Mitigating oxygen stress enhances aged mouse hematopoietic stem cell numbers and function.

Bone marrow (BM) hematopoietic stem cells (HSCs) become dysfunctional during aging (i.e., they are increased in number but have an overall reduction in long-term repopulation potential and increased myeloid differentiation) compared with young HSCs, suggesting limited use of old donor BM cells for hematopoietic cell transplantation (HCT). BM cells reside in an in vivo hypoxic environment yet are evaluated after collection and processing in ambient air. We detected an increase in the number of both young and aged mouse BM HSCs collected and processed in 3% O2 compared with the number of young BM HSCs collected and processed in ambient air (~21% O2). Aged BM collected and processed under hypoxic conditions demonstrated enhanced engraftment capability during competitive transplantation analysis and contained more functional HSCs as determined by limiting dilution analysis. Importantly, the myeloid-to-lymphoid differentiation ratio of aged BM collected in 3% O2 was similar to that detected in young BM collected in ambient air or hypoxic conditions, consistent with the increased number of common lymphoid progenitors following collection under hypoxia. Enhanced functional activity and differentiation of old BM collected and processed in hypoxia correlated with reduced "stress" associated with ambient air BM collection and suggests that aged BM may be better and more efficiently used for HCT if collected and processed under hypoxia so that it is never exposed to ambient air O2.

Ractopamine-induced fiber type-specific gene expression in porcine skeletal muscles is independent of growth.

Feeding ractopamine (RAC), a ß-adrenergic agonist (BAA), to pigs increases type IIB muscle fiber type-specific protein and mRNA expression. However, increases in the abundance of these fast-twitch fiber types occur with other forms of muscle hypertrophy and thus BAA-induced changes in myosin heavy chain (MyHC) composition may simply be associated with increased muscle growth known to occur in response to BAA feeding. The objective of this study was to determine whether RAC feeding could change the MyHC gene expression in the absence of maximal muscle growth. Pigs were fed either an adequate diet that supported maximal muscle hypertrophy or a low nutrient diet that limited muscle growth. RAC was included in diets at 0 or 20 mg/kg for 1, 2, or 4 wk. Backfat depth was less (P < 0.05) in pigs fed the low nutrient diet compared with the adequate diet but was not affected by RAC. Loin eye area was greater (P < 0.05) in pigs fed an adequate diet plus RAC at 1 wk but did not differ among remaining pigs. At 2 and 4 wk, however, pigs fed the adequate diet had greater loin eye areas (P < 0.05) than pigs fed the low nutrient diet regardless of RAC feeding. Gene expression of the MyHC isoforms, I, IIA, IIX, and IIB, as well as glycogen synthase, citrate synthase, ß 1-adrenergic receptor (AR), and ß 2-AR were determined in longissimus dorsi (LD) and red (RST) and white (WST) portions of the semitendinosus muscles. MyHC type I gene expression was not altered by RAC or diet. Feeding RAC decreased (P < 0.01) MyHC type IIA gene expression in all muscles, but to a greater extent in WST and LD. MyHC type IIX gene expression was lower (P < 0.05) in WST and LD muscles in response to RAC but was not altered in RST muscles. RAC increased (P < 0.05) MyHC type IIB gene expression in all muscles, but to a greater extent in RST. ß 1-AR gene expression was unaffected by RAC or diet, whereas the expression of the ß 2-AR gene was decreased (P < 0.001) by RAC. No significant RAC * diet interactions were observed in gene expression in this study, indicating that RAC altered MyHC and ß 2-AR gene expression in porcine skeletal muscles independent of growth.

CD166 Engagement Augments Mouse and Human Hematopoietic Progenitor Function via Activation of Stemness and Cell Cycle Pathways.

Hematopoietic stem (HSC) and progenitor (HPC) cells are regulated by interacting signals and cellular and noncellular elements of the hematopoietic niche. We previously showed that CD166 is a functional marker of murine and human HSC and of cellular components of the murine niche. Selection of murine CD166+ engrafting HSC enriched for marrow repopulating cells. Here, we demonstrate that CD166-CD166 homophilic interactions enhance generation of murine and human HPC in vitro and augment hematopoietic function of these cells. Interactions between cultured CD166+ Lineage- Sca-1+ c-Kit+ (LSK) cells and CD166+ osteoblasts (OBs) significantly enhanced the expansion of colony-forming units (CFUs). Interactions between CD166+ LSK cells and immobilized CD166 protein generated more CFU in short-term cultures than between these cells and bovine serum albumin (BSA) or in cultures initiated with CD166- LSK cells. Similar results were obtained when LSK cells from wildtype (WT) or CD166 knockout (KO) (CD166-/- ) mice were used with immobilized CD166. Human cord blood CD34+ cells expressing CD166 produced significantly higher numbers of CFUs following interaction with immobilized CD166 than their CD166- counterparts. These data demonstrate the positive effects of CD166 homophilic interactions involving CD166 on the surface of murine and human HPCs. Single-cell RNA-seq analysis of CD150+ CD48- (signaling lymphocyte activation molecule (SLAM)) LSK cells from WT and CD166-/- mice incubated with immobilized CD166 protein revealed that engagement of CD166 on these cells activates cytokine, growth factor and hormone signaling, epigenetic pathways, and other genes implicated in maintenance of stem cell pluripotency-related and mitochondria-related signaling pathways. These studies provide tangible evidence implicating CD166 engagement in the maintenance of stem/progenitor cell function. Stem Cells 2019;37:1319-1330.

Cefsulodin Inspired Potent and Selective Inhibitors of mPTPB, a Virulent Phosphatase from Mycobacterium tuberculosis.

mPTPB is a virulent phosphatase from Mycobacterium tuberculosis and a promising therapeutic target for tuberculosis. To facilitate mPTPB-based drug discovery, we identified α-sulfophenylacetic amide (SPAA) from cefsulodin, a third generation ß-lactam cephalosporin antibiotic, as a novel pTyr pharmacophore for mPTPB. Structure-guided and fragment-based optimization of SPAA led to the most potent and selective mPTPB inhibitor 9, with a K i of 7.9 nM and more than 10,000-fold preference for mPTPB over a large panel of 25 phosphatases. Compound 9 also exhibited excellent cellular activity and specificity in blocking mPTPB function in macrophage. Given its novel structure, modest molecular mass, and extremely high ligand efficiency (0.46), compound 9 represents an outstanding lead compound for anti-TB drug discovery targeting mPTPB.

Exploring the Existing Drug Space for Novel pTyr Mimetic and SHP2 Inhibitors.

Protein tyrosine phosphatases (PTPs) are potential therapeutic targets for many diseases. Unfortunately, despite considerable drug discovery efforts devoted to PTPs, obtaining selective and cell permeable PTP inhibitors remains highly challenging. We describe a strategy to explore the existing drug space for previously unknown PTP inhibitory activities. This led to the discovery of cefsulodin as an inhibitor of SHP2, an oncogenic phosphatase in the PTP family. Crystal structure analysis of SHP2 interaction with cefsulodin identified sulfophenyl acetic amide (SPAA) as a novel phosphotyrosine (pTyr) mimetic. A structure-guided and SPAA fragment-based focused library approach produced several potent and selective SHP2 inhibitors. Notably, these inhibitors blocked SHP2-mediated signaling events and proliferation in several cancer cell lines. Thus, SPAA may serve as a new platform for developing chemical probes for other PTPs.

A potent and selective inhibitor for the UBLCP1 proteasome phosphatase.

The ubiquitin-like domain-containing C-terminal domain phosphatase 1 (UBLCP1) has been implicated as a negative regulator of the proteasome, a key mediator in the ubiquitin-dependent protein degradation. Small molecule inhibitors that block UBLCP1 activity would be valuable as research tools and potential therapeutics for human diseases caused by the cellular accumulation of misfold/damaged proteins. We report a salicylic acid fragment-based library approach aimed at targeting both the phosphatase active site and its adjacent binding pocket for enhanced affinity and selectivity. Screening of the focused libraries led to the identification of the first potent and selective UBLCP1 inhibitor 13. Compound 13 exhibits an IC50 of 1.0µM for UBLCP1 and greater than 5-fold selectivity against a large panel of protein phosphatases from several distinct families. Importantly, the inhibitor possesses efficacious cellular activity and is capable of inhibiting UBLCP1 function in cells, which in turn up-regulates nuclear proteasome activity. These studies set the groundwork for further developing compound 13 into chemical probes or potential therapeutic agents targeting the UBLCP1 phosphatase.

Therapeutic potential of targeting the oncogenic SHP2 phosphatase.

The Src homology 2 domain containing protein tyrosine phosphatase-2 (SHP2) is an oncogenic phosphatase associated with various kinds of leukemia and solid tumors. Thus, there is substantial interest in developing SHP2 inhibitors as potential anticancer and antileukemia agents. Using a structure-guided and fragment-based library approach, we identified a novel hydroxyindole carboxylic acid-based SHP2 inhibitor 11a-1, with an IC50 value of 200 nM and greater than 5-fold selectivity against 20 mammalian PTPs. Structural and modeling studies reveal that the hydroxyindole carboxylic acid anchors the inhibitor to the SHP2 active site, while interactions of the oxalamide linker and the phenylthiophene tail with residues in the ß5-ß6 loop contribute to 11a-1's binding potency and selectivity. Evidence suggests that 11a-1 specifically attenuates the SHP2-dependent signaling inside the cell. Moreover, 11a-1 blocks growth factor mediated Erk1/2 and Akt activation and exhibits excellent antiproliferative activity in lung cancer and breast cancer as well as leukemia cell lines.

Hydroxyindole carboxylic acid-based inhibitors for receptor-type protein tyrosine protein phosphatase beta.

AIMS: Protein tyrosine phosphatases (PTPs) play an important role in regulating a wide range of cellular processes. Understanding the role of PTPs within these processes has been hampered by a lack of potent and selective PTP inhibitors. Generating potent and selective probes for PTPs remains a significant challenge because of the highly conserved and positively charged PTP active site that also harbors a redox-sensitive Cys residue. RESULTS: We describe a facile method that uses an appropriate hydroxyindole carboxylic acid to anchor the inhibitor to the PTP active site and relies on the secondary binding elements introduced through an amide-focused library to enhance binding affinity for the target PTP and to impart selectivity against off-target phosphatases. Here, we disclose a novel series of hydroxyindole carboxylic acid-based inhibitors for receptor-type tyrosine protein phosphatase beta (RPTPß), a potential target that is implicated in blood vessel development. The representative RPTPß inhibitor 8b-1 (L87B44) has an IC50 of 0.38 µM and at least 14-fold selectivity for RPTPß over a large panel of PTPs. Moreover, 8b-1 also exhibits excellent cellular activity and augments growth factor signaling in HEK293, MDA-MB-468, and human umbilical vein endothelial cells. INNOVATION: The bicyclic salicylic acid pharmacophore-based focused library approach may provide a potential solution to overcome the bioavailability issue that has plagued the PTP drug discovery field for many years. CONCLUSION: A novel method is described for the development of bioavailable PTP inhibitors that utilizes bicyclic salicylic acid to anchor the inhibitors to the active site and peripheral site interactions to enhance binding affinity and selectivity.

A potent and selective small-molecule inhibitor for the lymphoid-specific tyrosine phosphatase (LYP), a target associated with autoimmune diseases.

Lymphoid-specific tyrosine phosphatase (LYP), a member of the protein tyrosine phosphatase (PTP) family of signaling enzymes, is associated with a broad spectrum of autoimmune diseases. Herein we describe our structure-based lead optimization efforts within a 6-hydroxy-benzofuran-5-carboxylic acid series culminating in the identification of compound 8b, a potent and selective inhibitor of LYP with a K(i) value of 110 nM and more than 9-fold selectivity over a large panel of PTPs. The structure of LYP in complex with 8b was obtained by X-ray crystallography, providing detailed information about the molecular recognition of small-molecule ligands binding LYP. Importantly, compound 8b possesses highly efficacious cellular activity in both T- and mast cells and is capable of blocking anaphylaxis in mice. Discovery of 8b establishes a starting point for the development of clinically useful LYP inhibitors for treating a wide range of autoimmune disorders.

A facile hydroxyindole carboxylic acid based focused library approach for potent and selective inhibitors of Mycobacterium protein tyrosine phosphatase B.

Focused on Mtb: A facile hydroxyindole carboxylic acid based focused amide library was designed to target both the PTP active site and a unique nearby pocket for enhanced affinity and selectivity. HTS of the library led to the identification of a highly potent and selective inhibitor, 11 a, of mPTPB, an essential virulence factor for Mycobacterium tuberculosis. Compound 11 a shows high cellular activity and is capable of reversing the altered immune responses induced by mPTPB in macrophages.

Discovery and evaluation of novel inhibitors of mycobacterium protein tyrosine phosphatase B from the 6-Hydroxy-benzofuran-5-carboxylic acid scaffold.

Mycobacterium tuberculosis (Mtb) protein tyrosine phosphatase B (mPTPB) is a virulence factor secreted by the pathogen and mediates mycobacterial survival in macrophages by targeting host cell immune responses. Consequently, mPTPB represents an exciting new target to combat tuberculosis (TB) infection. We describe a medicinal chemistry oriented approach that transforms a benzofuran salicylic acid scaffold into a highly potent (IC(50) = 38 nM) and selective mPTPB inhibitor (>50 fold against a large panel of PTPs). Importantly, the inhibitor is capable of reversing the altered host immune responses induced by the bacterial phosphatase and restoring the macrophage's full capacity to secrete IL-6 and undergo apoptosis in response to interferon-γ stimulation, validating the concept that chemical inhibition of mPTPB may be therapeutically useful for novel TB treatment. The study further demonstrates that bicyclic salicylic acid pharmacophores can be used to deliver PTP inhibitors with high potency, selectivity, and cellular efficacy.

SHP2 is a target of the immunosuppressant tautomycetin.

SHP2 phosphatase is a positive transducer of growth factor and cytokine signaling. SHP2 is also a bona fide oncogene; gain-of-function SHP2 mutations leading to increased phosphatase activity cause Noonan syndrome, as well as multiple forms of leukemia and solid tumors. We report that tautomycetin (TTN), an immunosuppressor in organ transplantation, and its engineered analog TTN D-1 are potent SHP2 inhibitors. TTN and TTN D-1 block T cell receptor-mediated tyrosine phosphorylation and ERK activation and gain-of-function mutant SHP2-induced hematopoietic progenitor hyperproliferation and monocytic differentiation. Crystal structure of the SHP2⋅TTN D-1 complex reveals that TTN D-1 occupies the SHP2 active site in a manner similar to that of a peptide substrate. Collectively, the data support the notion that SHP2 is a cellular target for TTN and provide a potential mechanism for the immunosuppressive activity of TTN. Moreover, the structure furnishes molecular insights upon which therapeutics targeting SHP2 can be developed on the basis of the TTN scaffold.

Double click reaction for the acquisition of a highly potent and selective mPTPB inhibitor.

Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), is a major worldwide threat to public health. Mycobacterium protein tyrosine phosphatase B (mPTPB) is a virulent phosphatase secreted by Mtb, which is essential for the survival and persistence of the bacterium in the host. Consequently, small-molecule inhibitors of mPTPB are expected to serve as anti-TB agents with a novel mode of action. Herein, we report the discovery of highly potent and selective mPTPB inhibitors using a novel, double Click chemistry strategy. The most potent mPTPB inhibitor from this approach possesses a K(i) value of 160 nM and a >25-fold selectivity for mPTPB over 19 other protein tyrosine phosphatases (PTBs). Molecular docking study of the enzyme-inhibitor complex provides a rationale for the high potency and selectivity of the lead compound and reveals an unusual binding mode, which may guide further optimization effort.

Salicylic acid based small molecule inhibitor for the oncogenic Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2).

The Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) plays a pivotal role in growth factor and cytokine signaling. Gain-of-function SHP2 mutations are associated with Noonan syndrome, various kinds of leukemias, and solid tumors. Thus, there is considerable interest in SHP2 as a potential target for anticancer and antileukemia therapy. We report a salicylic acid based combinatorial library approach aimed at binding both active site and unique nearby subpockets for enhanced affinity and selectivity. Screening of the library led to the identification of a SHP2 inhibitor II-B08 (compound 9) with highly efficacious cellular activity. Compound 9 blocks growth factor stimulated ERK1/2 activation and hematopoietic progenitor proliferation, providing supporting evidence that chemical inhibition of SHP2 may be therapeutically useful for anticancer and antileukemia treatment. X-ray crystallographic analysis of the structure of SHP2 in complex with 9 reveals molecular determinants that can be exploited for the acquisition of more potent and selective SHP2 inhibitors.

Targeting inactive enzyme conformation: aryl diketoacid derivatives as a new class of PTP1B inhibitors.

There has been considerable interest in protein tyrosine phosphatase 1B (PTP1B) as a therapeutic target for diabetes, obesity, as well as cancer. Identifying inhibitory compounds with good bioavailability is a major challenge of drug discovery programs targeted toward PTPs. Most current PTP active site-directed pharmacophores are negatively charged pTyr mimetics which cannot readily enter the cell. This lack of cell permeability limits the utility of such compounds in signaling studies and further therapeutic development. We identify aryl diketoacids as novel pTyr surrogates and show that neutral amide-linked aryl diketoacid dimers also exhibit excellent PTP inhibitory activity. Kinetic studies establish that these aryl diketoacid derivatives act as noncompetitive inhibitors of PTP1B. Crystal structures of ligand-bound PTP1B reveal that both the aryl diketoacid and its dimeric derivative bind PTP1B at the active site, albeit with distinct modes of interaction, in the catalytically inactive, WPD loop open conformation. Furthermore, dimeric aryl diketoacids are cell permeable and enhance insulin signaling in hepatoma cells, suggesting that targeting the inactive conformation may provide a unique opportunity for creating active site-directed PTP1B inhibitors with improved pharmacological properties.