Amantadine inhibits the M2 proton channel of influenza A virus, yet most of the currently circulating strains of the virus carry mutations in the M2 protein that render the virus amantadine-resistant. While most of the research on novel amantadine analogues has revolved around the synthesis of novel adamantane derivatives, we have recently found that other polycyclic scaffolds effectively block the M2 proton channel, including amantadine-resistant mutant channels. In this work, we have synthesized and characterized a series of pyrrolidine derivatives designed as analogues of amantadine. Inhibition of the wild-type M2 channel and the A/M2-S31N, A/M2-V27A, and A/M2-L26F mutant forms of the channel were measured in Xenopus oocytes using two-electrode voltage clamp assays. Most of the novel compounds inhibited the wild-type ion channel in the low micromolar range. Of note, two of the compounds inhibited the amantadine-resistant A/M2-V27A and A/M2-L26F mutant ion channels with submicromolar and low micromolar IC50, respectively. None of the compounds was found to inhibit the S31N mutant ion channel.

No abstract

The androgen receptor is a transcription factor that plays a key role in the development of prostate cancer, and its interactions with general transcription regulators are therefore of potential therapeutic interest. The mechanistic basis of these interactions is poorly understood due to the intrinsically disordered nature of the transactivation domain of the androgen receptor and the generally transient nature of the protein-protein interactions that trigger transcription. Here, we identify a motif of the transactivation domain that contributes to transcriptional activity by recruiting the C-terminal domain of subunit 1 of the general transcription regulator TFIIF. These findings provide molecular insights into the regulation of androgen receptor function and suggest strategies for treating castration-resistant prostate cancer.

2The androgen receptor is a transcription factor that plays a key role in the development of prostate cancer and its interactions with general transcription regulators are therefore of potential therapeutic interest. The mechanistic basis of these interactions is poorly understood due to the intrinsically disordered nature of the transactivation domain of the androgen receptor and the generally transient nature of the protein-protein interactions that trigger transcription. Here we identify a motif of the transactivation domain that contributes to transcriptional activity by recruiting the C-terminal domain of subunit 1 of the general transcription regulator TFIIF. These findings provide new molecular insights into the regulation of androgen receptor function and suggest new strategies for treating prostate cancer.3 Highlights -A short motif in transactivation unit 5 recruits the transcription machinery to the AR -The motif is intrinsically disordered but folds into a helix upon binding -Phosphorylation of Ser 424 enhances the interaction and is essential for transcription -The interaction is a potential therapeutic target for castration-resistant prostate cancer 4 Graphical abstract 5 IntroductionThe activation of transcription relies on interactions between specific transcription factors and general transcription regulators that can mediated by transcriptional co-activators (Fuda et al., 2009). It is important to characterize these interactions because their inhibition by small molecules or other biological tools offers opportunities for therapeutic intervention in many disease areas, including oncology (Darnell, 2002). Since they involve intrinsically disordered transactivation domains the associated complexes are however transient, marginally stable and challenging to study (Wright and Dyson, 2015).One case where inhibiting these interactions is appealing is castration resistant prostate cancer (CRPC). This condition is suffered by prostate cancer patients that are refractory to hormone therapy, which is based on preventing the activation of the androgen receptor (AR). The mechanisms that allow cell proliferation under these conditions are not yet fully characterized but it is becoming clear that they include expression of constitutively active AR isoforms lacking the ligand binding domain (Miyamoto et al., 2015;Robinson et al., 2015).The complexes formed by the transactivation domain of AR (Lavery and McEwan, 2008a) and general transcription regulators are targets to interfere with CRPC (Sadar, 2011) because inhibiting their formation can lead to a decrease in AR transcriptional activity and in the proliferation of prostate cancer cells. Here we report the structural basis for the interaction of the transactivation domain of AR and the C-terminal domain of subunit 1 of the general transcription regulator TFIIF (RAP74-CTD), which involves the partial folding upon binding of a ca 10-residue motif in this receptor and contributes to the initiation of transcription (Choudhry et al., 2006;McEwan and Gustafsson, 1...

The synthesis of several [4,4,3], [4,3,3], and [3,3,3]azapropellanes is reported. Several of the novel amines displayed low-micromolar activities against an amantadine-resistant H1N1 strain, but they did not show activity against an amantadine-sensitive H3N2 strain. None of the tested compounds inhibit the influenza A/M2 proton channel function. Most of the compounds did not show cytotoxicity for MDCK cells. KEYWORDS: Amantadine, influenza, M2 channel, hemagglutinin, propellane H uman influenza A virus, a member of the Orthomyxoviridae, causes significant morbidity and mortality, particularly in infants, elderly people, and those suffering from previous pathology or immunodeficiency. 1 Its capacity to cause widespread epidemics is related to its fast droplet transmission and ability to escape from existing immunity.The life cycle of influenza viruses is well documented and most viral proteins are regarded as potential therapeutic targets. 2−6 However, presently available drugs for the treatment of influenza virus infections only comprise the M2 ion channel blockers amantadine (Amt) and rimantadine (Chart 1), 7,8 and the neuraminidase inhibitors oseltamivir, zanamivir, peramivir, and laninamivir. 5 Taking into account that most of the currently circulating influenza strains are resistant to the M2 ion channel blockers and as resistance to the neuraminidase inhibitors (in particular oseltamivir) is also rising, 9,10 novel antiinfluenza virus drugs, preferably with a novel mechanism of action, are urgently needed.The influenza virus enters its target cells by receptormediated endocytosis, which is followed by acid-induced fusion of the viral and endosomal membranes. This fusion event is mediated by a conformational change of the influenza hemagglutinin (HA) protein, triggered by the low pH in the endosomal lumen. 11 The endosomal acidic pH also activates the membrane-spanning A/M2 protein, which acts as a proton channel to conduct protons into the virion interior. The decrease in intravirion pH results in uncoating of the viral ribonucleoprotein, which is transferred, through the fusion pore, to the cytoplasm. 12 Since this endosomal escape of the virus critically depends on the activity of the M2 proton channel and the HA protein, dually acting agents which combine blockade of the M2 channel with an inhibitory effect on HA refolding, appear highly attractive. This dual approach could also, at least in theory, increase the barrier for selecting Amt resistance. 13 Optimized Amt analogues might be able to exert this dual pharmacological effect, provided that both the M2 and HA inhibition occur at similar and clinically relevant compound