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Using small molecules to idenf y cel lu a k n es im or tant fo d l r i as p r engue virus infecon
We are interested in using small molecules of known biological funcon to id y ho st factors enf required by dengue virus. Small molecules that inhibit (or enhance) dengue virus replicaon can be used to probe viral processes in vitro and potenal ly in vivo as well. Protein kinases provided an opm l system fo a yi n thi s chem cal b o o strat eg y. First, a r ppl g i i l gy protein kinases play central roles in intracellular signal transducon and regul a e many t n, etc. Second, protein kinases have been widely studied, providing us with many biochemical tools on the heavy investment by both industry and academia in developing small molecule kinase inhibitors that are highly selecve, p ent , c l p m abl e o al ly b o ot el er e , r i avai la e a d non-toxic. bl , n We assembled a library of ~120 inhibitors of ~40 different Ser/Thr and Tyr kinases. The library e. We screened this inhibitor library in an image-based assay in which we used immunofluorescent staining for dengue envelope protein (Env) as a marker of dengue virus infecon.

We then used viral plaque assays to measure the effect of "hits" from the primary screen on viral t er s in vitro. We conducted these experiments in two modes, "pre-inoculaon" m de o and "post-inoculaon" m de to id y kina e i ni bit o th m h be ac on s t e o enf s h rs at ig t ng ps in the viral life cycle (i.e., entry) versus those that act later in the viral life cycle. early

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From this original screen, we idenfied sever al in b to s o Sr c and A ki n es th t that hi i r f bl as a

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Home | Yang Lab | Harvard Medical School http://yanglab.html Using a combinaon o b o hem cal a vi ro o cal m t h .harvard. Likewise. Src inhibitors appear to prevent viral assembly. the Src inhibitors do not appear to block dengue virus genome replicaon si n e com a rable c p levels of dengue virus plus-stranded RNA accumulate in dengue-infected cells in the presence and absence of Src inhibitors (B) and since we can detect vesicle packets. c y ctro i s Rather.med. w h f i c i nd l gi e ods e ave d er m n et i ned that Src inhibitors like AZD0530 and dasani b d n in b t d o ot hi i engue v ral g i ene e es si o ( A) because xpr n dengue Env protein is expressed in a subset of cells in the presence of Src inhbitors. as evidenced by the failure to detect mature viral parc l e b scanni n e e n m cro copy ( C). s y g l ctro i s 2 of 3 10/8/2011 10:06 AM .edu/inhibitors. the structures in which flavivirus genome replicaon is thought to o cur . b el e n m cro copy ( C. right).

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