(2-Hydroxy-4-methoxy)benzyl Aminoadamantane Conjugates as Probes to Investigate Specificity Determinants in Blocking Influenza M2 S31N and M2 WT Channels with Binding Kinetics and Simulations

<p>In an attempt to synthesize potent blockers of the influenza A M2 S31N proton channel with modifications of amantadine, we used MD simulations and MM-PBSA calculations to project binding modes of compounds <b>2-5,</b> which are analogues of <b>1</b>, a dual blocker. Blocking both the S31N mutant and the wild type (WT) M2, <b>1 </b>is composed of amantadine linked to an aryl head group, (4-methoxy-2-hydroxy)-benzyl. Compound <b>6</b>, used as control, has an 3-(thiophenyl)isoxazolyl aryl head group, and selectively blocks M2 S31N (but not WT) in an aryl head group “out” (i.e. N-ward) binding orientation. We then tested <b>1</b>-<b>6</b> as anti-virals in cell culture and for M2 binding efficacy with electrophysiology (EP). The new molecules <b>2-5</b> have a linker between the adamantane and amino group which can be as small as a CMe₂ in rimantadine derivative <b>2</b>, or longer like phenyl in <b>3</b>. Alternatively, we explored the impact of expanding the diameter of adamantane with diamantyl or triamantyl in <b>4 </b>and<b> 5</b>, respectively. Antiviral effects against A/WSN/33 and its M2 WT revertant (M2 N31S) were seen for all six compounds except for <b>5</b> vs. the native (S31N) virus and (as predicted from previous studies) <b>6</b> vs. the WT revertant. Compounds <b>1-5, </b>projected to bind<b> </b>in a polar head group “in” (C-ward) orientation, strongly block<b> </b>proton currents through M2 WT expressed in voltage-clamped oocytes with fast association rate constants (k_on), and slow dissociation rate constants (k_off). Surprisingly,<b> 2-5, </b>projected to bind<b> </b>in a polar head group out orientation, do not effectively block M2 S31N-mediated proton currents in EP. The results from MD and MM-PBSA calculations suggested that compounds <b>2</b>-<b>5</b> can be fully effective at blocking the M2 channel when present. The low degree of blocking in M2 S31N is due to their kinetics of binding observed in EP, i.e. two orders of magnitude reduction in k_oncompared to <b>6</b>, and a fast off rate constant similar to that of <b>6</b>,<b> </b>which is consistent with<b> </b>steered-MDsimulations. The low k_on values can be interpreted from MD simulations, which suggest distortions to V27 cluster of the M2 S31N caused by the longer (even by one methylene) hydrophobic segment from adamantane to aryl head group, appropriate to fit from G34 to V27. The deformations in the N-terminus may be sufficiently energetic for <b>2-5</b> (compared to <b>6</b>)<b> </b>to cause the observed low k_on. <br></p>

(2-Hydroxy-4-methoxy)benzyl Aminoadamantane Conjugates as Probes to Investigate Specificity Determinants in Blocking Influenza M2 S31N and M2 WT Channels with Binding Kinetics and Simulations

<p>In an attempt to synthesize potent blockers of the influenza A M2 S31N proton channel with modifications of amantadine, we used MD simulations and MM-PBSA calculations to project binding modes of compounds <b>2-5,</b> which are analogues of <b>1</b>, a dual blocker. Blocking both the S31N mutant and the wild type (WT) M2, <b>1 </b>is composed of amantadine linked to an aryl head group, (4-methoxy-2-hydroxy)-benzyl. Compound <b>6</b>, used as control, has an 3-(thiophenyl)isoxazolyl aryl head group, and selectively blocks M2 S31N (but not WT) in an aryl head group “out” (i.e. N-ward) binding orientation. We then tested <b>1</b>-<b>6</b> as anti-virals in cell culture and for M2 binding efficacy with electrophysiology (EP). The new molecules <b>2-5</b> have a linker between the adamantane and amino group which can be as small as a CMe₂ in rimantadine derivative <b>2</b>, or longer like phenyl in <b>3</b>. Alternatively, we explored the impact of expanding the diameter of adamantane with diamantyl or triamantyl in <b>4 </b>and<b> 5</b>, respectively. Antiviral effects against A/WSN/33 and its M2 WT revertant (M2 N31S) were seen for all six compounds except for <b>5</b> vs. the native (S31N) virus and (as predicted from previous studies) <b>6</b> vs. the WT revertant. Compounds <b>1-5, </b>projected to bind<b> </b>in a polar head group “in” (C-ward) orientation, strongly block<b> </b>proton currents through M2 WT expressed in voltage-clamped oocytes with fast association rate constants (k_on), and slow dissociation rate constants (k_off). Surprisingly,<b> 2-5, </b>projected to bind<b> </b>in a polar head group out orientation, do not effectively block M2 S31N-mediated proton currents in EP. The results from MD and MM-PBSA calculations suggested that compounds <b>2</b>-<b>5</b> can be fully effective at blocking the M2 channel when present. The low degree of blocking in M2 S31N is due to their kinetics of binding observed in EP, i.e. two orders of magnitude reduction in k_oncompared to <b>6</b>, and a fast off rate constant similar to that of <b>6</b>,<b> </b>which is consistent with<b> </b>steered-MDsimulations. The low k_on values can be interpreted from MD simulations, which suggest distortions to V27 cluster of the M2 S31N caused by the longer (even by one methylene) hydrophobic segment from adamantane to aryl head group, appropriate to fit from G34 to V27. The deformations in the N-terminus may be sufficiently energetic for <b>2-5</b> (compared to <b>6</b>)<b> </b>to cause the observed low k_on. <br></p>

Voltage-gated proton channels exist in the plasma membrane of human oocytes

STUDY QUESTION Do human oocytes express voltage-gated proton channels? SUMMARY ANSWER Human oocytes exhibit voltage-gated proton currents. WHAT IS KNOWN ALREADY Voltage-gated proton currents have been reported in human sperm, where they contribute to capacitation and motility. No such studies of human oocytes exist. STUDY DESIGN, SIZE, DURATION Voltage-clamp studies were undertaken using entire oocytes and vesicles derived from oocytes and in excised patches of membrane from oocytes. PARTICIPANTS/MATERIALS, SETTING, METHODS Frozen, thawed human metaphase II oocytes were obtained from material donated to the gamete repository at the Rush Center for Advanced Reproductive Care. Prior to patch clamping, oocytes were warmed and equilibrated. Formation of an electrically tight seal requires exposing bare oolemma. Sections of the zona pellucida (ZP) were removed using a laser, followed by repeated pipetting, to further separate the oocyte from the ZP. Patch-clamp studies were performed using the whole-cell configuration on oocytes or vesicles derived from oocytes, and using inside-out patches of membrane, under conditions optimized to detect voltage-gated proton currents. MAIN RESULTS AND THE ROLE OF CHANCE Proton currents are present at significant levels in human oocytes where they exhibit properties similar to those reported in other human cells, as well as those in heterologous expression systems transfected with the HVCN1 gene that codes for the voltage-gated proton channel. LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION Human oocytes are large cells, which limits our ability to control the intracellular solution. Subtle effects of cryopreservation by vitrification and subsequent warming on properties of HVCN1, the HVCN1 gene product, cannot be ruled out. WIDER IMPLICATIONS OF THE FINDINGS Possible functions for voltage-gated proton channels in human oocytes may now be contemplated. STUDY FUNDING/COMPETING INTEREST(S) NIH R35GM126902 (TED), Bears Care (DM). No competing interests. TRIAL REGISTRATION NUMBER N/A.

An update of the chemiosmotic theory as suggested by possible proton currents inside the coupling membrane

Understanding how biological systems convert and store energy is a primary purpose of basic research. However, despite Mitchell's chemiosmotic theory, we are far from the complete description of basic processes such as oxidative phosphorylation (OXPHOS) and photosynthesis. After more than half a century, the chemiosmotic theory may need updating, thanks to the latest structural data on respiratory chain complexes. In particular, up-to date technologies, such as those using fluorescence indicators following proton displacements, have shown that proton translocation is lateral rather than transversal with respect to the coupling membrane. Furthermore, the definition of the physical species involved in the transfer (proton, hydroxonium ion or proton currents) is still an unresolved issue, even though the latest acquisitions support the idea that protonic currents, difficult to measure, are involved. Moreover, F o F 1 -ATP synthase ubiquitous motor enzyme has the peculiarity (unlike most enzymes) of affecting the thermodynamic equilibrium of ATP synthesis. It seems that the concept of diffusion of the proton charge expressed more than two centuries ago by Theodor von Grotthuss is to be taken into consideration to resolve these issues. All these uncertainties remind us that also in biology it is necessary to consider the Heisenberg indeterminacy principle, which sets limits to analytical questions.