we screened 
 a collection of sponge- and marine fungi - derived extracts 
 ( n = 2500 ) and pure compounds ( n = 400 ) using a primary cell - based in vitro model of hiv-1 latency 
 that is re - engineered for optimal sensitivity and miniaturized for 
 high - throughput screening .
two hits were initially 
 given high priority for further investigation : ( 1 ) the extract of 
 a sponge putatively identified as pseudoceratina purpurea ( ucsc coll .
06135 ) , based on morphological similarity to previously 
 identified sponges , and ( 2 ) the extract of a marine - derived fungus 
 that was not taxonomically identified ( ucsc strain no .
briefly , 
 the known compounds psammaplin a and 
 apicidin , shown in figure 1 , were isolated from the sponge and fungal extracts , respectively , 
 and retested to show significant potencies ( ec50 = 0.2 
 m and ec50 = 0.3 m , respectively ) .
a critical 
 first step in these outcomes was the application of our recently published 
 method for natural product peak library fractionation , whereby hplc coupled with ms and evaporative 
 light scattering detection ( elsd ) maps the distribution of metabolites 
 into biological screening wells as a function of retention time ( see experimental section and figure s1 , supporting information ) .
subsequent operations 
 involved scale - up isolation via peak libraries annotated with m / z ms data and bioassay hit information . 

apicidin has previously been shown to function as an hiv-1 reactivator 
 via an hdaci mechanism .
the successful 
 workflow described above was employed to investigate 
 the active extract of a marine - derived humicola fuscoatra ( ucsc strain no .
108111a ) , whose peak library elsd profile is shown 
 in figure 2 , panel a. the major component ( tr = 2728 min ) showed a positive response 
 in the latent hiv-1 reactivation assay . the mass spectrum ( esitof+ ) 
 of this analyte displayed a chlorinated ion at m / z = 387.1:389.1 ( 3:1 intensity , figure 2 , panel b ) , which we duly assigned as the sodiated molecular 
 ion [ m + na ] in conjunction with an ancillary [ m + h ] signal observed at m / z 365.1 . 

isolation of this active material ( ec50 = 9.1 m ) 
 was accomplished by retrieving a pure sample from an archived library 
 plate , and an accurate mass was measured ( esitof+ ) as 387.0637 ( [ m 
 + na ] ) , consistent with the molecular formula of c18h17clo6 , un = 10 . conducting a literature 
 search based upon taxonomy and this molecular formula yielded radicicol 
 ( 1 ) , a well - known fungal 
 metabolite and potent in vitro hsp90 inhibitor , as a solitary dereplication hit .
indeed , the h nmr spectrum of the active extract ( figure 2 , panel c ) was dominated by the resonances of this metabolite .
additionally , 
 the specific rotation of + 203 for our sample from h. fuscoatra matched the literature value for 1 ( + 216 ) ; therefore , we assigned the absolute configuration 
 of the three asymmetric carbon atoms in 1 as all r , based on a previously reported single - crystal xrd assignment . analytical data for the 
 active extract of humicola fuscoatra ( ucsc strain 
 no .
annotations 
 indicate the distribution of compounds 17 ( see experimental section for elution 
 conditions ) .
( b ) esitof(+ ) m / z data 
 collected during peak library fractionation .
expansion of [ m + na ] ion for radicicol ( 1 ) , at tr = 27 min ( see panel a ) . ( c ) 600 mhz h nmr 
 spectrum in acetone - d6 .
the expanded region shows the aromatic singlet of 
 the most abundant minor congener , compound 4 .
we were inspired to further mine the extract 
 for radicicol congeners by two pieces of information : ( 1 ) the h nmr spectrum shown in figure 2 , panel 
 c , displayed a characteristic aromatic singlet of considerably less 
 intensity than that of the major metabolite , and ( 2 ) the mass chromatogram 
 generated during the peak library fractionation was rich with chlorinated 
 ions in the region of 2026 min ( figure 2 , panel a ) .
successive rounds of ms and nmr - guided hplc purification 
 yielded minor quantities of several additional radicicol congeners : 
 three known compounds , pochonins b ( 2 ) , c18h19clo7 , c ( 3 ) , c18h18cl2o6 , and n ( 4 ) , c18h21clo7 , and three 
 new natural products , radicicols b  d ( 57 ) . the molecular formulas and nmr properties of the known 
 compounds
radicicol b ( 5 , c18h19clo7 , un = 9 ) contains an additional molecule of h2o and one less degree of unsaturation relative to radicicol 
 ( 1 ) .
the h and c nmr data of 5 closely matched those of 1 with the following 
 exceptions : ( i ) the h-4 and h-5 proton resonances of 1 were transposed downfield from h 3.05 and 3.32 
 to h 3.62 and 4.50 , respectively , and ( ii ) the c-4 
 and c-5 carbon resonances of 1 were transposed downfield 
 from c 56.1 and 55.8 to c 71.9 
 and 69.4 , respectively . taken together with the gcosy and ghmbc correlations 
 shown in figure 3 , the data suggest the epoxide 
 of 1 is a 1,2-diol in 5 .
the planar structure 
 of 5 is identical to semisynthetic epoxide - opened diasteromers 
 of natural radicicol described in both patent and peer - reviewed literature .
the configuration of 
 position c-2 is proposed to be analogous to that of radicicol ( 1 ) , and the anti relative configuration for the 1,2-diol of 5 is assigned based on a vicinal proton coupling constant 
 of j4,5 = 5.4 hz , which matched the reported 
 value of the synthetic anti diastereomer ( lit.j4,5 = 6.1 hz ) .
the molecular 
 formula of radicicol c ( 6 , c18h21clo8 , un = 8) contains two additional h2o molecules 
 in comparison to 1 . the c nmr data ( table 1 ) matched those of 1 for all positions , 
 apart from c-3 to c-10 , and suggested 
 the two equivalents of h2o were positioned in the macrocyclic 
 chain . with the aromatic ring functionality and macrocyclic ester 
 linkage deemed intact , the two remaining degrees of unsaturation were 
 assigned to resonances of a ketone ( c 204.1 ) and 
 a trans double bond ( c 135.7 and 131.7 ; h 5.47 and 5.39 , jhh = 15.6 hz ) .
additionally , three secondary alcohols were detected 
 at h 3.60 , 3.93 , and 4.27 ( c 78.7 , 
 69.1 , and 70.2 ) .
a single extended spin system in the gcosy spectrum 
 was delineated from h3 - 1 to h2 - 9 , and long - range 
 ghmbc correlations finalized the planar structure of 6 as shown in figure 3 .
the stereochemistry 
 of position c-2 is proposed to be analogous to that of radicicol ( 1 ) ; however due to a lack of both epoxide and ,,,-unsaturated 
 ketone functionalities , radicicol c ( 6 ) is too flexible 
 to assign the relative configurations of the three secondary alcohols 
 by noesy correlation spectroscopy .
radicicol d ( 7 ) was assigned the same molecular formula 
 as 2 and 5 based on accurate mass measurement 
 and required nine degrees of unsaturation .
comparison of c chemical shifts to those of 1 indicated the aromatic 
 ring functionality and macrocyclic ester linkage remained intact , 
 and positions c-4 to c-10 were modified .
two of the three remaining 
 degrees of unsaturation were assigned to a ketone detected at c 203.7 and a cis double bond at c 129.9 
 and 131.6 ( h 5.98 and 6.09 , jhh = 6.2 hz ) .
additionally , three oxygenated 
 carbons were detected at c 72.3 , 84.1 , and 89.7 . 

the connectivity from positions 1 through 9 was established from the 
 gcosy and ghmbc data ( figure 3 ) , and on the 
 basis of the vicinal coupling constant of 6.2 hz between olefinic 
 protons h-6 and h-7 , the final unit of unsaturation was assigned to 
 a 2,5-disubstituted-2,5-dihydrofuran ring system .
the stereochemistry 
 of position c-2 in radicicol d ( 7 ) is proposed to be 
 analogous to that of radicicol ( 1 ) ; the relative configuration 
 of positions 4 , 5 , and 8 could not be assigned using data sets collected 
 in this study .
the possibility that radicicols b  d ( 57 ) are artifacts produced during the 
 isolation process was 
 ruled out , as they were detected in the lcms analysis of the original 
 etoac extract .
furthermore , compounds 57 were not detected by lcms or h nmr after prolonged 
 exposure of both natural ( 1 ) and commercial ( sigma ) radicicol 
 samples to the isolation conditions .
selected gcosy ( bold ) and ghmbc ( arrow ) 
 correlations for radicicols 
 b  d ( 57 ) .
reported values include % reactivation 
 relative to saha and potency ( ec50 in m ) .
the screening data obtained in this 
 study are summarized in table 2 , and some of 
 the patterns deserve additional discussion . 

radicicol ( 1 ) was isolated as the most abundant metabolite 
 of h. fuscoatra and exhibited 98% reactivation efficiency 
 of latent hiv-1 relative to the tool compound saha ( figure 4 ) and an ec50 of 9.1 m .
radicicol 
 activation of hiv-1 was confirmed by qpcr of hiv-1 rna in the same 
 latency model ( see supplementary experimental 
 procedures and figure s15 , supporting information ) .
of the 
 additional congeners 27 , isolated 
 from the fungal extract , pochonins b ( 2 ) and c ( 3 ) and radicicol b ( 5 ) were reactivators ( figure 4 ) , while pochonin n ( 4 ) and radicicols 
 c ( 6 ) and d ( 9 ) were found to be inactive . 

comparison of the structures of the active compounds suggests the 
 epoxide functionality is not required for activation in our assay ; 
 however , the active compounds all contain a michael acceptor functionality , 
 something the inactive compounds 4 , 6 , and 7 do not possess . while the potencies in reactivating latent 
 hiv-1 for the active radicicols and pochonins are lower than that 
 of romidepsin , it appears that their mechanism of action is unique 
 and remains to be elucidated .
indeed , radicicol was previously reported 
 to lack in vitro activity against histone deacetylase .
although radicicol is a potent in vitro hsp90 
 inhibitor ( ic50 = 19 nm ) , a 
 structurally unrelated hsp90 inhibitor , geldanamycin , is inactive 
 in our assay , indicating that the mechanism of hiv-1 reactivation 
 by 1 is unlikely to involve hsp90 inhibition .
in addition , 
 the most potent analogue , pochonin c ( 3 ) , is unlikely 
 to act as an hsp90 inhibitor based on the conformational hypothesis 
 advanced by winssinger : that hsp90 inhibition by radicicol - type resorcylides 
 is closely tied to a specific bioactive molecular topology attenuated 
 by modifications at the epoxide moiety .
finally , radicicol 
 did not induce detectable nfkb reporter gene activation in jurkat 
 cells ( see supplementary experimental procedures 
 and figure s15 , supporting information ) , indicating it reactivates 
 latent hiv-1 by a pkc - independent mechanism . in vitro latent hiv-1 
 reactivation assay : dose 
response 
 profiles of radicicol ( 1 ) , pochonin b ( 2 ) pochonin c ( 3 ) , and radicicol b ( 5 ) relative 
 to the maximum activation of the positive control saha .
. 
 a screening paradigm has been created consisting of a sensitive and 
 high - throughput primary cell - based hiv-1 latency assay that was well 
 suited to investigate diverse marine natural product collections for 
 novel reactivators of latent hiv-1 .
the assay was robust with highly 
 reproducible ec50 values for reference compound saha in 
 all donors used in this screen .
starting from an extract of the marine - derived 
 fungus h. fuscoatra and followed by successive rounds 
 of purification , dereplication , and bioassay , we isolated seven compounds 
 in the radicicol / pochonin class , of which four display latent hiv-1 
 reactivation activity , providing a preliminary understanding of the 
 structure  activity relationship of these compounds . at this 
 stage
we believe that the new hiv-1 reactivators summarized in table 2 can be either the seeds for further scaffold modification 
 or useful in combination with agents possessing a distinct mechanism 
 of action , such as romidepsin , to achieve a higher magnitude of latent 
 hiv-1 reactivation .
standard pulse sequences 
 were used for all nmr experiments , which were run on either a varian 
 unity inova spectrometer ( 600 and 150 mhz for h and c , respectively ) equipped with a 5 mm triple resonance ( hcn ) 
 cold probe or a varian spectrometer ( 500 and 125 mhz for h and c , respectively ) equipped with an inverse detection 
 probe .
residual solvent shifts for acetone - d6 were referenced to h 2.05 and c 29.84 , respectively .
accurate mass measurements were obtained 
 on a mariner esitof instrument for molecular formula determinations . 

all chromatographic work was done in reversed - phase and utilized hplc 
 grade ch3cn ( solvent a ) and milli - q h2o ( solvent 
 b ) , both adjusted to contain 0.1% formic acid .
the analytical lcms 
 system was composed of waters hplc components ( i.e. , solvent pumps 
 and autosampler ) and controlled by empower software .
a 150  
 4.60 mm 5 m luna c18 column ( phenomenex ) was utilized , 
 and the system operated at a flow rate of 1 ml / min .
the postcolumn 
 flow of the eluent was first through a photodiode array ( waters ) and 
 then split ( 1:1 ) between an evaporative light - scattering detector 
 ( sedex model 55 ) and an esitof mass spectrometer ( applied biosystems 
 mariner ) .
the semipreparative hplc system was composed of waters hplc 
 components ( i.e. , solvent pumps and gradient controller ) and equipped 
 with a 250  10 mm 5 m luna c18 column ( phenomenex ) 
 and tunable uv - absorbance detector ( waters ) . the humicola fuscoatra strain ( ucsc coll .
108111a ) 
 was isolated from sediment collected 
 at <33 m depth by scuba in tutuila , american samoa ( s 1416.600 , 
 w 17036.719 ) , in 2010 .
the strain was taxonomically identified 
 by molecular ( 100% by its and d1/d2 regions of rdna ) and morphological 
 methods at the university of texas fungus testing laboratory .
the 125 ml and 5 l cultures of 108111a 
 were grown in media that consisted of the following : cornmeal ( 4.5 
 g/150 ml ) and 5xasw ( 200
after autoclave sterilization , the cultures 
 were inoculated and shaken at 75 rpm for 21 days at ambient temperature . at the time of harvest ,
the 
 fungal cultures ( 5  1 l ) were homogenized , and the liquid was 
 extracted three times with equal volumes of etoac .
the combined organic 
 extracts were concentrated in vacuo to yield 2 g of oil .
a portion 
 of the 108111a cma extract ( 194.9 mg ) was separated into 15 fractions 
 ( fraction codes h1h15 , figure s2 ) using the semipreparative hplc system utilizing a flow rate of 
 3 ml / min , 310 nm detection , and the following elution conditions : 
 15 min isocratic 25:75 , 25 min gradient from 25:75 to 70:30 , 3 min 
 gradient from 70:30 to 100:0 , 10 min isocratic 100:0 , and 3 min gradient 
 from 100:0 to 25:75 .
fraction h5 was pochonin n ( 4 , 5.0 
 mg ) , fraction h9 was pochonin b ( 2,1.8 mg ) , and fraction 
 h9 was pochonin c ( 3 , 1.9 mg ) .
fraction h12 was semipure 
 radicicol , and an additional run under identical hplc conditions yielded 
 pure radicicol ( 1 , 11.2 mg ) and three new analogues : 
 radicicol b ( 5 , 1.2 mg ) , radicicol c ( 6 , 
 2.2 mg ) , and radicicol d ( 7 , 1.0 mg ) .
the instrumentation 
 setup for our natural product peak library generation has been recently 
 published .
the hplc stage utilized two 
 waters 510 pumps and a waters 717plus autosampler , both controlled 
 with empower 2 software .
separation was performed on a 250  
 10 mm 5 m luna c18 column ( phenomenex ) .
spectra 
 from three detectors were acquired during peak library fractionation : 
 waters 996 photo diode array , sedex 55 elsd , and mariner 5054 esi - tof - ms . 

the mobile phase parameters are ch3cn ( a ) and h2o ( b ) with a flow rate of 2 ml / min and the following elution conditions : 
 30 min gradient from 10:90 , 10 min isocratic 100:0 , 1 min gradient 
 from 100:0 to 10:90 , 9 min isocratic 10:90 .
sample collection was performed using a gilson 
 215 liquid handler controlled with gilson unipoint lc software .
samples 
 were collected into bd biosciences 96-deep - well plates , with a working 
 volume of 2 ml ( part number : 353966 ) .
after the lc - ms - uv - elsd library is collected , a duplicate 
 archive plate is generated for analytical reference using a 12-channel 
 pipet , creating an exact copy and counter balance for centrifugal 
 drying .
[ ]d + 216 ( c 1.00 , chloroform ) ; uv , h nmr , c nmr , and 
 hresitofms data were in accordance with reported values .
[ ]d + 20 ( c 1.0 , acetone ) ; uv , h nmr , c nmr , and hresitofms 
 data were in accordance with reported values .
( )-2r,4s,5r [ ]d 68.3 
 ( c 0.06 , chloroform ) ; uv , h nmr , c nmr , and hresitofms data were in accordance with reported 
 values .
[ ]d 77.3 ( c 1.00 , methanol ) ; uv , h nmr , c 
 nmr , and hresitofms data were in accordance with reported values .
[ ]d + 32 ( c 0.8 , methanol ) ; uv ( lc 
 eluent 
 h2o / ch3cn / formic acid , 1:1:0.1 ) max ( au ) 219 ( 0.87 ) , 276 ( 0.60 ) nm ; h and c nmr data , see table 1 ; hresitofms m / z 405.0739 [ m + na ] ( calcd 
 for c18h19clo7na , 405.0712 ) .
[ ]d 69 
 ( c 1.0 , methanol ) ; uv ( lc 
 eluent h2o / ch3cn / formic acid , 1:1:0.1 ) max ( au ) 219 ( 0.94 ) 262 ( 0.36 ) 309 ( 0.20 ) nm ; h 
 and c nmr data , see table 1 ; hresitofms m / z 423.0826 [ m + na ] ( calcd 
 for c18h21clo8na , 423.0817 ) .
[ ]d 53 
 ( c 0.4 , methanol ) ; uv ( lc 
 eluent h2o / ch3cn / formic acid , 1:1:0.1 ) max ( au ) 217 ( 0.48 ) 257 ( 0.12 ) 297 ( 0.08 ) nm ; h 
 and c nmr data , see table 1 ; hresitofms m / z 405.0668 [ m + na ] ( calcd 
 for c18h19clo7na , 405.0712 ) . an nl4 - 3-based vector with frame - shift 
 mutations in vpr and env genes
was 
 engineered with a codon - optimized firefly luciferase gene in place 
 of nef to generate the resulting construct pks13 . 

the nl4 - 3-luc virus was generated by co - transfection of hek-293 t cells 
 with pks13 and a plasmid containing the hiv-1 env gene using lipofectamine 2000 ( life technologies ) .
briefly , naive cd4 + t cells were purified 
 by negative selection using pbmcs from healthy hiv-1 negative donors . 

purified naive cd4 + t cells were activated by incubation with anti - cd3/cd28 
 magnetic dynabeads ( 1 bead:2 cells ratio , life technologies ) , 1 mg / ml 
 anti - il-4 ( r&d systems ) , 2 mg / ml anti - il-12p70 antibodies ( r&d 
 systems ) , and 10 ng / ml tgf- ( r&d systems ) for 3 days .
cells 
 were maintained in 30 u / ml il-2 ( life technologies ) for 2 days followed 
 by infection with nl4 - 3-luc .
seven days postinfection , 20 l 
 of latently infected cells at 10  000 cells / well was dispensed 
 into 384-well plates containing 350 nl of compound solutions in 50 
 l of culture medium .
after a 48 h incubation , 40 l / well 
 briteglo ( promega ) was added , and luminescence measured using the 
 envision plate reader ( perkin - elmer ) .
 an extract of humicola fuscoatra ( ucsc strain 
 no . 
 108111a ) was shown to reactivate latent hiv-1 expression in an 
 in vitro model of central memory cd4 + t cells . 
 we report the bioassay - guided 
 isolation and structure determination of several resorcyclic acid 
 lactones , including four known compounds , radicicol ( 1 , aka . 
 monorden ) and pochonins b ( 2 ) , c ( 3 ) , and n ( 4 ) , and three new analogues , radicicols b  d 
 ( 57 ) . 
 compounds 13 and 5 showed moderate activities in the memory 
 t cell model of hiv-1 latency . 
 radicicol ( 1 ) displayed 
 lower potency in reactivating latent hiv-1 ( ec50 = 9.1 
 m ) relative to the hdac inhibitors apicidin ( ec50 = 0.3 m ) , romidepsin ( ec50 = 0.003 m ) , 
 and saha ( ec50 = 0.6 m ) ; however , it achieved equivalent 
 maximum efficacy relative to the positive control compounds ( 98% of 
 saha and romidepsin ) . 
Results and Discussion
Experimental Section