Human immunodeficiency virus type 1 (HIV-1) is the causative agent of Acquired Immunodeficiency Syndrome (AIDS). Current therapies are capable of controlling viral infection but do not represent a definitive cure. The virus has proven to be capable of developing resistance to therapy, evading the immune response, altering cellular immune function and protecting infected cells from apoptosis. HIV-1 is inherently capable of accomplishing these functions with a limited genome that expresses only nine proteins. As such, the HIV-1 must make extensive use of cellular pathways and subvert native molecular processes for its own purposes.
Expression of the HIV-1 proviral genome requires host cell transcription factors as well as the Tat viral transactivator (reviewed in [1–3]). Tat stimulates formation of full-length transcripts from the HIV-1 promoter [4, 5] by promoting efficient transcriptional elongation (reviewed in [1, 2]). Tat interacts with the bulge of the transactivation response element (TAR) RNA, a hairpin-loop structure at the 5'-end of all nascent viral transcripts [6–9]. Full functional activity of Tat requires host cell cofactors, which interacts with the loop of TAR RNA hairpin (reviewed in [1, 2]) as well as other site on the LTR. Tat also associates with a protein kinase that phosphorylates the C-terminal domain (CTD) of RNA Polymerase II (RNA Pol II) called Tat associated kinase (TAK) . The CTD consists of heptapeptide repeats, Tyr1-Ser2-Pro3-Tyr4-Ser5-Pro6-Ser7, which are phosphorylated on serine 2 (Ser-2) and serine 5 (Ser-5) during transcription [11, 12]. Recently, serine 7 (Ser-7) has been shown to be phosphorylated by cdk7 [13, 14]. Previously, it has also been shown that partially purified TAK and the loop-binding factor represent the same protein complex, cdk9/cyclin T1 [15–17]. Tat associates with cdk9 [16, 17] through interaction with cyclin T1 which interacts with the TAR RNA loop structure . Tat interacts with human cyclin T1 through a critical cysteine and the presence of a different amino acid in this position in rodent cells renders Tat transactivation inefficient [18, 19]. In an in vitro transcription system, Tat stimulates additional phosphorylation of the hyperphosphorylated RNA Pol II . In kinase assays, Tat induces phosphorylation of CTD by cdk9, which requires the N-terminal domain (amino acids 1 to 48) and the arginine-rich motif (amino acids 49-57) of Tat . Tat may also induce TFIIH-associated cdk7 to phosphorylate Ser-5 in the pre-initiation complex [22, 23]. Subsequently, TFIIH dissociates from the preinitiation complex and this dissociation relieves inhibition of cdk9 autophosphorylation , which is required for efficient binding of cdk9/cyclin T1 to TAR RNA .
Recently, a growing body of evidence has indicated the role of yet another cyclin/cdk complex, namely cyclin E/cdk2, in Tat activated transcription. Cyclin E/cdk2 is the major cyclin/cdk complex whose maximal activity is observed at the late G1/S boundary. Cyclin E/cdk2 has been shown to be important in the transition of G1/S by regulating the release of Rb sequestered factors, including E2F . Given the importance that the G1/S checkpoint plays in viral replication, it is not surprising that HIV-1 viral proteins, like Tat, have been shown to modulate G1/S activity. From our own studies, we have observed the increased kinase activity of cyclin E/cdk2 complexes in HIV-1 latently infected cells due to the loss of the natural cdk inhibitor p21/waf1 . Cdk inhibitor p21/waf1 is normally induced by p53 upon cellular stress and regulates the G1/S transition by inhibiting the activity of cyclin/cdk complexes. Studies from our lab have shown that HIV-1 latently infected T cells do not induce expression of p21/waf1 after injury to the host cell. For instance, flow cytometric analysis revealed that upon γ-irradiation, these cells proceeded into the S phase and apoptosed. The lack of p21/waf1 expression was attributed to the physical and functional interaction of Tat with p53, resulting in the inactivation of p53 [26, 27]. To further validate the significance of the G1/S and cdk2 in HIV-1 transcription in vivo, HLM-1 cells (HIV-1+/Tat-), were first transfected with wild type Tat and were subsequently blocked with either hydroxyurea (a general G1/S blocker) or nocodazole (a general M phase blocker). Supernatants were collected every third day and analyzed for the presence of the gag/p24 antigen. HIV-1 attained peak viral replication between days 9 and 12 for those cells blocked with nocodazole, while G1/S blockage by hydroxyurea resulted in the dramatic inhibition of virion production . Collectively, these studies pointed to two important findings. One, that HIV-1 in latently infected cells down modulates the natural cdk inhibitor p21/waf1 (i.e., by Tat binding to p53 and/or other related mechanisms), and in turn is able to control the primary cdk target such as cyclin E/cdk2 complex, and second, that G1/S kinases, such as cdk2/cyclin E, could be targeted for inhibition of HIV-1 replication using drugs that mimic the natural cdk inhibitors.
Over the past few years, pharmacological cdk inhibitors (PCIs) have been reported to prevent viral replication in vitro . The underlying mechanism of action, inhibition of cellular rather than viral targets, is unlikely to favor the appearance of resistant strains and could potentially be efficient against several unrelated viruses. Numerous viruses require active cdks for their replication and some viruses actually encode their own cyclins, thereby regulating their host cell cycle . Cdks are required for replication of viruses that multiply only in dividing cells, such as adeno- and papillomaviruses. Recently, cdks have also been shown to be required for the replication of viruses that multiply in non-dividing cells, such as HIV-1 and herpes simplex virus types 1 and 2 (HSV-1 and -2) [31, 32]. In these experiments PCIs were shown to have potent antiviral activity in vitro against HIV-1, HSV-1 and -2, human cytomegalovirus, varicella-zoster virus, and to inhibit specific functions of other viruses . Since two PCIs, flavopiridol and roscovitine, have been proven to be non-toxic in human clinical trials against cancer , PCIs, therefore may be useful as antivirals. As significant advantage of PCI are its activity against many viruses, including drug-resistant strains of HIV-1 and HSV-1 [35, 36]. Furthermore, the antiviral effects of a PCI and a conventional antiviral drug could have an additive effect. Roscovitine is the second-best-studied PCI in vivo (after flavopiridol) and it has proven non-toxic in several animal models [37, 38]. The purified r-enantiomer of roscovitine (cyc202) has entered human clinical trials. In phase I clinical trials, r-roscovitine has proven to be orally bioavailable and to have no acute toxicity .
Other class of inhibitors including paullones represents a novel class of small molecule cdk inhibitors. Paullones constitute a new family of benzazepinones with promising antitumoral properties. They were described as potent, ATP-competitive, inhibitors of the cell cycle regulating cdks . Alsterpaullone, the most active paullone, was demonstrated to act by competing with ATP for binding to GSK-3β. Alsterpaullone inhibits the phosphorylation of tau in vivo at sites which are typically phosphorylated by GSK-3β in Alzheimer's disease . Alsterpaullone also inhibits the cdk5/p35-dependent phosphorylation of DARPP-32 in mouse striatum slices in vitro . This dual specificity of paullones may turn these compounds into very useful tools for the study and possibly treatment of neurodegenerative and proliferative disorders . Replacement of the 9-bromo substituent of kenpaullone by a 9-cyano or 9-nitro group produced a substantial increase in enzyme-inhibiting potency . Interestingly, alsterpaullone has been selected for preclinical development in a NCI program .
In this study, we identified alsterpaullone having a potent inhibitory effect on HIV-1 infected cells. Its mechanism of action has previously been attributed to inhibition of cdk2/cyclin A complex at the G1/S as well as few other kinases. Here, the primary mode of the inhibition in infected cells appears to be at the protein levels of cyclins which ultimately result in apoptosis of HIV-1 infected cells. Finally, low concentration of two drugs combined, alsterpaullone and r-roscovitine, favor inhibition of the HIV-1 transcription in primary cells.