Synthesis of Reblastatin, Autolytimycin, and Non-Benzoquinone Analogues: Potent Inhibitors of Heat Shock Protein 90

A full account of an asymmetric synthesis of reblastatin (1) and the first total synthesis of autolytimycin (2) and related structural compounds is described. The syntheses expand the utility of a highly regio- and diastereoselective hydrometalation aldehyde addition sequence to assemble the fully functionalized ansa chain of the natural products. Also documented is an intramolecular copper-mediated amidation reaction to close the 19-membered macrolactams. The amidation reaction was also employed for the generation of structural derivatives (6−9) of phenolic ansamycins. Ansamycin natural products and selected structural analogues were evaluated in a competitive binding assay to breast cancer cell lysate and a cytotoxicity assay. Both reblastatin (1) and autolytimycin (2) were shown to bind the heat shock protein 90 with enhanced binding activity (∼25 nM) than 17-allylamino-17-demethoxygeldanamycin (17-AAG, 4), a geldanamycin (3) derivative currently under evaluation for treatment of cancer (∼100 nM)

Synthesis of Reblastatin, Autolytimycin, and Non-Benzoquinone Analogues: Potent Inhibitors of Heat Shock Protein 90

A full account of an asymmetric synthesis of reblastatin (1) and the first total synthesis of autolytimycin (2) and related structural compounds is described. The syntheses expand the utility of a highly regio- and diastereoselective hydrometalation aldehyde addition sequence to assemble the fully functionalized ansa chain of the natural products. Also documented is an intramolecular copper-mediated amidation reaction to close the 19-membered macrolactams. The amidation reaction was also employed for the generation of structural derivatives (6−9) of phenolic ansamycins. Ansamycin natural products and selected structural analogues were evaluated in a competitive binding assay to breast cancer cell lysate and a cytotoxicity assay. Both reblastatin (1) and autolytimycin (2) were shown to bind the heat shock protein 90 with enhanced binding activity (∼25 nM) than 17-allylamino-17-demethoxygeldanamycin (17-AAG, 4), a geldanamycin (3) derivative currently under evaluation for treatment of cancer (∼100 nM)

A Purine Analog Synergizes with Chloroquine (CQ) by Targeting <i>Plasmodium falciparum</i> Hsp90 (PfHsp90)

<div><p>Background</p><p>Drug resistance, absence of an effective vaccine, and inadequate public health measures are major impediments to controlling <i>Plasmodium falciparum</i> malaria worldwide. The development of antimalarials to which resistance is less likely is paramount. To this end, we have exploited the chaperone function of <i>P. falciparum</i> Hsp90 (PfHsp90) that serves to facilitate the expression of resistance determinants.</p><p>Methods</p><p>The affinity and activity of a purine analogue Hsp90 inhibitor (PU-H71) on PfHsp90 was determined using surface plasmon resonance (SPR) studies and an ATPase activity assay, respectively. In vitro, antimalarial activity was quantified using flow cytometry. Interactors of PfHsp90 were determined by LC-MS/MS. <i>In vivo</i> studies were conducted using the <i>Plasmodium berghei</i> infection mouse model.</p><p>Results</p><p>PU-H71 exhibited antimalarial activity in the nanomolar range, displayed synergistic activity with chloroquine <i>in vitro</i>. Affinity studies reveal that the PfHsp90 interacts either directly or indirectly with the <i>P. falciparum</i> chloroquine resistance transporter (PfCRT) responsible for chloroquine resistance. PU-H71 synergized with chloroquine in the <i>P.berghei</i> mouse model of malaria to reduce parasitemia and improve survival.</p><p>Conclusions</p><p>We propose that the interaction of PfHsp90 with PfCRT may account for the observed antimalarial synergy and that PU-H71 is an effective adjunct for combination therapy.</p></div

Biochemical evaluation of the affinity of PU-H71 for PfHsp90.

<p>(A) Illustration of geldanamycin (GA, left) and PU-H71 (right) docked within the ATP-binding site of PfHsp90. The models were generated using the PfHsp90 crystal structure (PDB ID: 3K60) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075446#pone.0075446-Corbett1" target="_blank">[45]</a>. By convention, the electrostatic potential surface in the background denotes acidic residues in red and basic residues in blue. (B) Surface plasmon resonance (SPR) measurements for PU-H71 binding to the ATP-binding domain of PfHsp90. The colors on the sensorgrams represent varying concentrations of the respective drug (5–1000 µM) injected over the surface with the immobilized PfHsp90. The steady state responses were fitted using non-linear regression to a single binding site model (as shown on the right) to obtain the K_d value indicated. The number in parentheses represents standard error on the K_d obtained from fits. The sensorgrams shown have been double referenced as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075446#pone.0075446-Zhao1" target="_blank">[34]</a>. (C) 17-AAG was used as a positive control drug. (D) SPR measurements for PU-H71 binding of the R98K mutant ATP-binding domain of PfHsp90. (E) Full-length PfHsp90 was expressed and ATPase activity tested in the presence of PU-H71. Results are shown as a percentage of total ATPase activity in the absence of drug and IC₅₀ indicated (511 nM) for a single experiment. Positive control drug treatments included radicicol (144 nM) and 17-AAG (146 nM). The inset shows the logarithmic curve fitting of ATPase activity with increasing concentration of PU-H71.</p

Assessment of the <i>in vitro</i> antimalarial activity of PU-H71.

<p>(A) Antimalarial activity of PU-H71 using a standard SYBR Green cell-based assay using the laboratory strain 3D7 resulted in an IC₅₀ of 89.4±15.3 (SEM) nM (n = 3, performed in duplicate). A single representative experiment performed in duplicate is depicted here resulting in a IC₅₀ of 111±8 nM (SD). Antimalarial activity of PU-H71 using the strain W2 resulted in an IC₅₀ of 94.3±6.3 (SEM) nM. (B) PU-H71 acts synergistically with chloroquine (CQ) in a CQ sensitive strain (3D7). Synergistic activity was defined by calculations of the average sum fractional inhibitory concentration ratio (FIC) FIC₅₀ and by the FIC₅₀ ratios lying under the line corresponding to additive activity. (C) PU-H71 acts synergistically with chloroquine (CQ) and potentiates CQ activity in the CQ resistant strain W2. The dots on the isobolograms represent the average of technical duplicates. The calculations for the FIC ratios are enclosed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075446#pone.0075446.s002" target="_blank">Table S2</a>. The response modification index (RMI) was calculated for each of the doses tested in both 3D7 and W2 strains and has been summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075446#pone-0075446-t001" target="_blank">Table 1</a>.</p

Summary of the drug combinations tested in the <i>P.berghei</i> mouse model trials.

<p>Summary of the drug combinations tested in the <i>P.berghei</i> mouse model trials.</p

Synergy studies conducted with current antimalarials used in malaria endemic regions in the CQ sensitive strain 3D7.

*<p>Shaded areas indicate the concentration of the drug that was kept constant, while the combination drug concentration was varied to determine the corresponding IC50.</p

The effect of intra-peritoneal PU-H71 administration on the <i>Plasmodium berghei</i> mouse model.

<p>Mice were infected on day 0 and treatment was started on day 5 at a parasitemia of 1%. (A–D) Average parasitemia levels in response to drug treatments alone or in combination with CQ are indicated (n = 4 animals). The asterix (*) identifies significant differences in parasitemia relative to the vehicle control (A) and PU-H71 treatment alone (B–D) (Student’s t-test p≤0.05). The double asterix (**) indicates significant differences in parasitemia relative to the CQ treatment alone (Student’s t-test p≤0.05). (E-H) Kaplan-Meier survival plots for PU-H71 alone or in combination with CQ versus placebo.</p

A step-wise selection method obtained the PfHsp90 Thr163Pro mutant (strain Dd2).

<p>(A) The Thr163Pro mutation is depicted computationally in the crystal structure of the PfHsp90 ATP-binding domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075446#pone.0075446-Corbett1" target="_blank">[45]</a>. (B) The presence of this mutation resulted in doubling of the PU-H71 IC₅₀ in the cell-based assay (a single representative experiment performed in duplicate is shown). (C) Synergy between PU-H71 and CQ in the Thr163Pro mutant was still observed based on sum FIC ratio ≤0.5. The inset provides a magnified view of the FIC₅₀ ratios for the different PU-H71 and CQ drug combinations in the mutant strain.</p

Summary of response modification indexes (RMI) for various combinations of PU-H71 and chloroquine <i>in vitro</i> based on the checkerboard assay.

*<p>A response modification index (RMI) of ≈1 correspond with no effect of one drug on another when used in combination; RMI <<1 potentiation of antimalarial activity (ie. synergistic activity); RMI>>1 antagonistic activity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075446#pone.0075446-Oduola1" target="_blank">[38]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075446#pone.0075446-Pereira1" target="_blank">[39]</a>. Standard error of the mean is indicated.</p