Small Molecule Inhibitors of Ca2+-S100B Reveal Two Protein Conformations

Article abstract of DOI:10.1021/acs.jmedchem.5b01369

The drug pentamidine inhibits calcium-dependent complex formation with p53 (^CaS100B·p53) in malignant melanoma (MM) and restores p53 tumor suppressor activity in vivo. However, off-target effects associated with this drug were problematic in MM patients. Structure-activity relationship (SAR) studies were therefore completed here with 23 pentamidine analogues, and X-ray structures of ^CaS100B·inhibitor complexes revealed that the C-terminus of S100B adopts two different conformations, with location of Phe87 and Phe88 being the distinguishing feature and termed the "FF-gate". For symmetric pentamidine analogues (^CaS100B·5a, ^CaS100B·6b) a channel between sites 1 and 2 on S100B was occluded by residue Phe88, but for an asymmetric pentamidine analogue (^CaS100B·17), this same channel was open. The ^CaS100B·17 structure illustrates, for the first time, a pentamidine analog capable of binding the "open" form of the "FF-gate" and provides a means to block all three "hot spots" on ^CaS100B, which will impact next generation ^CaS100B·p53 inhibitor design.

Full text of DOI:10.1021/acs.jmedchem.5b01369

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Article
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Small Molecule Inhibitors of Ca-S100B Reveal Two Protein Conformations
Michael Christopher Cavalier, Mohd. Imran Ansari, Adam D. Pierce, Paul T Wilder, Laura E
McKnight, E. Prabhu Raman, David B Neau, Padmavani Bezawada, Milad J Alasady, Thomas H.
Charpentier, Krisin Varney, Eric A Toth, Alexander D. MacKerell, Andrew Coop, and David J. Weber
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01369 • Publication Date (Web): 04 Jan 2016
Downloaded from http://pubs.acs.org on January 9, 2016
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Journal of Medicinal Chemistry
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Small Molecule Inhibitors of Ca2+-S100B Reveal Two Protein Conformations
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Michael C. Cavalier†, Mohd. Imran Ansari#, Adam D. Pierce†, Paul T. Wilder†§, Laura E. McKnight†, E. Prabhu
Raman#, David B. Neau‡, Padmavani Bezawada#, Milad J. Alasady†, Thomas H. Charpentier†, Kristen M.
Varney†, Eric A. Toth†§¥, Alexander D. MacKerell, Jr.#§, Andrew Coop#§, and David J. Weber†§*
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† Center for Biomolecular Therapeutics (CBT), Department of Biochemistry and Molecular Biology, University
of Maryland School of Medicine, Baltimore, Maryland 21201, United States.
§Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore,
Maryland 21201, United States.
#Computer Aided Drug Design Center, University of Maryland, School of Pharmacy, Baltimore, Maryland
21201, United States.
¥Institute for Bioscience and Biotechnology Research, 9600 Gudelsky Drive Rockville, MD 20850, USA
‡ NEꢀCAT, Cornell University, Argonne IL 60439, USA.
Funding Source: This work was supported by grants from the NIH (GM58888 and CA107331) to D.J.W. and
(GM070855) to A.D.M. Jr. The training of M.C.C. is supported by a NIH T32 grant (CA154274).
*To whom correspondence should be addressed; D.J.W., University of Maryland School of Medicine,
Department of Biochemistry and Molecular Biology, Biomedical Research Facility Rm. 439, 108 N. Greene St.,
Baltimore, MD 21201. Phone (410) 706ꢀ4354, Fax (410) 706ꢀ0438, Eꢀmail: dweber@som.umaryland.edu.
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ABSTRACT
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The drug pentamidine inhibits calciumꢀdependent complex formation with p53 (CaS100B•p53) in malignant
melanoma (MM), and restores p53 tumor suppressor activity in vivo. However, offꢀtarget effects associated
with this drug were problematic in MM patients. Structureꢀactivity relationship (SAR) studies were therefore
completed here with 23 pentamidine analogues, and Xꢀray structures of CaS100B•inhibitor complexes revealed
that the Cꢀterminus of S100B adopts two different conformations, with location of Pheꢀ87 and Pheꢀ88 being the
distinguishing feature and termed the “FFꢀGate”. For symmetric pentamidine analogues (CaS100B•5a,
CaS100B•6b) a channel between Sites 1 and 2 on S100B was occluded by residue Pheꢀ88, but for an asymmetric
pentamidine analogue (CaS100B•17), this same channel was open. The CaS100B•17 structure illustrates, for the
first time, a pentamidine analog capable of binding the “open” form of the “FFꢀgate” and provides a means to
block all three “hot spots” on CaS100B, which will impact next generation CaS100B•p53 inhibitor design.
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INTRODUCTION
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Longꢀtime survival (> 3 years) remains extremely poor for most malignant melanoma (MM) patients (>70%
mortality), and side effects from treatments are often severe1ꢀ3. Prognosis is even more problematic after the
onset of metastasis and/or when drugꢀresistant mechanisms arise4, 5. While advances in immune checkpoint
inhibitors can have remarkable outcomes for some MM patients, these therapies have limited or small effects in
others (50%), so trials with immune checkpoint inhibitors are often done in drug combination trials including
with chemotherapy regimens, radiation, and/or with other targeted approaches 6, 7. One alternative target in MM
is a calciumꢀbinding protein, S100B, which is an important biomarker for this and other cancers8. S100B is
elevated in >90% of MM patients and its protein level correlates directly with poor survival (<1 yr.) and
relapse, and it is especially predictive as a biomarker when used in combination with other diagnostic
indicators9ꢀ11. For example, MM vaccines are only effective in a small number of patients (5ꢀ10%) and they
nearly all have low levels of S100B12, 13. While this correlation is not fully understood, mechanistic studies
show that lowering S100B levels via siRNA or via small molecule inhibitors restore p53 levels and its tumor
suppressor activities including UVꢀactivated apoptosis pathways found in normal melanocytes14ꢀ18. Studies
suggest that S100B blocks p53 oligomerization, promotes p53 phosphorylation in its Cꢀterminus, and
contributes to p53 degradation in concert with the E3 ubiquitin ligase, hdm28. Elevated S100B and
overexpression of the receptor for advanced glycation end products (RAGE) confers a metastatic phenotype19,
so learning how S100B affects tumor progression and metastasis are ongoing including the development of
novel S100B inhibitors (termed SBiXs) to ablate S100Bꢀdependent effects in cancer.
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One of the first compounds found to inhibit S100B was the FDAꢀapproved drug pentamidine16. In these
studies, pentamidine, 4b (SBi1) binds two pockets in the p53 binding site on Ca2+ꢀS100B (termed Sites 2 & 3,
Fig. 1), and it was repurposed for use in a phase II clinical trial for treatment of relapsed and/or refractory
melanoma in patients with detectable levels of S100B protein (www.clinicaltrials.gov Identifier:
NCT00729807). While p53 levels were often restored in these patients after treatment, the toxicity profile for
pentamidine was not ideal, likely in part since 4b binds to several cellular targets, including DNA20ꢀ22. For
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these reasons, structureꢀactivity relationship (SAR) studies are ongoing to improve the affinity and selectivity of
pentamidine analogues for binding Ca2+ꢀS100B. Thus far, modifying the chemical scaffold of pentamidine first
yielded heptamidine, 4a (SBi4211), which occupies the same two persistent binding sites as pentamidine (i.e.
Sites 2 & 3), only it does so with a single molecule bound to the protein versus two pentamidines/monomer 23.
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However, improvements to this drug scaffold are still needed to increase its affinity and a provide a meaningful
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therapeutic effect
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Therefore, in the present study, structureꢀactivity relationships (SARs) of novel
pentamidine/heptamidine derivatives were explored.
The SBiX compounds synthesized here bind Sites 2 and 3, as found for pentamidine and heptamidine;
however, few such analogues interact with Site 1 (Fig. 1), or showed potential for efficient extension into Site 1.
This is due to conformational exchange in this region involving Phe87 and Phe88, which serve as a gate to
access a narrow channel linking Sites 1 & 2 (termed the “FFꢀgate”). In one case, structural results with
compound 17 (SBi29), indicate that the Site 2/3 interactions in the S100B•17 complex are made in a unique
way, as compared to the other pentamidine analogues. Importantly, the FFꢀgate is “open” in the S100B•17
complex, such that it can accommodate modifications that would possibly extend into Site 1 from Site 2
efficiently while retaining an amidine moiety like those in the pentamidine diamidine scaffold. Importantly, the
gained understanding of the FFꢀgate now permits a more rational design of novel S100B inhibitor(s) that can
simultaneously occupy Sites 1 & 2 and perhaps all three targetꢀbinding pockets in the CaS100B•p53 complex
(i.e. Sites 1, 2, and 3). Although chemical scaffolds occupying Site 1 have been successfully identified24, 25
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combining moieties or tethering with pentamidine derivatives towards Sites 2 and 3 have not yet been achieved.
Therefore, the results described here represent a significant advance towards developing such SBiX molecules
that achieves a threshold required for a useful therapeutic window.
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LIGAND DESIGN STRATEGY:
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The development of small molecule inhibitors that block the CaS100B•p53 interaction requires inhibition of
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large proteinꢀprotein interfaces (PPIs) that were once thought to be difficult, if not impossible, to block. It is
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now recognized that the interfaces of most PPIs have smaller amino acid domain(s), termed “hot spots” or
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“persistent sites” that contribute more energetically to the PPIs than the surrounding protein surface
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Successful strategies to target small molecule PPI inhibitors were developed and, as a result, several PPIs have
been successfully inhibited, including those involving bclꢀ6, bclꢀ2, p53, ERK, tubulin, and several SH2
domains, to name but a few27, 28. For PPI’s involving S100B, two inhibitors, pentamidine (4b) and heptamidine
(4a), were discovered to block the CaS100B•p53 complex and occupy persistent binding Sites 2 and 3
simultaneously within Ca2+ꢀS100B23, 29 (Fig. 1). Efforts were made here to find the most efficient means to
extend the heptamidine scaffold towards Site 1 and create compounds capable of binding all three persistent hot
spots in S100B simultaneously (i.e. Sites 1, 2, and 3).
To facilitate this design strategy, the site identification by ligand competitive saturation (SILCS)
approach was applied to map the functional group requirements of the threeꢀtargeted sites. From the SILCS
calculations, 3D FragMaps are obtained that indicate the different functional group requirements of the protein,
information that can be used to identify moieties of a compound that are contributing to binding as well as to be
used to indicate how modifications of a compound can be used to improve affinity. During the design stage,
what is termed a “Tier 1” SILCS approach was used. This approach includes aromatic maps based on benzene,
aliphatic maps based on propane, and hydrogen bond donors and acceptors maps based on water hydrogen
atoms and oxygen atoms, respectively. Shown in Figure 2 are the SILCS FragMaps overlaid on the crystal
structure of the CaS100B•SBi132 complex (PDB 3GK1) along with the crystallographic orientation of
heptamidine (PDB 4FQO). The approximate locations of the three hot spots are labeled. The occupancy of
Sites 2 and 3 by heptamidine is evident, with aromatic and aliphatic FragMaps in the vicinity of the aromatic
rings and aliphatic linker of heptamidine indicating the contribution of those moieties to ligand binding. The
aliphatic and aromatic FragMaps cover a relatively wide range indicating that variations in the linker length
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between the aromatic rings could be tolerated. Notably, only small hydrogen bond donor FragMaps are directly
adjacent to either amidine moiety, indicating that the positive charges may not be essential for binding. In
addition, in regions beyond the amidine moieties in Sites 1 and 3 are hydrogen bond donor FragMaps that are
adjacent to acidic residues (Fig. 2B), suggesting that extending those moieties with additional positively
charged groups with linkers of varying lengths may lead to improved binding. Therefore, functionalities were
added to the amidine moieties of both heptamidine (4a) and pentamidine (4b)23 in an attempt to improve
affinity. In addition, the extended aromatic/aliphatic FragMaps around the linker region in the P2 site,
supported by other CaS100B•SBiX Xꢀray structures23, 29, 30, indicated that increased diversity of the linker could
facilitate binding.
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Based on these design criteria and synthetic considerations, a number of novel analogs were developed.
Compounds of series 5, 6 and 7 (Scheme 2) include modification of the amidine moiety, yielding neutral
species in aliphatic rings ranging in size from 5 to 7 nonꢀhydrogen atoms that may have favorable interactions
as evidenced by the aliphatic FragMaps in the vicinity of the amidine moieties and the adjacent aromatic rings.
Alternatively, the amidine moieties were substituted with phenylamine and amide in 10 and 11. In addition, in
the 5, 6 and 7 series the linker length between the aromatic rings was varied from n = 3, as in pentamidine, to n
= 6, which is one methylene group longer than heptamidine. To take advantage of the donor FragMaps in the
vicinity of acid groups (Fig. 2B) compounds 8 and 9 were designed. These include a primary amino group
terminating aliphatic linkers of 5 and 7 methylene groups in 8 and 9, respectively, with different linker lengths
between the aromatic rings, such that 8 represents a shorter compound as compared to the two 9 analogs. To
address the presence of the aliphatic and aromatic FragMaps in the vicinity of the linker in heptamidine (Fig.
2A), compounds 16a and b were designed, in which a linker of the same length as that in heptamidine is present
that contains a tertiary amine to which a benzyl or propyl moiety is attached. An additional compound, 17, was
identified later in the project based on the developed SAR model, as described below.
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CHEMICAL SYNTHESIS
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Symmetric molecules flanked by diamidine moieties were synthesized using short, convergent syntheses
strategies as shown in schemes 1 to 4. For example, pentamidine derivatives 4a-c were synthesized by the
Pinner reaction as described (Scheme 1) starting from 4ꢀcyanophenol23.
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Scheme 1: Preparation of diamidines 4a-c
Reagents and conditions: (a) CsCO3, acetone, reflux, 14 hrs.; (b) methanol:dioxane (1:1), HCl gas; (c)
ethanolic ammonia, ammonium chloride, 80 °C, 32 hrs. Cyclic amidines (5a-d, 6a-d, and 7a-d) were
synthesized according to literature with some modification (Scheme 2). Specifically, treatment of
dibenzimidate with diaminoethane, diaminopropane and diaminobutane at room temperature in DMF
accomplished cyclic amidine in satisfactory yield31ꢀ33. Increasing the chain length of the diamine results in
open chain products (8 and 9a, 9b), which were useful for testing aliphatic chain derivatives of varying lengths
(Scheme 2).
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Scheme 2: Preparation of cyclic and fatty amine substituted diamidine analogs
Reagents and conditions: (a) 1,2ꢀdiaminoethane, DMF, rt; (b) 1,3ꢀdiaminopropane, DMF, rt; (c) 1,4ꢀ
diaminobutane, DMF, rt; (d) 1,2ꢀdiaminopentane, DMF, rt; (e) 1,2ꢀdiaminoheptane, DMF, rt.
For further synthesis of Nꢀderived diamidine compound 10, compound 4a was coupled with phenyl isocyanate
to furnish final diamidine compound in good yield. Compound 11 was obtained through the carbomylation of
compound 4a with NꢀsuccinamidylꢀNꢀmethyl carbamate at 40 oC for 2 hours (Scheme 3)34.
Scheme 3: Preparation of N-acyl substituted diamidine analogs
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Reagents and conditions: (a) phenyl isocyanate, DBU, DMF, 40 °C; (b) NꢀsuccinamidylꢀNꢀmethyl carbamate,
DBU, DMF, 40 °C.
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Compounds 16a-b were synthesized to further diversify the structureꢀactivity relationship series, Nꢀ
ligated. For these compounds, 1,3ꢀdibromopropane was reacted with benzylamine and propylamine to obtain
compound 13a-b, which then reacted with 4ꢀhydroxybenzonitrile to furnish the intermediate 14a-b. After that,
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the wellꢀknown Pinner reaction was used to prepare the target compounds 16a-b (Scheme 4)35, 36
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Scheme 4: Preparation of analogs with central amine substitutions
Reagents and conditions: (a) For 13a, Benzylamine, 13b, Propylamine, K2CO3, acetone; (b) 1, CsCO3, acetone;
(c) methanol, HCl gas; (d) ethanolic ammonia, ammonium chloride. One additional commercial compound, 17,
was included in the study. Commercially available compound 17 maintains many of the advantageous
functionalities of the pentamidine scaffold but is a more rigid scaffold since it has fewer rotatable bonds.
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RESULTS & DISCUSSION:
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Characterization of cyclic diamidine analogs bound to Ca2+-S100B (CaS100B). Twelve compounds
prepared in Scheme 2 retained the linker scaffold as found for compounds in Scheme 1; however, the diamidine
moiety for these twelve molecules was modified to form a five membered ring (compounds 5a-5d), a six
membered ring (compounds 6a-6d), or a seven membered ring (compounds 7a-7d; Scheme 2). These
compounds (Scheme 1) were designed to retain hydrogen bond donor capacity and to include aliphatic
hydrocarbon regions on the compounds that could potentially provide hydrophobic interactions with residues
Leu44, Ile80, and Ala83 of Ca2+ꢀS100B at or nearby the lower portion of Site 1 as indicated by the aliphatic
FragMaps adjacent to the amidine moiety (Fig. 2).
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Using an established fluorescent polarization competition assay (FPCA)1, compounds 5a-5d, 6a-6d, and
7a-7d were tested to determine if they were able to compete with a probe, TAMRAꢀTRTK, which binds Site 1
in Ca2+ꢀS100B (Table 1). Of the twelve compounds in this series of cyclic diamidine analogues, they all
interacted with Ca2+ꢀS100B as measured using NMR; however, none were able to compete for binding with the
TAMRAꢀTRTK probe at low concentrations in the FPCA (i.e. < 1mM). In cellular assays (Table 2), EC50
values for these compounds were all in the micromolar range (EC50: 5a-d ≈ 20ꢀ60 ꢁM; 6a-6b ≈ 200ꢀ600 ꢁM;
6c-6d ≈ 25ꢀ45 ꢁM; 7a, 7b, 7d ≈ 5ꢀ20 ꢁM; 7c ≈ 250ꢀ300 ꢁM; Table 2) but the EC50 values for the compounds in
all four of these series were all higher than those observed for comparable studies with pentamidine (EC50:
4b=17±2 ꢁM; Table 2) and none showed improvement over that observed for heptamidine, which also
demonstrated an S100B specific affect (High S100BEC50: 4b=11±2 ꢁM; Low S100BEC50: 4b=26±4 ꢁM; Table 2).
While the NMR spectra of Ca2+ꢀS100B upon binding to 5a, 6a, 6c, and 6d showed similar chemical shift
perturbations as pentamidine and heptamidine (Table 3), the binding of the other compounds in this series (5b,
5c, 5d, 6b, 7a-7d) caused significant broadening to the NMR spectra either due to an intermediate exchange
binding regime and/or because of protein aggregation. The NMR and FPCA results provided indication that the
cyclic diamidine moieties in 5a-5d, 6a-6d, or 7a-7d did not interact with Site 1 of Ca2+ꢀS100B. Nonetheless,
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Xꢀray crystallography experiments were initiated and structure determinations were completed for Ca2+ꢀS100B
complexes with compounds 5a (SBi4225) and 6b (SBi4214) to further explore this possibility.
Global Conformation. Xꢀray structures of Ca2+ꢀS100B bound to either 5a or 6b consists of four helixes
(helix 1, E2ꢀG19; helix 2, K28ꢀL40; helix 3, E49ꢀD61; helix 4, D69ꢀF86) and is assembled as an active
symmetric homodimer (Fig. 1a), as found previously in other structures of S100B. Both the typical and nonꢀ
canonical EFꢀhand calcium binding domains in each subunit were found to coordinate a Ca2+ ion in the two
structures29, 30, 37, 38. A comparison with the structure of Ca2+ꢀS100B in the absence of compound (PDB ID:
1MHO39) shows a strong conservation of the global fold (see Supporting Information Table S1) with the only
significant deviation observed at Cꢀterminal residues His85ꢀGlu91 involving, most notably, residues Phe87 and
Phe88 (Fig. 3d,4d). This segment of the polypeptide consistently conforms to accommodate the binding of the
SBiX molecules, as similarly found for the binding of pentamidine and heptamidine.
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CaS100B•5a. The Xꢀray structure of 5a bound, calcium loaded S100B (CaS100B•5a) was solved at 1.53 Å
resolution (Fig. 3a). The asymmetric unit contained one S100B monomer with the second monomer forming
the biologically active dimeric form of S100B contained within the crystal symmetry. Residues Met0 to His90
were modeled. The monomer in the CaS100B•5a structure bound two calcium ions and a 5a molecule. In the
final refined model (Rcrys/Rfree of 0.195/0.216), all residues were in the favored (100%) region of the
Ramachandran Plot with no outliers (see Table 4).
CaS100B•6b. The Xꢀray structure of 6b bound, calcium loaded S100B (CaS100B•6b) was solved at 1.59 Å
resolution (Fig. 4a). The asymmetric unit contained one S100B monomer with the second monomer forming the
biologically active dimeric form of S100B contained within the crystal symmetry. Residues Met0 to His90 were
modeled. The monomer in the CaS100B•6b structure bound two calcium ions and a 6b molecule. In the final
refined model (Rcrys/Rfree of 0.207/0.230), all residues were in the favored (100%) region of the Ramachandran
Plot with no outliers (see Table 4).
Structural comparisons of CaS100B•5a, CaS100B•6b, CaS100B•Pentamidine, and CaS100B•Heptamidine. As
both 6b and 5a are derivative compounds based on the previously published SBiX molecules pentamidine (4b)
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and heptamidine (4a), they share similar binding sites and interactions as their predecessors23, 29 (Fig. 3b,c and
Fig. 4b,c). These derivatives bind within close proximity of several residues of S100B in a pocket formed by
Helix 4, Loop2, and Helix1´ (Fig. 3d and Fig. 4d). The diazacyclohexene and the imidazoline groups of 6b and
5a, respectively, are able to participate in hydrogen bonds with the backbone carbonyl His85 (Helix 4) while
participating in stacking interactions with Phe88 and Phe87 (Helix4). Such interactions were previously
described in pentamidine and heptamidine structures. Similar to both pentamidine and heptamidine, 5a and 6b
occupy both binding Sites 2 and 3. However, like heptamidine and unlike pentamidine, the derivatives occupy
both sites with a single molecule. When compared to the preceding compounds, the larger substituent groups of
5a and 6b within Site 2 do not extend into Site 1, consistent with the FPCA results above. Rather, in both cases,
the ring starts to protrude into solvent with few if any interactions with the protein. On the other hand, within
Site 3 the larger ring displaces the benzyl moiety 0.9ꢀ1.2 Å from the center in both cases (i.e. for 5a and 6b)
while the second benzyl moieties occupy the same hydrophobic site formed by the sidechains of Leu40, Phe43,
and Phe87. However, neither derivative compound makes the hydrogen bond with the backbone carbonyl of
Phe43 as observed with heptamidine.
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Characterization of non-cyclic aliphatic amine substituted diamidine analogs. Three additional
compounds (8, 9a, 9b) prepared in Scheme 2 retained the linker scaffold as in the Scheme 1 compounds;
however, the diamidine moiety for these three molecules was modified to include longꢀchain aliphatic primary
amine moieties. It was thought that these compounds would also retain a hydrogen bond donor as found in the
original diamidines (in Scheme 1), but extend into Sites 1 and 3 to occupy regions indicated by donor SILCS
FragMaps adjacent to acidic residues (Fig 2B). In addition, the linkers may provide hydrophobic interactions
with residues of Ca2+ꢀS100B at or nearby the lower portion of Site 1, also termed here as a channel connecting
Sites 1 and Sites 2 (i.e. with residues Phe43, Leu44, Ile80, and Ala83) as indicated by the SILCS FragMaps
(Fig. 2).
The in vitro fluorescence polarization competition assay (FPCA) was completed with these three compounds
and they were all (8, 9a, 9b) found to compete with the Site 1 probe, so IC50’s were measured and the
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Journal of Medicinal Chemistry
dissociation constants determined to be in the low micromolar range in all cases (<5 ꢁM; see Table 1).
However their EC50 values in cells were significantly higher than their KD values (EC50: 8 ≈ 35ꢀ40 ꢁM; EC50:
9a-b ≈ 25ꢀ50 ꢁM), so offꢀtarget affects are likely in each of these cases. Likewise, all three of these compounds
interacted with Ca2+ꢀS100B as measured using NMR and in the case of compound 8, it showed similar chemical
shift perturbations as pentamidine and heptamidine, as well as numerous additional perturbations. The other
two compounds (9a, 9b) caused significant broadening to the NMR spectra either due an intermediate exchange
and/or because of protein aggregation. In these cases, the NMR and FPCA results provided indication that the
longꢀchain primary amine moiety did indeed interact with Site 1 (Table 1). Nonetheless, Xꢀray crystallography
experiments were initiated and structure determinations were attempted for Ca2+ꢀS100B complexes with
compounds 8, 9a, and 9b to further explore this possibility.
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Coꢀcrystals of 8, 9a, and 9b were obtained from conditions similar to that of 6b and 5a. Although an
examination of electron density maps could confirm the presence of smallꢀmolecule ligands occupying the
predicted binding sites, this subꢀfamily of compounds maintained low occupancy despite various attempts at
improvement. Amongst these compounds, the S100B•9a crystal diffraction data provided the best ligand
electron density, and the atoms of benzamidineꢀlike chemical groups could be accurately modeled. However,
the acyl chains terminated with amino groups could not be tracked in the electron density with the same
confidence. Therefore, in silico methods were used to predict the positions of atoms with weak and/or missing
electron density (see Supporting Information Fig. S1). Both AutoDock and MCꢀSILCS sampling similarly
place the linker alkyl chain. The location of one of the terminal alkyl chains predicted by AutoDock places the
amino group such that it hydrogen bonds with Glu86 and His85. The location of the second amino group does
not allow hydrogen bonding with the protein. The only favorable interactions would be with the hydrophobic
environment provided by the sidechains of Leu44, Ala83, and Phe88. MCꢀSILCS, on the other hand, places the
first amino group in the vicinity of Glu2 (of the other S100B chain), and the second group close to Glu46,
forming hydrophilic interactions in both cases. These locations are associated with the positive donor SILCS
FragMap adjacent to these residues leading to favorable placement of the basic group (see Supporting
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Information Fig. S1). The MCꢀSILCS docking also point to the diversity of conformations populated by the
terminal groups. The additional hydrogen bonding predicted by AutoDock and/or MCꢀSILCS would explain the
increased affinity of this subꢀfamily for S100B as measured by FPCA. The diversity of orientations found by
the two methods is also consistent with the alkyl tails not being resolved in the crystal structure. The small
differences in affinity between the amino group containing compounds are likely due to the varying lengths of
linkers and associated positions of the amino groups, which would likely impact the hydrogen bond network
between the ligands and the protein. Importantly, the SILCS modeling nicely explains why these molecules
compete with TRTK12 since an interaction at Glu46 would compete with the interactions between TRTK12 and
S100B as seen in the coꢀcrystal structure37.
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Characterization of N-acyl substituted diamidine analogs. Two compounds (10, 11) were prepared as
shown in Scheme 3 that retained the heptamidine linker scaffold; however, the diamidine moieties for these
molecules were designed to retain two hydrogen bond donors as found in heptamidine, but also have some
aliphatic character either via a phenyl ring (compound 10) or with a methyl group (compound 11). Both of
these compounds had an affect the cellular assays with compound 10 having contributions from general toxicity
effects (EC50: 10 < 1 ꢁM; EC50: 11 ≈ 130ꢀ150 ꢁM). The in vitro fluorescence polarization competition assay
(FPCA) was completed with these compounds and neither was able to compete with TAMRAꢀTRTK indicating
that they do not interact with Site 1 despite their ability to bind Ca2+ꢀS100B as determined by NMR (see
Supporting Information Fig. S2ꢀ5). 11 showed a significant number of chemical shift perturbations that
mimicked those found for pentamidine and heptamidine (see Supporting Information Fig. S6). 10 did not
perturb chemical shifts at the concentrations tested. Although, Xꢀray crystallography experiments were
initiated, crystallization of Ca2+ꢀS100B complexes with compounds 10 and 11 were not successful. While the
atomic level detail of compounds 10 and 11 bound to Ca2+ꢀS100B remains somewhat elusive, the NMR and
FPCA data indicate that neither of these compounds extends into Site 1.
Characterization of diamidine analogs with a central substituted amine. Two compounds (16a, 16b)
were prepared as shown in Scheme 4 that retained the symmetric diamidine moieties; however, the heptamidine
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Journal of Medicinal Chemistry
linker was designed to include a substituted secondary amine. Such a substitution was designed to complement
a hydrophobic pocket that is indicated in the SILCS FragMaps and observed in the heptamidine Xꢀray crystal
structure 23. For these complexes, the in vitro fluorescence polarization competition assay (FPCA) was
completed and as expected, neither was able to compete with TAMRAꢀTRTK. Unexpectedly, the compounds
bound weakly and the chemical shift perturbations were unlike those observed for pentamidine and heptamidine
suggesting an alternativeꢀbinding mode. The EC50 values for these compounds were measured in cellular
assays and found to affect cell death quite differently with 16b having little if any effect on cellular functions
(EC50: 16a ≈ 40ꢀ50 ꢁM; EC50: 16b > 500 ꢁM).
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Characterization of an asymmetric diamidine molecule. In all of the structural studies completed thus far
with symmetric diamidines, it was becoming clear that the position of Phe88 in the pocket between Sites 1 and
2 was a common feature. Such conformation of the protein target necessitated the synthesis of very large
molecules to circumvent this occlusion and to compete with a Site 1 binding peptide (see Supporting
Information Fig. S1). This requires a compound that eliminates the symmetric nature of the
pentamidine/heptamidine scaffold such that only Site 3 interactions would occur. In addition, it was of interest
to determine whether it was possible to have the channel between Sites 1 and 2 open while occupying Sites 2
and 3 with an inhibitor. Using chemical similarity searching with the program MOE (Chemical Computing
Group) we searched an in house database of commercially available molecules for those that contain a single
aromatic amidine moiety as present in heptamidine. One commercial molecule, compound 17, fit these criteria
and was tested for its ability to bind to Ca2+ꢀS100B. Remarkably, 17 was found to bind Ca2+ꢀS100B via NMR,
has a binding affinity in the low micromolar range (KD=6.0 ± 0.2 ꢁM; Table 1), and had a low EC50 in cellular
assays (EC50 < 1 ꢁM; Table 2) likely due to contributions from general toxicity affects. Therefore, compound
17 is not itself an endpoint for a pharmaceutical, but it turned out to provide very important information for
structureꢀbased drug design. Specifically, 17 was found to inhibit TAMRAꢀTRTK binding into Site 1 in the
FPCA, despite being designed to interact with Sites 2 and 3. Notably, this is the first small molecule with a
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pentamidineꢀlike scaffold (M.W. < 500 Da) shown to block the Site 1 binding peptide TAMRAꢀTRTK. Xꢀray
crystal determination of CaS100B•17 was thus undertaken to identify the binding site of 17.
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CaS100B•17. The Xꢀray structure of 17 bound, calcium loaded S100B (CaS100B•17) was solved at 1.74 Å
resolution (Fig. 5a). The asymmetric unit contained two S100B monomers. Residues Met0 to Phe88 were
modeled in monomer A while residues Met0 to Glu89 were modeled in monomer B. The monomers in the
CaS100B•17 structure bound two calcium ions. Only one molecule 17 was modeled as the symmetrical binding
cleft formed by the dimer remained vacant. In the final refined model (Rcrys/Rfree of 0.209/0.247), all residues
were in the favored (100%) region of the Ramachandran Plot with no outliers (see Table 4). The occupation of
the binding cleft by 17 in Sites 2 & 3 was discovered to indeed be the case, but it required an entirely different
conformation of helix 4 versus that observed in the structures of the other pentamidine/heptamidine scaffold
compounds. In fact, 17 binds a more nativeꢀlike conformation (unliganded) of Ca2+ꢀS100B while other
pentamidine derivatives require a breaking of the last turn of helix 4 coinciding with a repositioning of Phe87
and Phe88 in stacking position with the cyclic moieties (Fig. 1c). Although the global positioning of 17 in Site 2
& 3 remains similar (Fig. 4b,c), there are other major differing interactions with the protein. The
carbamimidoylꢀphenoxyl group of 17, which would be expected to hydrogen bond with the backbone carbonyl
of His85, as occurs with pentamidine, lies in the hydrophobic pocket formed by Val8, Phe88, and Met0, where
it makes bidentate hydrogen bonds with the sidechain carbonyl of Asp12 (Fig. 5d). In addition, the indoleꢀ
carboximidamide of 17 is in the hydrophobic pocket formed by Ala83, Phe43, and Phe87 and makes hydrogen
bonds with not only the backbone carbonyl of Phe43 but also the backbone carbonyl His42 and the sidechain
carbonyl of Glu45. Notably, the binding orientation of 17 is such that the FFꢀgate residue, Phe88, assumes an
open conformation that allows access between Sites 1 and 2. Thus, when comparing all of the inhibitor
complex structures, so far available for Ca2+ꢀS100B, there are two major conformations observed for the
position of the Cꢀterminal residues. One is with Phe88 within the small channel connecting Sites 1 & 2 (i.e. the
“closed” FFꢀgate) and the other is with this small channel open and Phe88 outside this narrow channel (i.e. the
“open” FFꢀgate) and part of helix 4 (Fig. 5).
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Journal of Medicinal Chemistry
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CONCLUSIONS
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Three binding sites or “hot spots” on Ca2+ꢀbound S100B were recognized in structural studies with several
different CaS100B•peptide and CaS100B•SBiX complexes38, 40, 41. Pentamidine, which binds to Sites 2 and 3,
exhibits enough potency to be used in clinical trials for malignant melanoma. However, its low efficacy, poor
specificity, and toxicity issues limit its therapeutic potential and needed to be addressed. The development of
heptamidine was a reasonable improvement as indicated by increased efficacy in cellular assays, with the
efficacy shown to be dependent on S100B levels (Table 2). In the evaluation of heptamidine, a more stable
knockdown of S100B was developed and used, and selectivity in killing melanoma cells with high S100B over
those with low S100B was clearly observed.
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In this study, we attempted to extend the heptamidine scaffold from binding Sites 2 and 3 towards Site 1. The
addition of aliphatic amine groups indeed showed significant competition with the Site 1 and can be used as a
molecular probe of the S100B binding pocket. Also, the results here indicate the optimal length of linker region
between the aromatic moieties and from the amidine to amine are seven methylene groups. From the available
crystallographic structure of S100B in complex with heptamidine 16, the importance of hydrogen bonding made
with the backbone carbonyl of Phe43 may be assumed. Although higher affinity binders were observed in vitro,
it did not improve the efficacy in cellular assays when compared to many of the other diamidine derivatives (see
Table 2); this result may be due to decreased cellular absorption or unfavorable solubility.
Further analysis of the crystal structures of S100B in complex with symmetric pentamidine analogs revealed a
consistent reorganization of the Cꢀterminal helix from the native state towards the closing of the “FFꢀgate”. This
includes heptamidine even though such a reorganization of the Cꢀterminal helix was not reported in the original
coordinates deposited in the PDB. A reexamination of the deposited raw data for CaS100Bꢀheptamidine
(structure factors) and newly calculated maps, in light of these new structures, showed that the heptamidineꢀ
bound state of S100B does indeed exhibit the same reorganization of the Cꢀterminal helix associated with a
“closed” FFꢀgate, with Pheꢀ88 blocking the cavity connecting Sites 1 & 2, as found here for structures of
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CaS100B•5a and CaS100B•6b. Thus, the protein conformational change necessary for the binding of symmetric
pentamidine analogs leads to closing of the FFꢀgate, which pushes the inhibitors away from Site 1 and towards
bulk solvent. As the occupation of Site 1 (the p53ꢀpeptide binding site) is predicted to be important for
successful inhibition of the targeted PPI, the dependence on this conformation change for binding has proven to
be problematic. The inability of pentamidine/heptamidine and many of the analogs to compete effectively with
the peptide probe of Site 1 may, at least partially, be explained by the need for the FFꢀgate to be in the “open”
conformation with a more nativeꢀlike orientation of Pheꢀ87 and Pheꢀ88. Relaxation dispersion curves of S100B
in the absence and presence of peptides or small molecule inhibitors indicate that the Cꢀterminus of Ca2+ꢀS100B
exists in at least two slowly exchanging conformations in the millisecond time regime in solution, consistent
with the FFꢀgating mechanism proposed here 42 (Fig. 6). Notably, by breaking the symmetry of heptamidine and
its other analogs we identified a compound, 17, that binds to the “open” FFꢀgate while maintaining several
desirable features of the pentamidine scaffold. Unfortunately, the killing efficacy of 17 far exceeds in binding
affinity and shows no S100Bꢀdependent killing in S100Bꢀknockdown cell lines. Thus, compound 17 does not
resolve the toxicity problems of pentamidineꢀlike compounds, but its structure bound to CaS100B represents an
important advance in S100B inhibitor design, perhaps as a linker, since it enables the design of inhibitors that
can take advantage of all three hot spots in CaS100B for the first time (i.e. Sites 1, 2 and 3; Figure 1).
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Journal of Medicinal Chemistry
EXPERIMENTAL PROCEDURES
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All reactions were performed under an atmosphere of nitrogen, and all solvents were removed on a rotary
evaporator under reduced pressure. TLC was performed on plates coated with silica gel GHLFꢀ0.25 mm plates
(60 F254) (Analtech). Mass spectra were obtained on a ThermoFinnigan LCQ Classic. 1H and 13C NMR spectra
were obtained using a 500 MHz Varian NMR Spectrometer. Melting points were determined in open capillary
tubes using a MelꢀTemp melting point apparatus; melting points are uncorrected. Combustion analysis was
performed by Atlantic Microlab, Inc. (Norcross, GA). Synthesis follows that of Hisashi et al. (Scheme 1ꢀ4)
Purity of all of the compounds synthesized and/or used in this study were determined to be >95% by HPLC
analyses (see Supporting Information Fig. S7ꢀ29).
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Heptamidine (4a), pentamidine (4b), hexamidine (4c): Compounds 4aꢀc were synthesized by reported
procedure23 as shown in scheme 1.
1,7-bis (4-(4, 5-dihydro-1H-imidazol-2-yl)phenoxy)heptane (5a): 3a (0.1 g, 0.25 mmol) and 1,2ꢀ
diaminoethane (0.075 g, 1.25 mmol) were dissolved in DMF (2 mL) and stirred at room temperature for
overnight. DMF was removed under vacuum (Scheme 2). The crude reaction mixture was purified on neutral
alumina column (6:94 Methanol/Chloroform) to yield the pure product 5a as a white solid Yield 61%; Mp (250ꢀ
252 °C). 1H NMR (400 MHz, DMSOꢀd6): δ 7.98 (d, J = 8.8 Hz, 4H), 7.11 (d, J = 8.8 Hz, 4H), 4.07 (t, J = 6.2
Hz, 4H), 3.85 (m, 8H), 1.72ꢀ1.74 (m, 4H), 1.41 (m, 6H); 13C NMR (100 MHz, DMSOꢀd6): δ 163.8, 162.6,
130.3, 116.0, 114.7, 67.9, 45.3, 28.3, 25.3; MS (ESI) m/z: 421 [M+H]
Compounds 5b-d were similarly synthesized as per the above procedure (Scheme 2).
1,5-bis(4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy)pentane (5b): White solid; Yield 61%; Mp (250ꢀ252 °C);
1H NMR (400 MHz, DMSOꢀd6): δ 8.01 (d, J = 8.8 Hz, 4H), 7.13 (d, J = 8.4 Hz, 4H), 4.11 (t, J = 6.4 Hz, 4H),
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3.83 (m, 8H), 1.77ꢀ1.82 (m, 4H), 1.57ꢀ1.59 (m, 2H); C NMR (100 MHz, DMSOꢀd6): δ 164.3, 162.2, 130.9,
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1,6-bis(4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy)hexane (5c): Off white solid, Yield 63%; Mp (221ꢀ223
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°C); H NMR (400 MHz, DMSOꢀd6): δ 8.00 (d, J = 8.4 Hz, 4H), 7.12 (d, J = 8.4 Hz, 4H), 4.09 (t, J = 6.2 Hz,
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4H), 3.85 (m, 8H), 1.76 (m, 4H), 1.48 (m, 4H); C NMR (100 MHz, DMSOꢀd6): δ 164.3, 163.3, 130.9, 115.8,
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115.3, 68.4, 45.4, 28.8, 25.5; MS (ESI) m/z: 407 [M+H]
1,8-bis (4-(4, 5-dihydro-1H-imidazol-2-yl) phenoxy) octane (5d): Off white solid, Yield 61%; Mp (198ꢀ200
°C); 1H NMR (400 MHz, DMSOꢀd6): δ 8.01 (d, J = 8.4 Hz, 4H), 7.14 (d, J = 8.4 Hz, 4H), 4.08 (t, J = 6.4 Hz,
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4H), 3.91 (m, 8H), 1.71ꢀ1.75 (m, 4H), 1.35ꢀ1.42 (m, 8H); C NMR (100 MHz, DMSOꢀd6): δ 163.9, 163.1,
130.6, 114.9, 68.1, 44.6, 28.6, 28.3, 25.3; MS (ESI) m/z: 435 [M+H]
1,7-bis (4-(1,4,5,6-tetrahydropyrimidin-2-yl)phenoxy)heptane (6a): 3a (0.1 g, 0.25 mmol) and 1,3ꢀ
diaminopropane (0.092 g, 1.25 mmol) were dissolved in DMF (2 mL) and stirred at room temperature for
overnight. DMF was removed under vacuum (Scheme 2). The crude reaction mixture was purified on neutral
alumina column (6:94 Methanol/Chloroform) to yield the pure product 6a as a white solid , Yield 65%; Mp
(183ꢀ185 °C); 1H NMR (400 MHz, DMSOꢀd6): δ 7.72 (d, J =8.4 Hz, 4H), 7.12 (d, J = 8.4 Hz, 2H), 4.06 (t, J =
13
6.4 Hz, 4H), 3.45ꢀ3.47 (m, 8H), 1.94 (m, 4H), 1.72ꢀ1.74 (m, 4H), 1.41 (m, 6H); C NMR (100 MHz, DMSOꢀ
d6): δ 162.8, 158.8, 129.8, 120.4, 115.1, 68.4, 28.8, 25.8, 18.3; MS (ESI) m/z: 449 [M+H]
Compounds 6b-d were similarly synthesized as per the above procedure (Scheme 2).
1,5-bis(4-(1,4,5,6-tetrahydropyrimidin-2-yl)phenoxy)pentane (6b): White solid, Yield 62%; Mp (99ꢀ101
°C); 1H NMR (400 MHz, DMSOꢀd6): δ 7.83 (d, J = 8.8 Hz, 4H), 7.23 (d, J = 8.4 Hz, 4H), 4.19 (t, J = 6.2 Hz,
4H), 3.55 (m, 8H), 1.92ꢀ2.05 (m, 4H), 1.87ꢀ1.92 (m, 4H), 1.67ꢀ1.9 (m, 2H); 13C NMR (100 MHz, DMSOꢀd6):
δ 162.8, 158.8, 129.8, 120.5, 115.1, 68.3, 28.6, 22.5, 18.3; MS (ESI) m/z: 421 [M+H]
1,6-bis(4-(1,4,5,6-tetrahydropyrimidin-2-yl)phenoxy)hexane (6c): 3c (0.1 g, 0.26 mmol); and 1,3ꢀ
diaminopropane (0.096 g, 1.3 mmol) were reacted using the same procedure as the one described for the
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1
synthesis of 6a, providing 6c as white solid, Yield 61%; Mp (274ꢀ276 °C); H NMR (400 MHz, DMSOꢀd6): δ
1
2
3
7.72 (d, J = 8.4 Hz, 4H), 7.13 (d, J = 9.2 Hz, 4H), 4.08 (t, J = 6.2 Hz, 4H), 3.44ꢀ3.48 (m, 8H), 1.94ꢀ1.97 (m,
4
5
6
13
4H), 1.76 (m, 4H), 1.49 (m, 4H); C NMR (100 MHz, DMSOꢀd6): δ 162.8, 158.8, 129.8, 120.5, 115.1, 68.4,
7
8
28.8, 25.6, 18.3; MS (ESI) m/z: 435 [M+H]
9
1,8-bis(4-(1,4,5,6-tetrahydropyrimidin-2-yl)phenoxy)octane (6d): White solid, Yield 64%; Mp (260ꢀ262
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°C); H NMR (400 MHz, DMSOꢀd6): δ 7.73 (d, J = 8.8 Hz, 4H), 7.13 (d, J = 8.8 Hz, 4H), 4.06 (t, J = 6.6 Hz,
4H), 3.34ꢀ3.47 (m, 8H), 1.94ꢀ1.96 (m, 4H), 1.72ꢀ1.75 (m, 4H), 1.36ꢀ1.42 (m, 8H); 13C NMR (100 MHz,
DMSOꢀd6): δ 162.8, 158.8, 129.8, 115.1, 68.4, 29.1, 28.9, 25.8, 18.3; MS (ESI) m/z: 463 [M+H]
1,7-bis(4-(4,5,6,7-tetrahydro-1H-1,3-diazepin-2-yl)phenoxy)heptane (7a): 3a (0.1 g, 0.25 mmol) and 1,4ꢀ
diaminobutane (0.11 g, 1.25 mmol) were dissolved in DMF (2 mL) and stirred at room temperature for
overnight. DMF was removed under vacuum (Scheme 2). The crude reaction mixture was purified on neutral
alumina column (6:94 Methanol/Chloroform) to yield the pure product 7a as a white solid, Yield 60%; Mp
1
(169ꢀ171 °C); H NMR (400 MHz, DMSOꢀd6): δ 7.71 (m, 4H), 7.1 (m, 4H), 4.06 (m, 4H), 3.62 (m, 8H), 1.92
(m, 8H), 1.73 (m, 4H), 1.42 (m, 6H); 13C NMR (100 MHz, DMSOꢀd6): δ 164.5, 163.3, 131.5, 121.6, 114.9,
68.4, 28.8, 25.9, 25.7; MS (ESI) m/z: 477 [M+H]
Compounds 7b-d were similarly synthesized as per the above procedure (Scheme 2).
1,5-bis (4-(4,5,6,7-tetrahydro-1H-1,3-diazepin-2-yl)phenoxy)pentane (7b): Off white solid, Yield 59%; Mp
(153ꢀ155 °C); 1H NMR (400 MHz, DMSOꢀd6): δ 7.74 (d, J = 6.00 Hz, 4H), 7.10 (d, J = 6.00 Hz, 4H), 4.10 (m,
4H), 3.63 (m, 8H), 1.92 (m, 8H), 1.80 (m, 4H), 1.57 (m, 2H); 13C NMR (100 MHz, DMSOꢀd6): δ 164.4, 163.2,
131.5, 121.6, 115.0, 68.4, 43.9, 28.5, 25.9, 22.4; MS (ESI) m/z: 449 [M+H]
1,6-bis(4-(4,5,6,7-tetrahydro-1H-1,3-diazepin-2-yl)phenoxy)hexane (7c): White solid, Yield 57%; Mp (174ꢀ
1
176 °C); H NMR (500 MHz, DMSOꢀd6): δ 7.57ꢀ7.71 (m, 4H), 7.09ꢀ7.21 (m, 4H), 4.06ꢀ4.21 (m, 4H), 3.56ꢀ
3.69 (m, 4H), 3.12ꢀ3.22 (m, 4H), 1.81ꢀ2.01 (m, 4H), 1.62ꢀ1.79 (m, 6H), 1.51ꢀ1.59 (m, 2H), 1.40ꢀ1.51 (m, 4H).
MS (ESI) m/z: 463 [M+H]
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1,8-bis (4-(4,5,6,7-tetrahydro-1H-1,3-diazepin-2-yl)phenoxy)octane (7d): 3d (0.1 g, 0.24 mmol) and 1,4ꢀ
diaminobutane (0.106 g, 1.21 mmol) were reacted using the same procedure as the one described for the
synthesis of 7a, providing 7d as off white solid, Yield 62%; Mp (223ꢀ225 °C); 1H NMR (400 MHz, DMSOꢀd6):
δ 7.69 (d, J = 7.6 Hz, 4H), 7.11 (d, J = 8.8 Hz, 4H), 4.05ꢀ4.08 (m, 4H), 3.63 (m, 8H), 1.93 (m, 8H), 1.72ꢀ1.74
(m, 4H), 1.36ꢀ1.42 (m, 8H); 13C NMR (100 MHz, DMSOꢀd6): δ 164.5, 163.3, 131.5, 130.8, 121.6, 115.5, 114.9,
68.4, 43.9, 29.0, 28.8, 25.9, 25.7; MS (ESI) m/z: 491 [M+H]
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4,4'-(pentane-1,5-diylbis(oxy))bis(N-(5-aminopentyl)benzimidamide) (8): 3b (0.1 g, 0.25 mmol) and 1,2ꢀ
diaminopentane (0.128 g, 1.25 mmol) were dissolved in DMF (2 mL) and stirred at room temperature for
overnight. DMF was removed under vacuum, the crude reaction mixture was washed with chloroform (5 mL
each). Residue was extracted with water and chloroform, water layer was separated and evaporated to obtain
solid residue (Scheme 2). It was dissolved in methanol (1 mL) and product was precipitated by the addition of
diꢀethyl ether (5 mL). Solvent was decanted and dried in vacuum to obtain pure product 8a as a white solid
Yield 51%; Mp (109ꢀ111 °C); 1H NMR (400 MHz, DMSOꢀd6): δ 7.80 (d, J = 7.2 Hz, 4H), 7.09 (d, J = 7.6 Hz,
4H), 4.08 (m, 4H), 3.41 (m, 4H), 2.73 (m, 4H), 1.40ꢀ1.79 (18); 13C NMR (100 MHz, DMSOꢀd6): δ 162.8,
162.2, 130.6, 120.9, 114.9, 68.3, 42.7, 28.6, 27.3, 27.2, 23.4, 22.4; MS (ESI) m/z: 511 [M+H]
4,4'-(heptane-1,7-diylbis(oxy))bis(N-(7-aminoheptyl)benzimidamide) (9a): Procedure is similar as described
1
for 8. White solid , Yield 47%; Mp (98ꢀ101 °C); (Scheme 2); H NMR (400 MHz, CD3OD): δ 7.89 (d, J = 8
Hz, 4H), 7.30 (d, J = 8.4 Hz, 4H), 4.28 (m, 4H), 3.3 (m, 4H), 2.99 (m, 2H), 1.64ꢀ2.03 (m, 32H); 13C NMR (100
MHz, DMSOꢀd6): δ 162.8, 162.1, 130.5, 121.1, 114.9, 79.6, 68.4, 42.8, 28.8, 28.0, 27.2, 25.8, 23.5; MS (ESI)
m/z: 595 [M+H]
4,4'-(octane-1,8-diylbis (oxy))bis(N-(7-aminoheptyl)benzimidamide) (9b): Solid, Yield 42%; Mp (111ꢀ113
°C); 1H NMR (400 MHz, DMSOꢀd6): δ 7.79 (d, J = 8 Hz, 4H), 7.08 (d, J = 7.6 Hz, 4H), 4.04 (m, 4H), 2.68 (m,
13
4H), 1.53ꢀ1.70 (m, 12H), 1.25ꢀ1.39 (m, 24H); C NMR (100 MHz, DMSOꢀd6): δ 162.9, 162.1, 130.5, 120.8,
114.9, 68.4, 42.8, 40.3, 29.0, 28.8, 28.5, 28.4, 27.7, 26.4, 26.2, 26.1, 25.7; MS (ESI) m/z: 609 [M+H]
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4,4'-(heptane-1,7-diylbis(oxy))bis(N-(phenylcarbamoyl)benzimidamide) (10): 4a (0.1 g, 0.27 mmol) and
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DBU (0.16 mL, 1.1 mmol) were dissolved in DMF (2 mL) and stirred at 40 °C. A solution of phenyl isocyanate
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5
6
°
(0.06mL, 0.54mmol) in DMF (0.5 mL) was added to the above reaction mixture and was stirred at 40 C for 2
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hours. DMF was removed under reduced pressure, chloroform (20 mL) was added to the residue and washed
with water (10mL) (Scheme 3). The organic layer was separated, concentrated and purified on neutral alumina
column (4:96 Methanol/Chloroform) to yield the pure product 11 as a white solid , yield 68%; Mp (136ꢀ138
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°C); H NMR (400 MHz, DMSOꢀd6): δ 8.01 (d, J = 8.4 Hz, 4H), 7.65 (d, J = 8.0 Hz, 4H), 7.24 (t,J = 7.8 Hz,
4H) 7.03 (d, J = 9.6 Hz, 4H), 6.91ꢀ6.95 (m, 2H), 4.03ꢀ4.06 (m, 4H), 1.74 (m, 4H) 1.43 (m, 6H); 13C NMR (100
MHz, DMSOꢀd6): δ 163.9, 130.4, 129.1, 119.0, 114.7, 68.3, 28.9, 25.8; MS (ESI) m/z: 607 [M+H]
4,4'-(heptane-1, 7-diylbis (oxy))bis(N-(methylcarbamoyl)benzimidamide) (11): 4a (0.1 g, 0.27 mmol) and
DBU (0.20 mL, 1.35 mmol) were dissolved in DMF (2 mL) and stirred at 40 °C. A solution of Nꢀsuccinamidylꢀ
Nꢀmethyl carbamate (0.093 g, 0.54 mmol) in DMF (0.5 mL) was added to the above reaction mixture and was
°
stirred at 40 C for 2 hours (Scheme 3). DMF was removed under reduced pressure, and chloroform (20 mL)
was added to the residue and washed with water (10mL). The organic layer was separated, chloroform was
evaporated and purified on neutral alumina column (5:95 Methanol/Chloroform) to yield the pure product 13a
1
as a white solid , yield 61%; Mp (147ꢀ149 °C); H NMR (400 MHz, DMSOꢀd6): δ 7.90 (d, J = 8.4 Hz, 4H),
6.97 (d, J = 8.4 Hz, 4H), 4.02 (t, J = 6.4 Hz, 4H), 2.60 (s, 6H), 1.71ꢀ1.74 (m, 4H), 1.41 (m, 6H); 13C NMR (100
MHz, DMSOꢀd6): δ 162.8, 161.5, 129.3, 127.5, 114.2, 68.0, 28.9, 26.6, 25.8; MS (ESI) m/z: 483 [M+H]
N-benzyl-3-bromo-N-(3-bromopropyl)propan-1-amine (13a): Potassium carbonate (3.22 g/23.4 mmol/2.5
equivalents) and 1,3ꢀdibromopropane (12) (4.32 g/21.5 mmol/2.3 equiv.) was added to benzylamine (1.0
°
mL/9.3 mmol/1 equivalent) in acetone (50mL). The reaction mixture was refluxed for 48 hours at 60 C, and
then cooled to room temperature. Acetone was removed under reduced pressure to give a solid residue. Water
(75 mL) was added to the residue and extracted with dichloromethane (25 mL x 3). The organic layer was
separated and dried with sodium sulfate, filtered, and evaporated to give pure solid 13a (2.15 g, 69%) (Scheme
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4); 1H NMR (400 MHz, CDCl3): δ 7.14ꢀ7.54 (m, 5H), 3.29ꢀ3.61 (m, 6H), 2.21ꢀ2.46 (m, 4H), 1.92ꢀ2.14 (m, 4H);
MS (ESI) m/z: 350 [M+H]
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1
3-bromo-N-(3-bromopropyl)-N-propylpropan-1-amine (13b): H NMR (400 MHz, CDCl3): δ 3.53ꢀ3.41 (m,
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8
9
4H), 2.59ꢀ3.46 (m, 4H), 2.21ꢀ2.29 (m, 2H), 1.91ꢀ1.99 (m, 4H), 1.40ꢀ1.51 (m, 2H), 0.91ꢀ0.99 (m, 3H); MS (ESI)
m/z: 302 [M+H]
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4,4'-(((benzylazanediyl)bis(propane-3,1-diyl))bis(oxy))dibenzonitrile (14a): Cesium carbonate (10.9 g/33.6
mmoles/4 equivalents) and 15a (1.4 g/4.2 mmol/ 0.5 equiv.) was added to 1 (1 g/ 8.4 mmol/1 equiv.) in acetone
(50mL). The reaction mixture was refluxed for 48 hours at 60 °C, and then cooled to room temperature. Acetone
was removed under reduced pressure to give a solid residue (Scheme 4). Water (75 mL) was added to the
residue and extracted with dichloromethane (25 mL x 3). The organic layer was separated and washed with 10%
sodium hydroxide in water (10 mL x 3) and washed with water until neutral to pH paper. The organic layer was
dried with sodium sulfate, filtered, and evaporated to give pure solid 14a; 1H NMR (400 MHz, CDCl3): δ 7.40ꢀ
7.62 (m, 4H), 7.717ꢀ7.39 (M, 5H), 6.79ꢀ6.87 (m, 4H), 3.82ꢀ4.06 (m, 4H), 3.61 (s, 2H), 2.59ꢀ2.68 (m, 4H), 1.89ꢀ
1.99 (m, 4H); MS (ESI) m/z: 425 [M+H].
4,4'-(((propylazanediyl)bis(propane-3,1-diyl))bis(oxy))dibenzonitrile (14b): 1H NMR (400 MHz, CDCl3): δ
7.53ꢀ7.61 (m, 4H), 6.80ꢀ6.92 (m, 4H), 4.09ꢀ4.19 (m, 4H), 2.42ꢀ2.53 (m, 4H), 1.82ꢀ1.99 (m, 4H), 1.39ꢀ1.51 (m,
2H), 0.92ꢀ0.98 (m, 3H); MS (ESI) m/z: 378 [M+H].
4,4'-(((benzylazanediyl)bis(propane-3,1-diyl))bis(oxy))dibenzimidamide (16a): 1.0 g of 14a (2.3 mmole) in
anhydrous dioxane (25mL) and dry methanol (20mL) was saturated with hydrochloric acid gas at 0ꢀ5oC under
nitrogen atmosphere. The reaction mixture was stirred at room temperature for 36 hours, after which the solvent
was reduced by 50%. 30 mL of anhydrous ether was added and the resulting precipitate was filtered, washed
with anhydrous ether (10 mL x 5) and dried under vacuum to yield 15a (0.83 g, 73% yield). Dry solid 15a (0.8
g, 1.6 mmol) was dissolved in dry ethanol (50mL) and 2 M ethanolic ammonia (50mL), to which ammonium
chloride (0.025 g, 0.47 mmol) was added. This mixture was refluxed at 80oC for 32 hours under nitrogen
atmosphere, after which it was cooled and concentrated (Scheme 4). The solid obtained was filtered and washed
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with cold ethanol (5 mL x 3) and ether (10 mL x 3) to give diamidine product 16a as brown solid, Yield 75%;
Mp ( 183ꢀ185 °C); 1H NMR (400 MHz, DMSOꢀd6): δ 7.82ꢀ7.84 (m, 4H), 7.18ꢀ7.25 (m, 5H), 7.01ꢀ7.3 (m, 4H),
4.04ꢀ4.05 (m, 4H), 3.56 (s, 2H), 2.55 (m, 4H), 1.88 (m, 4H); 13C NMR (100 MHz, DMSOꢀd6): δ 165.0, 163.4,
130.5, 129.7, 128.9, 128.4, 127.1, 119.5, 119.3, 115.0, 66.4, 58.4, 49.7, 26.5; MS (ESI) m/z: 460 [M+H].
Compounds 16b was similarly synthesized as per the above procedure (Scheme 4).
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4,4'-(((propylazanediyl)bis(propane-3,1-diyl))bis(oxy))dibenzimidamide (16b): Off white solid, Yield 67%;
1
Mp (177ꢀ179 °C); H NMR (400 MHz, DMSOꢀd6): δ 7.88 (d, J = 8.4 Hz, 4H), 7.16 (d, J = 8.8 Hz, 4H), 4.21
(m, 4H), 3.25 (m, 4H), 3.07 (m, 2H), 2.24 (m, 4H), 1.73ꢀ1.75 (m, 2H), 0.92 (t, J = 6.4 Hz, 3H); 13C NMR (100
MHz, DMSOꢀd6): δ 165.0, 163.0, 130.6, 120.0, 115.2, 65.9, 23.2, 16.8, 11.3; MS (ESI) m/z: 412 [M+H].
2-(4-(4-carbamimidoylphenoxy)phenyl)-1H-indole-6-carboximidamide (17).
Purchased from Sigmaꢀ
Aldrich (St. Louis, MO, Catalog No. A8675). A purity of >95% was confirmed HPLC and by HPLCꢀMS.
Cellular Assays
Cell lines and cell culture. WM115 (American Type Tissue Collection (ATCC)) malignant melanoma cells
were cultured in Minimum Essential medium (Invitrogen) supplemented with 10% heatꢀinactivated fetal bovine
serum (FBS). Cells were maintained in a 37ºC incubator with 5% CO2. The WM115 have the activating BRAF
V600E mutation and wild type NRAS19. As shown by Western blot analysis, the WM115 cells have elevated
levels of endogenous S100B protein.
Growth inhibition assay. A collection of pentamidine analogs were tested for their ability to inhibit the
growth of WM115 human melanoma cells expressing high levels of S100B (WM115shSCRAMBLED) and low
levels of S100B (WM115shS100B 30, 43
)
using a modification of the highꢀthroughput screening assay performed by
44
Bachman et al
(Table 2). Using a Biomek FX Laboratory Automation Workstation (BeckmanꢀCoulter)
equipped with a 96ꢀchannel pipetting head, 20ꢁl of MEM (Invitrogen) supplemented with 10% fetal bovine
serum, were added to each well of a 384ꢀwell tissue culture plate (Corning) containing enough cells such that
growing uninhibited they reach 80% confluence in ~5 days. The cells are propagated in the presence of 0.5
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mg/ml puromycin to maintain the vector expressing S100B siRNA to knockdown (KD) the protein or the same
vector containing a scrambled control siRNA. After 24 hours of growth at 37 °C in 5% CO2 humidity
controlled incubator the cells were treated with 20 ꢁl of compound in the same culture media, while in the
control cultures, only an equivalent amount of DMSO added with the compound is added. Titrations of the
compounds were performed in triplicate using the Biomek NXP Laboratory Automation Workstation
(BeckmanꢀCoulter) equipped with an 8ꢀchannel pipetting head before being added to the cell plates. After four
additional days of incubation, the cells were lysed by the adding 20ꢁl of lysis buffer consisting of 1.8% Igepal
with 1:10,000 dilution of SYBR Green I (Invitrogen). The covered plates were incubated for another 24 hours
at 37 °C in a 5% CO2 humidity controlled incubator. The fluorescence intensity was then read through the
bottom of the plate using a POLARstar Optima fluorescent plate reader (BMG) with 485 nm excitation and 520
nm emission filters. The SYBRꢀgreen fluorescence is used to measure total DNA that in turn correlates with
cell number as previously described45. The EC50 of the compounds were determined using serial dilutions and
done in quadruplicate, as above, with no more than 1.0% DMSO or 1000 µM compound.
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Protein Purification
15Nꢀlabeled Bovine or Rat S100B was expressed and purified (> 99%) with methods similar to those
previously46, 47. The concentrations of S100B stock solutions were determined using the BioꢀRad Protein Assay
(BioꢀRad Inc., Hercules, CA) using bovine serum albumin as a standard. The S100B was concentrated to 10ꢀ15
mM using Amicon Ultra centrifugal filter units with a 10 kDa MWCO and stored at a concentration of ~ 10 mM
in 0.25mM Tris, pH 7.2 with 0.25 mM DTT at –20 °C until use.
Protein NMR Spectrometry
The Ca2+ꢀloaded S100B•SBiXs HSQC samples contained 0.1 mM S100B Rat subunit, 0.125 mM SBiX, 0.34
mM NaN3, 15 mM NaCl, 5% DMSOꢀd6, 10 mM CaCl2, 10% D2O, 0.2% TPEN and 10 mM HEPES, adjusted to
pH 7.2 with HCl. Heteronuclear singleꢀquantum coherence (HSQC) NMR data were collected at 37 °C with a
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Bruker Avance 800 US2 (800.27 MHz for protons) instrument equipped with pulsedꢀfield gradients, four
frequency channels, and a tripleꢀresonance, zꢀaxis gradient cryogenic probe. Data were processed with
NMRPipe, and proton chemical shifts were reported with respect to the H2O or HDO signal taken as 4.608 ppm
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relative to external TSP (0.0 ppm). The N chemical shifts were indirectly referenced as previously described
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using the following ratio of the zeroꢀpoint frequency: 0.10132905 for N to H. HSQC spectra were visually
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inspected for chemical shift perturbations (Table 1, Table 3, Fig. S2ꢀ6).
Fluorescence Polarization Competition Assay
The ability of the compounds to compete with a peptide probe was evaluated using an adaptation of Wilder et
al, 2010 (Table 1). Briefly, changes is fluorescence polarization were measured upon competition with the
TAMRAꢀlabeled version of the peptide TRTK12, which is derived from the CapZ protein residues 265–276
(TRTKIDWNKILS), for Ca2+ꢀloaded S100B. The S100B TAMRAꢀTRTK FPCA was performed in 384ꢀwell
black polypropylene microplate with a final volume of 20 ꢁL containing: 10 nM TAMRAꢀTRTK, 1 ꢁM S100B
Rat, 0.01% Triton Xꢀ100, 0.5% DMSO, 50 mM HEPEs, pH 7.2, 100 mM KCl, 15 mM NaCl, 10 mM CaCl2.
Titrations of each compound were done beginning from the highest concentration possible. The fluorescence
polarization was measured at room temperature in a BMG PHERAstar Plus multimode microplate equipped
with dual detection PMTs with an excitation of 544 nm and emission of 590 nm.
X-ray Crystallography
Crystallization. Crystallization experiments were conducted using vapor diffusion methods. CaS100B•5a
crystals were grown in hanging drops consisting of 3.0:3.0 ꢁL protein solution (40mg/mL S100B Bovine;
10mM Cacodylate, pH 7.2; 7.5mM CaCl2; and 4mM 5a prepared in DMSO) and mother liquor (10% Peg 3,350;
0.1M Cacodylate, pH 6.4; 7.5mM CaCl2) with no additional volume of mother liquor left in the crystallization
chamber. CaS100B•6b crystals were grown in sitting drops consisting of 0.75:0.75 ꢁL protein solution
(20mg/mL S100B Bovine; 10mM Cacodylate, pH 7.2; 7.5mM CaCl2; and 2mM 6b prepared in DMSO) and
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mother liquor (25% Peg 3,350; 0.1M BisꢀTris, pH 7.5; 7.5mM CaCl2; 5% glycerol). CaS100B•6b crystals were
grown with mother liquor left in the crystallization chamber. CaS100B•17 crystals were grown in sitting drops
consisting of 0.75:0.75ꢁL protein solution (20mg/mL S100B bovine; 10mM Cacodylate, pH 7.2; 7.5mM CaCl2;
and 2mM 10 prepared in DMSO) and mother liquor (40% 2ꢀmethylꢀ2,4ꢀpentanediol; 0.1M Hepes, pH 7.0; and
7.5mM CaCl2). The crystals were grown over a period of one to fourteen days at a temperature of 295K.
Crystals were not cryoꢀprotected before being flashꢀcooled in liquid nitrogen.
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Data Collection and Processing. Diffraction data for CaS100B•5a crystals were collected Northeastern
Collaborative Access Team (NEꢀCAT) beamline at the Advanced Photon Source (Argonne National
Laboratory). Data was recorded at 100K on an ADSC Q315 (315mm X 315mm) detector and processed by
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NEꢀCAT's RAPD automated processing (https://rapd.nec.aps.anl.gov/rapd), which uses XDS for integration
and scaling. A 1.53Å dataset was collected at a wavelength of 0.97920Å while oscillating the crystal 1.0° each
frame. The space group was determined to be P41212.
Diffraction data for CaS100B•6b and CaS100B•17 crystals were collected remotely48, 49 at the Stanford
Synchrotron Radiation Lightsource (SSRL) beamline 7ꢀ1. Data was recorded at 100K on an ADSC Q315
(315mm X 315mm) detector with collection strategies generated by BLUꢀICE50. Datasets were processed and
integrated by AUTOXDS51. A 1.59Å dataset was collected at a wavelength of 1.1271Å while oscillating the
crystal 0.74° or 0.75°, respectively, each frame. The space groups were determined to be P41212 and P212121,
respectively. Diffraction data statistics for both models are summarized in Table 4.
Structure Determination and Refinement. To determine the structure of S100B in complex with SBiXs,
molecular replacement (MR) was performed. A search model from a previously elucidated S100B structure
(PDB accession code 1MHO39) was generated by removing coordinates for ligands and water. Molecular
replacement was carried out using within the AUTOMR52, 53 function of the PHENIX54 software suite. The
models were finished by manual building within COOT55. The models was refined the PHENIX.REFINE56
function of the PHENIX54 software suite. Ligands and waters were incorporated into the models by referring to
the |Fo|ꢀ|Fc| omit maps. The structure refinement statistics for both models are summarized in Table 4.
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Journal of Medicinal Chemistry
Coordinates and structure factors have been deposited in the Protein Data Bank with the accession numbers:
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CaS100B•5a (PDB ID:5DKN), CaS100B•6b (PDB ID: 5DKQ), CaS100B•17 (PDB ID: 5DKR).
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In Silico Methods
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Ligand design was performed using the Site Identification by Ligand Competitive Saturation (SILCS)
approach57, 58. For ligand design the Tier 1 SILCS approach was applied in which the protein is immersed in an
aqueous solution containing 1 M benzene and propane. SILCS simulations, initiated from the SBi132•S100B
(PDB 3GK1) crystal structures, were performed as previously described yielding probability distributions for
aromatic groups based on benzene carbons, aliphatic groups based on propane carbons, hydrogen bond donors
based on water hydrogen waters and hydrogen bond acceptors based on water oxygens58. The probability
distributions in the presence of the protein are normalized with respect to probability distributions in the
absence of the protein, such the final distributions, termed FragMaps, take into account both functional group
and protein desolvation as well and protein flexibility and proteinꢀfunctional group interactions. FragMaps may
be visualized as 3D contour maps that indicate the regions around the protein favorable for the different classes
of functional groups (Fig. 2).
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The prediction of the location of the missing aminoꢀalkyl tails of 9a was accomplished by applying two
independent methods. In both approaches the initiator Met0 residue was retained since this residue is present in
the S100B coꢀcrystal data with 9a. In the first method, molecular docking was performed using the AutoDock
Vina program59. Standard parameters were applied to obtain 20 docked orientations. A consensusꢀbinding mode
was obtained based both on favorable AutoDock score and the overlap of the partially resolved part of the
ligand with the crystal data. The second approach involved the more recent Tier2 SILCS approach57, 60. Tier2
SILCS simulations were performed on the protein conformation obtained from the crystal data with compound
9a following the removal of the partially resolved ligand. The 10 simulation trajectories spanned a cumulative
sampling time of 400ns were used to calculate the protein functional group affinity FragMaps. In the Tier 2
approach a wider range of solute molecules are used in the aqueous solution including benzene, propane,
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