancer.

Exploring the Molecular Level Interaction of Human Serum Albumin with Calcium Oxalate Monohydrate Crystals.

BACKGROUND: Human serum albumin (HSA) is one of the most abundant proteins in the blood plasma, urine as well as in the organic matrix of renal calculi. Macromolecules present in the urine modulate kidney stone formation either by stimulating or inhibiting the crystallization process. OBJECTIVE: In the present study, the effect of HSA protein on the growth of calcium oxalate monohydrate crystal (COM) was investigated. METHODS: Crystal growth assay was used to measure oxalate depletion in the crystal seeded solution in the presence of HSA. HSA concentrations exhibiting effect on crystal growth were selected for FTIR and XRD analysis. In silico docking was performed on seven different binding sites of HSA. RESULTS: Albumin plays dual role in the growth of calcium oxalate crystallization. FTIR and XRD studies further revealed HSA exerted strain over crystal thus affecting its structure by interacting with amino acids of its pocket 1. Docking results indicate that out of 7 binding pocket in protein, calcium oxalate interacts with Arg-186 and Lys-190 amino acids of pocket 1. CONCLUSION: Our study confirms the role of HSA in calcium oxalate crystallization where acidic amino acids arginine and lysine bind to COM crystals, revealing molecular interaction of macromolecule and crystal in urolithiasis.

Diagnostic Significance of p38 Isoforms (p38α, p38ß, p38γ, p38δ) in Head and Neck Squamous Cell Carcinoma: Comparative Serum Level Evaluation and Design of Novel Peptide Inhibitor Targeting the Same.

PURPOSE: The p38 mitogen-activated protein kinase (MAPKs) play a crucial role in the production of pro-inflammatory cytokines and over-expression of it increase cytokines which promote cancer. Among four isoforms, p38α has been well studied in head and neck squamous cell carcinoma (HNSCC) and other cancers as a therapeutic target. p38δ has recently emerged as a potential disease-specific drug target. Elevated serum p38α level in HNSCC was reported earlier from our lab. This study aims to estimate the levels of p38 MAPK-isoforms in the serum of HNSCC and design peptide inhibitor targeting the same. MATERIALS AND METHODS: Levels of p38 MAPK isoforms in the serum of HNSCC and healthy controls were quantified by surface plasmon resonance technology. The peptide inhibitor for p38 MAPK was designed by molecular modeling using Grid-based Ligand Docking with Energetics tools and compared with known specific inhibitors. RESULTS: We have observed highly elevated levels of all four isoforms of p38 MAPK in serum of HNSCC patients compared to the control group. Further, serum p38α, p38ß, and p38δ levels were down regulated after therapy in follow-up patients, while p38γ showed no response to the therapy. Present study screened designed peptide WFYH as a specific inhibitor against p38δ. The specific inhibitor of p38δ was found to have no effect on p38α due to great structural difference at ATP binding pocket. CONCLUSION: In this study, first time estimated the levels of p38 MAPK isoforms in the serum of HNSCC. It can be concluded that p38 MAPK isoforms can be a diagnostic and prognostic marker for HNSCC and p38δ as a therapeutic target.

CID-6033590 inhibits p38MAPK pathway and induces S-phase cell cycle arrest and apoptosis in DU145 and PC-3 cells.

Metastatic prostate cancer, with no effective treatment, is among the leading causes of cancer-associated deaths in men. Overexpression of p38αMAPK has been observed in neuroendocrine prostate cancer patients and in both DU145 and PC-3 cell lines and represents a good drug target. Sulfonamide derivatives have shown biological activities against many human diseases, including cancer. CID-6033590, a sulfonylhydrazide compound, screened from PubChem database by molecular docking with p38αMAPK, was evaluated for anti-cancerous activities. CID-6033590 induced toxicity in both DU145 and PC-3 cells in a concentration and time-dependent manner with an IC50 value of 60 µM and 66 µM, respectively. Sub-cytotoxic concentrations of the compound significantly induced S-phase cell cycle arrest, inhibited cyclinA/CDK2 complex and blocked cell proliferation. Further, CID-6033590 downregulated phosphorylation of p38MAPK (P-p38) as well as its downstream targets, Activating transcription factor 2 (ATF-2) and Heat shock protein 27 (Hsp27). The compound increased ROS and decreased mitochondrial membrane potential (Δψm), downregulated Bcl-2 and survivin and cleaved poly ADP ribose polymerase (PARP) and caspase-3, indicating the induction of apoptosis. The evaluaion of the compound on noncancerous, human prostatic epithelial cell line RWPE-1, and healthy murine tissues yielded no significant toxicity. Taken together, we suggest CID-6033590 as a potential candidate for prostate cancer therapy.

Design, synthesis of allosteric peptide activator for human SIRT1 and its biological evaluation in cellular model of Alzheimer's disease.

Sirtuin 1 (SIRT1) is one of the member of the mammalian proteins of the Sirtuin family of NAD+ dependent deacetylases, has recently been shown to attenuate amyloidogenic processing of amyloid protein precursor (APP) in in-vitro cell culture studies and transgenic mouse models of Alzheimer's disease (AD). SIRT1 has been shown to have a protective role against (AD). It has been reported earlier that increasing SIRT1 activity can prevent AD in mice model. Tripeptide as an activator of SIRT1 were screened on the basis of structural information by molecular docking and synthesized by solid phase method. The enhancement of biochemical activity of pure recombinant SIRT1 as well as SIRT1 in serum of AD patients in presence of tripeptide was done by Fluorescent Activity Assay. The activity of SIRT1 by peptide was assessed in IMR-32 cell line by measuring acetylated p53 level. Further the protective effect of SIRT1 activator in cellular model of AD was analyzed by MTT assay. We find CWR tripeptide as a SIRT1 activator by molecular docking, enhanced the activity of SIRT1 protein by lowering the Michaelis constant, Km by allosteric mechanism. The activity of serum SIRT1 of AD was also increases by CWR. It also decreased the acetylation of p53 in IMR32 neuroblastoma cells and protected the cell death caused by Aß amyloid fragments in cell line model of AD. Thus, it can be concluded that CWR may serve as platform to elucidate further small molecule activator as a therapeutic agent for AD targeting SIRT1.

The rational design of specific peptide inhibitor against p38α MAPK at allosteric-site: a therapeutic modality for HNSCC.

p38α is a significant target for drug designing against cancer. The overproduction of p38α MAPK promotes tumorigenesis in head and neck squamous cell carcinoma (HNSCC). The ATP binding and an allosteric site referred as DFG are the key sites of the p38α mitogen activated protein kinase (MAPK) exploited for the design of inhibitors. This study demonstrated design of peptide inhibitor on the basis of allosteric site using Glide molecular docking software and the biochemical analysis of the best modeled peptide. The best fitted tetrapeptide (FWCS) in the allosteric site inhibited the pure recombinant and serum p38α of HNSCC patients by 74 and 72%, respectively. The potency of the peptide was demonstrated by its IC50 (4.6 nM) and KD (3.41×10-10 M) values, determined by ELISA and by surface plasmon resonance (SPR) technology, respectively. The cell viability of oral cancer i.e. KB cell line was reduced in dose dependent manner by 60 and 97% by the treatment of peptide and the IC50 was 600 and 210 µM after 24 and 72 h incubation, respectively. Our result provides an insight for the development of a proficient small peptide as a promising anticancer agent targeting DFG site of p38α kinase.