Chemical structure-guided design of dynapyrazoles, potent cell-permeable dynein inhibitors with a unique mode of action

Cytoplasmic dyneins are motor proteins in the AAA+ superfamily that transport cellular cargos toward microtubule minus-ends. Recently, ciliobrevins were reported as selective cell-permeable inhibitors of cytoplasmic dyneins. As is often true for first-in-class inhibitors, the use of ciliobrevins has in part been limited by low potency. Moreover, suboptimal chemical properties, such as the potential to isomerize, have hindered efforts to improve ciliobrevins. Here, we characterized the structure of ciliobrevins and designed conformationally constrained isosteres. These studies identified dynapyrazoles, inhibitors more potent than ciliobrevins. At single-digit micromolar concentrations dynapyrazoles block intraflagellar transport in the cilium and lysosome motility in the cytoplasm, processes that depend on cytoplasmic dyneins. Further, we find that while ciliobrevins inhibit both dynein's microtubule-stimulated and basal ATPase activity, dynapyrazoles strongly block only microtubule-stimulated activity. Together, our studies suggest that chemical-structure-based analyses can lead to inhibitors with improved properties and distinct modes of inhibition

Development of a Highly Selective Plasmodium falciparum Proteasome Inhibitor with Anti-malaria Activity in Humanized Mice.

Plasmodium falciparum proteasome (Pf20S) inhibitors are active against Plasmodium at multiple stages-erythrocytic, gametocyte, liver, and gamete activation stages-indicating that selective Pf20S inhibitors possess the potential to be therapeutic, prophylactic, and transmission-blocking antimalarials. Starting from a reported compound, we developed a noncovalent, macrocyclic peptide inhibitor of the malarial proteasome with high species selectivity and improved pharmacokinetic properties. The compound demonstrates specific, time-dependent inhibition of the β5 subunit of the Pf20S, kills artemisinin-sensitive and artemisinin-resistant P. falciparum isolates in vitro and reduces parasitemia in humanized, P. falciparum-infected mice

Antimalarial proteasome inhibitor reveals collateral sensitivity from intersubunit interactions and fitness cost of resistance.

We describe noncovalent, reversible asparagine ethylenediamine (AsnEDA) inhibitors of the Plasmodium falciparum proteasome (Pf20S) β5 subunit that spare all active subunits of human constitutive and immuno-proteasomes. The compounds are active against erythrocytic, sexual, and liver-stage parasites, against parasites resistant to current antimalarials, and against P. falciparum strains from patients in Africa. The β5 inhibitors synergize with a β2 inhibitor in vitro and in mice and with artemisinin. P. falciparum selected for resistance to an AsnEDA β5 inhibitor surprisingly harbored a point mutation in the noncatalytic β6 subunit. The β6 mutant was resistant to the species-selective Pf20S β5 inhibitor but remained sensitive to the species-nonselective β5 inhibitors bortezomib and carfilzomib. Moreover, resistance to the Pf20S β5 inhibitor was accompanied by increased sensitivity to a Pf20S β2 inhibitor. Finally, the β5 inhibitor-resistant mutant had a fitness cost that was exacerbated by irradiation. Thus, used in combination, multistage-active inhibitors of the Pf20S β5 and β2 subunits afford synergistic antimalarial activity with a potential to delay the emergence of resistance to artemisinins and each other

Adipose-Derived Mesenchymal Stem Cells Restore Impaired Mucosal Immune Responses in Aged Mice

<div><p>It has been shown that adipose-derived mesenchymal stem cells (AMSCs) can differentiate into adipocytes, chondrocytes and osteoblasts. Several clinical trials have shown the ability of AMSCs to regenerate these differentiated cell types. Age-associated dysregulation of the gastrointestinal (GI) immune system has been well documented. Our previous studies showed that impaired mucosal immunity in the GI tract occurs earlier during agingthan is seen in the systemic compartment. In this study, we examined the potential of AMSCs to restore the GI mucosal immune system in aged mice. Aged (>18 mo old) mice were adoptively transferred with AMSCs. Two weeks later, mice were orally immunized with ovalbumin (OVA) plus cholera toxin (CT) three times at weekly intervals. Seven days after the final immunization, when fecal extract samples and plasma were subjected to OVA- and CT-B-specific ELISA, elevated levels of mucosal secretory IgA (SIgA) and plasma IgG antibody (Ab) responses were noted in aged mouse recipients. Similar results were also seen aged mice which received AMSCs at one year of age. When cytokine production was examined, OVA-stimulated Peyer’s patch CD4⁺ T cells produced increased levels of IL-4. Further, CD4⁺ T cells from the lamina propria revealed elevated levels of IL-4 and IFN-γ production. In contrast, aged mice without AMSC transfer showed essentially no OVA- or CT-B-specific mucosal SIgA or plasma IgG Ab or cytokine responses. Of importance, fecal extracts from AMSC transferred aged mice showed neutralization activity to CT intoxication. These results suggest that AMSCs can restore impaired mucosal immunity in the GI tract of aged mice.</p></div

OVA-specific AFCs in lamina propria (LP) and spleen of aged mice.

<p>Aged mice with (○) or without (□) mAMSC transfer were orally immunized as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148185#pone.0148185.g001" target="_blank">Fig 1</a> legend. One week after the last immunization, mononuclear cells were isolated from the LP and spleen and were then subjected to an OVA-specific ELISPOT assay to determine the numbers of IgA and IgG AFCs. Naïve mice served as a control group and did not exhibit any anti-OVA AFCs (data not shown). The values shown are the mean ± SEM taken from 10 mice in each group. *<i>p</i> < 0.05 when compared with aged mice without adoptive transfer of mAMSCs.</p

CT-B-specific Ab responses in aged and young adult mice.

<p>Aged mice were adoptively transferred with mAMSCs or hAMSCs. Aged mice given mAMSCs (hatched bar), naïve aged mice (without hAMSC transfer, open bar) and young adult mice (closed bar) were orally immunized with 1 mg of OVA plus 10 μg of CT three times at weekly intervals. One week after the last immunization, levels of anti-CT-B fecal SIgA and plasma IgG Abs were determined by CT-B-specific ELISA. The values shown are the mean ± SEM taken from 10 mice in each group. *<i>p</i> < 0.05 when compared with aged mice without either mAMSC or hAMSC transfer.</p

OVA-specific Ab responses in aged mice given human AMSCs (hAMSCs).

<p>Aged mice were adoptively transferred with hAMSCs. Aged mice adoptively transferred with hAMSCs (hatched bar), naïve aged mice (without hAMSC transfer, open bar) and young adult mice (closed bar) were orally immunized with 1 mg of OVA plus 10 μg CT three times at weekly intervals. One week after the last immunization, levels of anti-OVA fecal SIgA and plasma IgG Abs were determined by an OVA-specific ELISA. The values shown are the mean ± SEM taken from 10 mice in each group. *<i>p</i> < 0.05 when compared with aged mice without AMSC adoptive transfer.</p

Interleukin-4 and IFN-γ production by CD4⁺ T cells from PPs, LP and spleen were determined by intracellular analysis.

<p>Mononuclear cells were isolated from aged mice given oral OVA plus CT with or without mAMSC adoptive transfer. Cells were incubated with ionomycin (1 μg/ml) and phorbol 12-myristate 13-acetate (PMA; 25 ng/ml) for 3 hr and then stained with FITC-labeled anti-CD4 mAb. Samples were further stained intracellularly with PE-labeled anti-IL-4 (A) or anti-IFN-γ (B) mAb. The frequencies of cytokine-producing cells were determined by flow cytometry. The profiles represent typical results and are taken from one of three separate experiments.</p