A Small-Molecule Inhibitor of Prion Replication and Mutant Prion Protein Toxicity

Full text of DOI:10.1002/cmdc.201700302

Accepted Article
Title: A NOVEL SMALL MOLECULE INHIBITOR OF PRION
REPLICATION AND MUTANT PRION PROTEIN TOXICITY
Authors: Tania Massignan, Valeria Sangiovanni, Silvia Biggi, Claudia
Stincardini, Saioa R. Elezgarai, Giulia Maietta, Ivan A.
Andreev, Nina K. Ratmanova, Dmitry S. Belov, Evgeny R.
Lukyanenko, Grigory M. Belov, Maria Letizia Barreca, Andrea
Altieri, Alexander V Kurkin, and Emiliano Biasini
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To be cited as: ChemMedChem 10.1002/cmdc.201700302
Link to VoR: http://dx.doi.org/10.1002/cmdc.201700302
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A NOVEL SMALL MOLECULE INHIBITOR OF PRION REPLICATION AND MUTANT PRION
PROTEIN TOXICITY
Dr. Tania Massignan,1± Valeria Sangiovanni,1± Silvia Biggi,1 Claudia Stincardini,1 Dr. Saioa R.
Elezgarai,1,2 Giulia Maietta,1 Dr. Ivan A. Andreev,4 Dr. Nina K. Ratmanova,4 Dr. Dmitry S. Belov,4 Dr.
Evgeny R. Lukyanenko,4 Dr. Grigory M. Belov,4,5 Prof. Maria Letizia Barreca,3 Dr. Andrea Altieri,4,5*
Prof. Alexander V. Kurkin,4 and Dr. Emiliano Biasini1,6*
1Dulbecco Telethon Laboratory of Prions and Amyloids, Centre for Integrative Biology (CIBIO),
University of Trento, 38123 Trento, Italy
2Department of Molecular Biochemistry and Pharmacology & 6Department of Neuroscience, IRCCS-
Istituto di Ricerche Farmacologiche Mario Negri, 20156 Milan, Italy;
3Department of Pharmaceutical Sciences, University of Perugia, 06123 Perugia, ITALY
4Department of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia
5EDASA Scientific srls., 66050 San Salvo (CH), Italy
±These authors contributed equally
*To whom correspondence should be addressed: aaltieri@edasascientific.com; biasinie@gmail.com
Keywords: Drug discovery, Neurotoxicity, Protein misfolding, Prion Diseases, Amino propargylic
alcohols
ABSTRACT.
Prion diseases are a class of neurodegenerative disorders characterized by the accumulation in the
brain of a self-replicating, misfolded isoform (PrPSc) of the cellular prion protein (PrPC), a cell-surface
glycoprotein of uncertain function. Emerging evidence suggests that PrPC may passively serve as a
substrate for the replication of PrPSc, and actively transduce its toxic effects to neuronal cells. The vast
majority of previous drug screening campaigns have only targeted PrPSc replication, with largely
unsuccessful results. We have previously described an experimental paradigm for detecting the
cytotoxic effects of mutant PrP molecules in cultured cells, called drug-based cell assay (DBCA).
Here, we employed this assay to screen a chemical library of diverse compounds, and identified a
small molecule (molecule 55) that counteracts the cytotoxic activity of a disease-associated point
mutant of PrP. Importantly, this compound also blocks the replication of two different prion strains.
Molecule 55 may represent the starting point for the development of a novel class of therapeutic
agents for prion diseases.
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MAIN TEXT.
Prion diseases are neurodegenerative conditions of human and animals characterized by dementia,
motor abnormalities and cerebral amyloidosis. The main pathogenic event in these diseases is the
accumulation in various brain areas of PrPSc, a misfolded and infectious isoform (prion) that
propagates by imposing its abnormal conformation onto molecules of PrPC, its endogenous
counterpart.[1] Despite the fact that prion diseases have an annual incidence of only 1 to 2 cases per 1
million population, their scientific and medical relevance has grown enormously in the last two
decades, for multiple reasons. In fact, human prion diseases can be transmissible, posing important
medical concerns for organ transplants or blood transfusions.[2] Prions have also shown the ability to
spread in domesticated as well as wild animals, and to cross species barriers, as in the case of the
transmission of bovine spongiform encephalopathy to humans through contaminated food supplies.[3]
Importantly, a prion-like mechanism has been proposed to explain the spread in brain tissues of
different amylodogenic proteins associated with common neurodegenerative disorders, such as
Alzheimer’s or Parkinson’s diseases, suggesting the alarming possibility that at least a subset of these
disorders could be transmissible.[4] Unfortunately, despite more than three decades of intensive
research, the molecular mechanisms laying at the root of prion infectivity and neurotoxicity are still
poorly understood, and effective therapies for prion diseases are still missing.[5]
The formation, stability or metabolism of PrPSc have represented the main pharmacological targets of
virtually all previous screening campaigns in prion diseases.[6] These approaches identified a number
of compounds capable of blocking prion replication in cells, and occasionally delay the disease in
mice. However, none of these molecules have shown efficacy in patients. Moreover, while multiple
structural PrPSc conformers (called prion strains) may coexist in a host and possibly cause resistance
to pharmacological treatments, the majority of anti-prion compounds identified so far act in a strain-
specific fashion.[7] Collectively, these data challenge the concept that targeting exclusively PrPSc
replication is a convenient therapeutic strategy in prion diseases. A possible complementary approach
could be to also target neurotoxic pathways mediated by PrPC.[8] We have previously described the
2
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DBCA, a cellular assay based on the intrinsic cytotoxic effects of PrP molecules carrying mutations in
the central region of the protein.[9] Multiple previous studies indicated that this assay is a useful tool to
study PrP-mediated neurotoxic pathways, as well as to identify and characterize novel anti-prion
compounds.[10] Here, we employed the DBCA to screen a representative diverse set of small
molecules from an original collection of chemicals developed at EDASA Scientific.
The DBCA is based on the ability of PrP molecules carrying artificial deletions or naturally-occurring
point mutations in the central region of the protein to highly increase the susceptibility of a variety of
cultured cells and primary neurons to the toxicity of several cationic antibiotics, such as
aminoglycosides (e.g. hygromycin or G418) or bleomycin analogues (e.g. Zeocin). Here, we employed
this assay to screen a set of 54 molecules (Figures S1.A and S1.B) as representative heterobicyclic
compounds
available
from
the
EDASA
Scientific
public
compound
library
(www.edasascientific.com/page/catalogue). HEK293 cells stably expressing a PrP mutant carrying a
20 amino acids deletion in the central region (Δ105-125, called ΔCR) were exposed to Zeocin (500
µg/mL) for 48h, in presence (10 µM) or absence of each compound. Cell viability in the different
conditions was then evaluated by MTT assay (Figure 1). As expected, cells exposed to Zeocin alone
showed a strong reduction in viability (< 70%, not shown). Conversely, co-treatment with the porphyrin
Fe(III)-TMPyP, previously shown to inhibit the activity of ΔCR PrP in the DBCA,[10] abrogated the effect
(not shown). Among the different compounds, we identified eight molecules (34, 38, 39, 43, 46 , 50, 53
and 54) capable of rescuing at least 20% of antibiotic-induced cell death (Figure 2). We selected these
compounds for subsequent analyses.
Previous evidence showed that compounds active in the DBCA could also possess the ability to inhibit
prion replication in cell cultures.[11] Thus, we sought to test the potential anti-prion effects of our newly
identified molecules. N2a cells chronically infected with the 22L mouse prion strain were exposed for
72h to Fe(III)-TMPyP (10 µM), vehicle control (DMSO, volume equivalent) or two different
concentrations (10 and 30 µM) of each hit compound.
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The levels of 22L prions were then evaluated by detecting the amount of proteinase-K (PK)-resistant
PrP species by slot blotting (Figure 3). Consistent with previous reports, Fe(III)-TMPyP showed a
potent (>80% inhibition) anti-prion effect. Among the eight different molecules identified by DBCA, five
(38, 39, 43, 46 and 50, Figure 3) showed an evident (>30%), statistically-significant inhibitory effect
against 22L prion replication, at least at one concentration. These molecules represented the starting
point for the assembly of an additional, focused collection of related compounds. For this purpose, we
selected several EDASA compounds representing derivatives and/or having high (>80%) structural
similarity with the eight molecules emerged in the primary screen. A second, focused library
(compounds 55-98, Figure 4.A and 4.B) exploring the chemical scaffold of each of the positive hits
was built and subjected to activity validation by the different cellular assays.
The DBCA was used again to screen each compound of the new chemical library at three different
concentrations (0.1, 1 and 10 µM) (Figure S2). We identified six compounds (55, 58, 59, 60, 68 and
95) capable of rescuing cell viability in the DBCA in a dose-dependent fashion (Figure 5). Interestingly,
five of these molecules (55, 58-60 and 68) share a high degree of chemical similarity, with a common
alkynylcyclohexenylamine-based scaffold.[12] These compounds showed relatively low intrinsic
cytotoxicity, as assayed in HEK293 expressing ΔCR or wild type (WT) PrP (Figure S3 and S4).
It is worth noting that the N-benzyl moiety was present in all the active compounds belonging to this
subset, i.e. 55, 58-60 and 68. The key role of the N-benzyl moiety was further confirmed by the
inactive analogues 57, 63, 64, 65, 66, where this unit was missing. Interestingly compound 62, which
presents the N-benzyl moiety ramified with a methyl group, lost its activity as well. This ramification of
the α carbon next to the amine was also present in the non-active compounds 63 and 64. Among the
active compounds, derivatives 58-60 presented an extra moiety on the amino group, that was an N-
allylic moiety (58 and 59) or a N,N-dibenzyl moiety (60). The elongation of the acetylene unit by a
phenyl ring seemed to be well tolerated in 55, 59 and 60. This observation may possibly lead to the
conclusion that such position could represent an interesting point for adding chemical groups, with the
objective of further exploring new potential anti-prions compounds.
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In order the test the anti-prion activity of the six identified hits, we carried out a seven-point (0.03-10
µM) dose-response experiment in N2a cells infected with either the 22L or the RML prion strains. The
levels of both prion strains were then evaluated by detecting the amount of proteinase-K (PK) resistant
PrP species by Western blotting (Figure 6 and Figure S5 and S6). One compound (an amino
propargylic alcohol derivative, called 55) showed the ability to robustly block the replication of both 22L
and RML prion strains, at concentrations in the low micromolar range. Collectively, these results
indicated that compound 55 effectively silences the cytotoxic effect of ΔCR PrP, and inhibits prion
replication in a strain-independent manner. Interestingly, a previous study reported only a partial,
although still surprising pharmacological correlation between compounds active in the DBCA and
against prion propagation in cell cultures.[11] Our data support a similar conclusion, since we identified
compounds active only in the DBCA (58, 59, 60, 68 and 95), or in both assays (molecule 55). A
possible, logic explanation for these observations is that the different pharmacological targets of the
identified molecules may play roles in just mutant PrP-dependent toxicity, or also in prion replication.
In its standard format, the DBCA relies on the expression of ΔCR PrP. However, the deletion carried
by this PrP mutant is artificial, and not associated with any human prion disease. In order to further
validate the activity of molecule 55 in a more disease-relevant experimental context, we turned to
another PrP mutant, carrying the mouse homologue (G113V) of a single point substitution (G114V)
which in humans is associated with an early-onset form of Gerstmann-Straüssler-Scheinker syndrome
(GSS). Consistent with previous reports, we found that the expression of G113V PrP in cultured cells
confers hypersensitivity to cationic antibiotics in the DBCA similarly to ΔCR PrP, although with a lower
efficiency (Figure 7). Importantly, co-treatment with molecule 55 (30 µM) significantly rescued the
effects. These results demonstrate that molecule 55 is also capable of inhibiting the cytotoxic effect of
a PrP mutant associated with an inherited prion disease.
In order to rule out the possibility that the rescuing effects shown by compound 55 in the different
DBCA experiments were due to an interaction of the molecule with Zeocin, we performed the assay in
two modified versions. In contrast to mutant PrP, expression of WT PrP does not confer
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hypersensitivity to Zeocin.[9,10] Therefore, we performed the DBCA in HEK293 cells stably expressing
WT PrP, with increasing concentrations of Zeocin (0-2.000 µg/mL), in presence or absence of
molecule 55 (Figure S7). In cells not treated with 55, we observed a dose-dependent toxicity of Zeocin
over 72h, with approximately 50% of cell death obtained at the highest concentration (2.000 µg/mL).
Importantly, we failed to observe any significant rescue upon incubation with molecule 55 (3-10 µM).
Next, we used HEK293 cells stably expressing ΔCR PrP, but replaced Zeocin with G418, an
aminoglycoside previously shown to produce similar mutant PrP-dependent effects in the DBCA.
Consistent with the previous data, molecule 55 induced a robust, dose-dependent rescue of cell
viability (Figure S9). Collectively, these results clearly indicate that the rescuing effects of compound
55 in the DBCA are antibiotic-independent, and specific for mutant PrP expression.
One possible mechanism by which molecule 55 may exert its different anti-prion effects is by targeting
directly or indirectly PrPC. We tested this possibility in three different ways. First, we checked whether
the compound suppresses the expression of PrPC. HEK293 cells stably expressing ΔCR PrP, or N2a
cells stably expressing WT PrP, were treated with raising concentrations of molecule 55, the anti-PrP
porphyrin TMPyP (10 µM), or vehicle control (volume-equivalent), and PrP expression was evaluated
by Western blotting (Figure 8). We found no difference in the levels of PrPC between treated vs
untreated cells.
Next, we tested whether molecule 55 acts by promoting the removal of PrPC from the cell surface. In
this case, we analyzed the distribution of an EGFP-tagged WT PrP stably expressed in HEK293 cells,
in presence or absence of different concentrations of molecule 55 (Figure 9). As a positive control, we
used chlorpromazine (CPZ), an antipsychotic known to inhibit clathrin-mediated endocytosis (CME),
and recently found to cause the redistribution of PrPC from the plasma membrane (manuscript under
review). Even in this case, we found no evidence that molecule 55 alters the cell surface distribution of
PrPC.
Finally, we directly tested the possibility that molecule 55 may act by directly binding to PrPC, by using
dynamic mass redistribution (DMR), a biophysical technique previously employed to test the
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interaction of small ligands to PrPC.[10] As a positive control for the technique, we confirmed the
interaction of TMPyP to PrPC, as previously reported (Figure 10). Conversely, we failed to detect a
specific interaction of molecule 55 to PrPC. These data were confirmed using surface plasmon
resonance (SPR), another biophysical technique previously umployed to detect the interaction of small
molecules to PrPC in a kinetic fashion.[10] Collectively, these results indicate that 55 does not exert its
anti-prion effects by targeting PrPC, but rather through an unknown pharmacological target, which
could play a role in both mutant PrP toxicity and prion replication.
On the basis of the promising results described in this manuscript, we calculated key descriptors
(Table 1 and Supporting Information) to evaluate drug-likeness for compound 55, and thus its potential
as a therapeutic agent for the central nervous system (CNS). Indeed, in the last few years compounds
active on the CNS have extensively been characterized. As a result, sets of physicochemical and
pharmacokinetic parameters that may be used for the identification of successful CNS molecules have
been proposed (examples are reported in Table 1).[13] These analyses suggest that compound 55
might possess a desirable drug-like profile for oral dosage and for targeting the CNS, as all calculated
properties are in line with the recommended guidelines for CNS-directed therapeutics.[14,15]
In summary, we have used the DBCA assay, a previously described method to detect the cytotoxic
activity of PrP mutants, to sequentially screen a highly diverse set of original compounds from the
EDASA Scientific public compound library, and a second more focused subset of molecules built
around the scaffold of the positive hits arising from the primary screen. This approach identified
compound 55, an amino propargylic alcohol derivative,[16] as capable of inhibiting the cytotoxic effect
of a PrP mutant carrying a deletion in central region (residues 105-125, called ΔCR PrP), as well as of
the mouse homologue of a human disease-associated point mutant (G114V). Importantly, the
molecule also blocked the replication of two different prion strains in neuroblastoma cells. These
results indicate that molecule 55 possesses the remarkable ability of acting against both prion
replication and mutant PrP toxicity. However, the compound does not seem to target PrPC, either
directly or indirectly, suggesting the intriguing possibility that its pharmacological target could be a
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molecular factor playing a role in both prion replication and mutant PrP toxicity. Thus, in addition to
provide a starting point for the development of a novel class of therapeutic agents for prion diseases,
molecule 55 could represent an important tool to gain insights into the mechanisms underlying prion
replication and toxicity.
FIGURE LEGENDS.
Figure 1. The DBCA was employed to evaluate the anti-ΔCR PrP effects of each individual molecule.
The bar graph illustrates the quantification of the rescuing ability of each molecule. Mean values were
obtained from two independent experiments, and expressed as percentage of cell viability rescue.
Compounds 27, 35, 40, 41, 42, 44, 52 were found to be insoluble at the lowest concentration used in
the assay, and thus were not analyzed.
Figure 2. The chemical structure of the positive hits from the primary DBCA-based screen are shown.
Numbers indicate (from left): (i) Sequential numbering (1 to 54); (ii) EDASA chemical library code; (iii)
Code of the in house chemical library of the Dulbecco Telethon Laboratory of Prions & Amyloids at
University of Trento.
Figure 3. Positive hits from the primary screen are validated by prion replication assay in 22L-infected
cells. N2a cells chronically infected with the 22L prion strain were incubated with two different
concentrations (10 and 30 µM) of each compound for 72h. The level of PK-resistant PrP before and
after treatment was estimated by slot blotting, using anti-PrPC antibody 6D11. Signals were quantified
by densitometric analysis of blots, normalized on the total amount of proteins (obtained by Ponceau
staining) in PK-untreated replicates, and expressed in the bar graph as mean percentage (%) of the
signal in vehicle-treated cells (± standard error). Statistical differences between each
molecule/concentration and vehicle control were estimated by Student t-test (*p < 0.05; *p < 0.01).
Figure 4.A. Chemical structures of molecules 55-67. These molecules were selected from the
EDASA Scientific public compound library for their similarity with compound 50.
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Figure 4.B. Chemical structures of molecules 69-98. These molecules were selected from the
EDASA Scientific public compound library for their similarity with compound 38, 39, 43 and 46.
Figure 5. Hits from the focused library were tested by DBCA. Graph shows mean values (± standard
error) expressed as percentage of cell viability rescue using the equation: R = (T-Z)/(U-Z) (R: rescuing
effect; T: cell viability in compound-treated samples; Z: cell viability in zeocin-treated samples; U: cell
viability in untreated samples). Data were obtained from a minimum of 4 independent experiments (n ≥
4).
Figure 6. Compound 55 showed the ability to inhibit the replication of both RML (picture in panel A,
quantification in panel C) and 22L (B) prion strains. The level of PK-resistant PrP before and after
treatment was estimated by western blotting, using anti-PrPC antibody D18. Signals were quantified by
densitometric analysis of images, normalized on the total amount of proteins (obtained by Ponceau
staining) in PK-untreated replicates, and plotted in the graphs as mean percentage (%) of the signal in
vehicle-treated cells (± standard error). Inhibitory concentrations at 50% (IC50) values for molecule 55
were: 8.25 µM against the 22L strain, and 3.21 µM against the RML strain.
Figure 7. Molecule 55 was tested in the DBCA against a disease-associated PrP mutant. The DBCA
was performed as before, with slight modification (see Supplementary Information). Mean values were
obtained from 8 independent experiments (n = 8), and expressed as percentage of cell viability in
vehicle (VHC)-treated control.
Figure 8. Western blotting analyses indicate that molecule 55 does not reduce the expression of PrP.
HEK293 cells expressing ΔCR PrP or N2a cells expressing WT PrP were treated with the indicated
concentrations of molecule 55 for 48h. PrP signals were detected by anti-PrP antibody D18. The
porphyrin TMPyP (TP) and vehicle (DMSO) were used as negative controls. The experiment was
repeated three independent times (n = 3).
Figure 9. Treatment with molecule 55 did not induce the relocalization of EGFP-PrP from the cell
surface. HEK293 cells stably expressing EGFP-PrP were grown to ~60% confluence on glass
coverslips, and then treated with the indicated concentrations of molecule 55 or CPZ for 48h. After
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fixation and washing, the intrinsic green signal of EGFP-PrP was acquired. In contrast to molecule 55,
CPZ produced EGFP-PrP relocalization from the cell surface.
Figure 10. DMR analyses indicate that molecule 55 does not bind to recombinant PrPC. Different
concentrations (0.03-100 µM) of molecule 55 or TMPyP were added to label-free microplate well
surfaces (EnSpire-LFB HS microplate, Perkin Elmer) on which full-length mouse recombinant PrPC
had previously been immobilized. Measurements were performed before (baseline) and after (final)
adding the compound. The response (pm) was obtained subtracting the baseline output to the final
output signals. The output signal for each well was obtained by subtracting the signal of the protein-
coated reference area to the signal of uncoated area. The data for TMPyP (black dots) were fitted
(blue line) to a sigmoidal function using a 4 parameter logistic (4PL) non-linear regression model (R2 =
0.96).
Table 1.a,b First value indicates the median value derived for a set of 119 marked CNS drugs [13c] and
a set of 108 Pfizer CNS clinical candidates, [13c] respectively; numbers in brackets is the range values
c
for the same set. Suggested values for increasing the potential for blood–brain barrier (BBB)
e
penetration and oral bioavailability.[13a,b] d Calculated octanol/water partition coefficient. Calculated
f
g
h
distribution coefficient. Molecular weight. Topological polar surface area. Number of hydrogen
I
l
bond donors. Most basic center. Intrinsic aqueous solubility.m Classification for human intestinal
absorption; n Classification of BBB penetration. o Predicted BBB penetration value. p Classification of P-
glycoprotein transport.
EXPERIMENTAL SECTION
See Supporting Information for experimental details.
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ACKNOWLEDGEMENTS.
The authors thank Alessandro Negro (University of Padova) for providing recombinant mouse PrPC,
Roberto Chiesa for the mouse prion inocula (RML and 22L) and BioSolveIT GmbH
(http://www.biosolveit.de) for providing academic license of SeeSAR program. The study was
supported by a Young Investigator Award from the Italian Ministry of Health to EB and MLB, by a grant
from the Russian Foundation for Basic Research (RFBR; Projects No. 14-03-01114), and a grant from
the CJD Foundation to EB (GR-2010-2312769). SRE was supported by a grant from the E-Rare Joint
Transnational Call (E-Rare-2). EB is an Assistant Telethon Scientist at the Dulbecco Telethon Institute
(TCP14009, Fondazione Telethon, Italy).
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REFERENCES.
[1] S. B. Prusiner, Science 1982, 216, 136-144.
[2] E. D. Belay, L. B. Schonberger, Annu. Rev. Public Health 2005, 26, 191-212.
[3] R. M. Anderson, C. A. Donnelly, N. M. Ferguson, M. E. J. Woolhouse, C. J. Watt, H. J. Udy, S.
MaWhinney, S. P. Dunstan, M. E. J. Southwood, J. W. Wilesmith, J. B. M. Ryan, L. J. Hoinville, J. E.
Hillerton, A. R. Austin, G. A. H. Wells, Nature 1996, 382, 779-788.
[4] Z. Jaunmuktane, S. Mead, M. Ellis, J. D. Wadsworth, A. J. Nicoll, J. Kenny, F. Launchbury, J.
Linehan, A. Richard-Loendt, A. S. Walker, P. Rudge, J. Collinge, S. Brandner, Nature 2015,
525(7568), 247-250.
[5] N. Iraci, C. Stincardini, M. L. Barreca, E. Biasini, Virus Res 2014.
[6] V. L. Sim, Infect Disord Drug Targets 2012, 12(2), 144-160.
[7] K. Giles, S. H. Olson, S. B. Prusiner, Cold Spring Harbor perspectives in medicine 2017.
[8] S. R. Elezgarai, E. Biasini, Biol Chem 2016, 397(11), 1115-1124.
[9] T. Massignan, R. S. Stewart, E. Biasini, I. H. Solomon, V. Bonetto, R. Chiesa, D. A. Harris, J Biol
Chem 2010, 285(10), 7752-7765.
[10] T. Massignan, S. Cimini, C. Stincardini, M. Cerovic, I. Vanni, S. R. Elezgarai, J. Moreno, M.
Stravalaci, A. Negro, V. Sangiovanni, E. Restelli, G. Riccardi, M. Gobbi, J. Castilla, T. Borsello, R.
Nonno, E. Biasini, Sci Rep 2016, 6, 23180.
[11] T. Imberdis, J. T. Heeres, H. Yueh, C. Fang, J. Zhen, C. B. Rich, M. Glicksman, A. B. Beeler, D.
A. Harris, J Biol Chem 2016, 291(50), 26164-26176.
[12] D. Lednicer, P. F. VonVoigtlander, D. E. Emmert, J Med Chem 1981, 24(4), 404-408.
[13] a) M. D. Segall, I. Yusof, E. J. Champness, J Med Chem 2016, 59(9), 4267-4277; b) S. A.
Hitchcock, L. D. Pennington, J Med Chem 2006, 49(26), 7559-7583; c) T. T. Wager, R. Y.
Chandrasekaran, X. Hou, M. D. Troutman, P. R. Verhoest, A. Villalobos, Y. Will, ACS Chem Neurosci
2010, 1(6), 420-434; d) Z. Rankovic, J Med Chem 2015, 58(6), 2584-2608.
[14] MoKa version 2.6.6; Molecular Discovery LTD; www.moldiscovery.com
[15] SeeSAR version 5.5, BioSolveIT GmbH, Sankt Augustin, Germany (http://www.biosolveit.de)
[16] For recent examples of the synthetic utilization of amino propargylic alcohols, see the following
references: a) I. A. Andreev, N. K. Ratmanova, A. M. Novoselov, D. S. Belov, I. F. Seregina, A. V.
Kurkin, Chem. – Eur. J. 2016, 22, 7262–7267; b) N. Kaushik-Basu, N. K. Ratmanova, D. Manvar, D.
S. Belov, O. Cevik, A. Basu, M. M. Yerukhimovich, E. R. Lukyanenko, I. A. Andreev, G. M. Belov, G.
Manfroni, V. Cecchetti, D. N. Frick, A. V. Kurkin, A. Altieri, M. L. Barreca, Eur. J. Med. Chem. 2016,
122, 319–325; c) I. A. Andreev, D. Manvar, M. L. Barreca, D. S. Belov, A. Basu, N. L. Sweeney, N. K.
Ratmanova, E. R. Lukyanenko, G. Manfroni, V. Cecchetti, D. N. Frick, A. Altieri, N. Kaushik-Basu, A.
V. Kurkin, Eur. J. Med. Chem. 2015, 96, 250–258;
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ChemMedChem
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700302
ChemMedChem
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700302
ChemMedChem
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700302
ChemMedChem
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700302
ChemMedChem
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700302
ChemMedChem
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700302
ChemMedChem
This article is protected by copyright. All rights reserved.