Iterative Structure-Based Peptide-Like Inhibitor Design against the Botulinum Neurotoxin Serotype A

The botulinum neurotoxin serotype A light chain (BoNT/A LC) protease is the catalytic component responsible for the neuroparalysis that is characteristic of the disease state botulism. Three related peptide-like molecules (PLMs) were designed using previous information from co-crystal structures, synthesized, and assayed for in vitro inhibition against BoNT/A LC. Our results indicate these PLMS are competitive inhibitors of the BoNT/A LC protease and their Ki values are in the nM-range. A co-crystal structure for one of these inhibitors was determined and reveals that the PLM, in accord with the goals of our design strategy, simultaneously involves both ionic interactions via its P1 residue and hydrophobic contacts by means of an aromatic group in the P2′ position. The PLM adopts a helical conformation similar to previously determined co-crystal structures of PLMs, although there are also major differences to these other structures such as contacts with specific BoNT/A LC residues. Our structure further demonstrates the remarkable plasticity of the substrate binding cleft of the BoNT/A LC protease and provides a paradigm for iterative structure-based design and development of BoNT/A LC inhibitors.

The seven known BoNT serotypes are designated A -G. Post secretion, they undergo proteolytic processing to provide the bioactive (i.e., poisonous) holotoxin [1]. The holotoxin is composed of a 100 kDa heavy chain (HC) subunit and a 50 kDa light chain (LC) subunit; these two components are tethered by a disulfide bridge [1,19,20]. Mechanistically, the HC binds to specific motor neuron receptors and induces endosomal internalization [1]. The LC (BoNT/LC) is a zinc (Zn)(II) metalloprotease that is released from the holotoxin into the neuronal cytosol [1]. Once inside the neuronal cytosol, the LC cleaves specific peptide bonds (depending on the serotype) of proteins composing the neuronal SNARE complex: the synapto-somal-associated protein of 25 kDa (SNAP-25), the vesicleassociated membrane protein (VAMP), also referred to as synaptobrevin, and syntaxin [1,21]. Botulinum neurotoxin serotypes A, C, and E cleave SNAP-25 [22,23,24]; serotypes B, D, F, and G cleave VAMP [25,26,27,28], and BoNT serotype C also cleaves syntaxin [23]. The BoNT/LC mediated proteolytic cleavage of any one of the three SNARE proteins prevents acetylcholine-filled vesicles in the neuron from fusing with the active zone at the synaptic cleft [1]. This inhibits the transmission of motor nerve impulses, and as indicated above, results in the flaccid paralysis that is characteristic of botulism [29].
At present, the only treatments available for BoNT intoxication involve antitoxin administration [1], followed by critical care mechanical respiration. However, this treatment would not be practical for treating even a modest number of poisoned individuals: antitoxin administration is ineffective after BoNT internalization (and it is likely that victims would seek medical attention only after the paralysis manifestation). Critical care mechanical respiration is costly and the small number of medical facilities in the U.S. equipped with such devices would more than likely be overwhelmed. Thus, there is an urgent need for the development of small-molecule inhibitors of BoNT LCs.
Of the seven BoNT serotype LCs, the BoNT serotype A LC (BoNT/A LC) possesses the longest duration of action in the neuronal cytosol [30]. Hence, there continues to be a significant effort to identify and develop both peptidic and small, drug-like molecule inhibitors [1] of this particular serotype LC. Previously, we identified and developed BoNT/A LC inhibitors involving the simultaneous identification, design, and generation of both small molecule, non-peptidic, inhibitors (SMNPIs) [29,31,32,33,34] and peptide-like molecules (PLMs) [35,36]. Such PLM design complements SMNPI development, as BoNT/A LC:PLM co-crystal structures form the bases for: 1) the design and synthesis of more potent, drug-like peptidomimetics, 2) the rational, structure-based modification of existing SMNPIs to improve inhibitory efficacies, and 3) the discovery and development of novel SMNPIs via database mining (employing PLM binding modes as search query templates). For example, the conformation and chemical contacts of a PLM bound to the BoNT/A LC can be used to generate threedimensional (3D) search queries to discover new SMNPI chemotypes via the database mining of virtual small molecule libraries.
Here, we describe three new PLMs ( Figure 1) that were designed to explore the BoNT/A LC substrate cleft based on the inhibitorprotease interactions found in a previously published co-crystal structure of BoNT/A LC with the inhibitor I1 [35]. The three new PLMs possess K i values in the nM range which, together with I1, place them among the most potent BoNT/A LC inhibitors characterized to date. One of the PLMs, JTH-NB72-39, was cocrystallized in complex with the BoNT/A LC protease, confirming the interactions aimed by our design strategy while revealing new, unforeseen inhibitor:enzyme contacts that will preface future studies to design more potent PLM and SMNPI inhibitors.

Inhibitor Design
Previously, we reported several nanomolar (nM)-range PLM inhibitors resembling the cleavage site sequence of SNAP-25 [35]. The seven-residue P1-P69 sequence QRATKML (residue positions 197-203 of human SNAP-25) was used to design the PLMs. Of these, a co-crystal structure of the BoNT/A LC with PLM I1 (Figure 1) was determined and its binding contacts and mechanism of inhibition (with respect to the LC's active site) were studied in detail [35]. Based on a general design strategy ( Figure 1) and the BoNT/A LC:I1 co-crystal structure, we attempted to increase the inhibitory potency of the PLM I1 'template' by replacing and incorporating different components to increase both ionic and hydrophobic contacts with residues in the enzymes's binding cleft, and to stabilize the 3 10 helical conformation of I1. We hypothesized that such conformational stabilization of the otherwise flexible inhibitor would decrease the binding entropy of the resulting inhibitors, and thus increase affinity. Along these lines, we restrained the conformation of I1, as it is bound to the BoNT/A LC [35], and thus attempted to reduce the inhibitor's binding entropy, by introducing an aminoisobutyric acid (Aib) residue (Figure 1), which is known to favor the type II' b-turn repeat in a 3 10 helix [37,38]. In addition, we replaced the redoxactive DNP-DAP functional group of I1 [35] with an Arg residue, which, as Kumaran et al. [39,40] demonstrated with co-crystal structures, provides direct electrostatic contacts with anionic residues in the BoNT/A LC substrate cleft (in contrast to the DNP-DAP residue of I1 [35]). Furthermore, we increased the hydrophobic nature of the C-terminus of I1 by replacing the DAB residue [35] with an Ala residue (as found in the P49 position of SNAP-25). Finally, we allowed for combinatorial exploration of the aromatic hydrophobic interactions of the I1 Trp residue (Figure 1), mainly by substituting this position with two sterically and electronically diverse benzyl and naphthylene methylene substituents. The resulting PLM designs JTH-NB72-35, JTH-NB72-38, and JTH-NB72-39 are shown in Figure 1.

Inhibitor Synthesis
The synthesis of the PLMs used manual microwave assisted solid phase peptide synthesis using Fmoc protected amino acids and Rink amide SS resin. After swelling the resin in dichloromethane solvent for 30 min, a stepwise synthesis was initiated by removal of the Fmoc protecting group from the Rink amide resin with a solution of 20% piperidine in DMF. The newly formed free amine was then coupled to the activated, protected amino acid corresponding to the C-terminus of the desired PLM. Initial attempts to activate the protected amino acids for coupling using PyBop and HOBt provided unsatisfactory yields; however, a brief screening of activating agents revealed that Goodman's reagent (DEPBT) provided the desired PLMs in good yield [41,42]. With the first amino acid successfully coupled to the Rink resin, the Fmoc group was again removed, and the subsequent, activated, amino acid was coupled to the freshly deprotected peptide chain. This process of deprotection and coupling was repeated until the amide-terminal residue of the desired PLM was appended. Following the final Fmoc deprotection, the PLMs were cleaved from the solid support by stirring in a modified version of Reagent K (87.5% TFA, 3.6% thioanisole, 2.3% EDT, 3.7% phenol, 1.8% H 2 O, 1.1% triisopropylsilane) for 2 h at RT. Precipitation with diethyl ether provided crude products, which were subsequently purified by preparative RP HPLC (34-42% yield).

In Vitro Inhibition
Using the methods described below, we obtained K i values in the nM range for the JTH-NB72-35, JTH-NB72-38, and JTH-NB72-39 PLMs (Figure 1), although none of them were as potent as I1. Therefore, co-crystallization experiments were conducted in order to collect any structural information that might explain this unexpected result.
Co-crystal Structure of PLM JTH-NB72-39 in complex with BoNT/A LC Of the co-crystallization experiments conducted with the three PLMs, only BoNT/A LC:JTH-NB72-39 produced diffracting crystals. We obtained a co-crystal structure of this complex at 2.4 Å resolution ( Table 1). The structure was determined by molecular replacement using the structure of BoNT/A LC as the search model (PDB reference code 3DSE [35]), but omitting the inhibitor coordinates, water molecules, and other ligands (i.e., Zn(II) and Ni(II) ions) from the search model [35]. Significant electron density for the PLM emerged next to the catalytic Zn(II) around the binding cleft defined by loops 70, 250 and 370 in the LC protease ( Figure 2).

Binding interactions between PLM JTH-NB72-39 and the BoNT/A LC
The electron density for the first six residues of the PLM inhibitor is well-defined (i.e., visible at a contour level of 2.0 s in the Fo-Fc difference electron density map), but is weaker for the last Leu residue. As discussed in detail below, most of the specific interactions observed between JTH-NB72-39 and the BoNT/A LC are mediated by the first four residues of the PLM. Briefly, JTH-NB72-39 also possesses the electrostatic contacts reported for the RRGC, RRGI, RRGM, and RRGL tetrapeptides, as well as for the RRATKM PLM. Moreover, our design resulted in some of the same hydrophobic interactions previously observed between I1 and BoNT/A LC [35], but to a lesser degree.
The carbonyl oxygen of the JTH-NB72-39 P1 residue (Arg) coordinates the enzyme's catalytic Zn(II) ion (distance is 2.4 Å ) and also engages in a hydrogen bond with the hydroxyl group of residue Tyr 366 (which is known to directly stabilize the tetrahedral intermediate formed during SNAP-25 catalysis [43]), while the amino terminal group of the P1 Arg also coordinates the enzyme's catalytic Zn(II) ( Figure 2C). Furthermore, the conformation of the backbone atoms of this residue is determined, in part, by electrostatic interactions between its side-chain and BoNT/A LC residues. Specifically, and in contrast to I1, the P1 Arg of the inhibitor engages, via a salt bridge, with the LC's Glu 164 residue while it also shares a hydrogen bond with the carbonyl oxygen of the LC's Cys 165 residue ( Figures 3A and 4). A similar ionic contact with Glu 164 has been proposed for Gln 197 in the P1 position of SNAP-25 substrate during the formation of the enzyme:substrate complex [44,45]. Moreover, the orientation of the JTH-NB72-39 P1 Arg resembles that of the P1 Arg residues found in previously reported tetrameric inhibitors RRGI, RRGC, RRGM, and RRGL and RRATKM [40] (Figure 3B), but is in closer contact with the enzyme's Glu 164, and it is the only PLM that interacts with Cys 165 of the BoNT/A LC ( Figure 4). In contrast to the binding of the P1 DNP-DAP component of I1 [35], the JTH-NB72-39 P1 Arg does not interact with BoNT/A LC residue Ser 259.
In the P19 position of JTH-NB72-39 there is a second Arg residue ( Figure 3). In SNAP-25, a corresponding P19 Arg plays a key role in facilitating hydrolysis by specifically interacting with Asp 370 in the S19 pocket [46]. This native interaction is also exploited by all structurally known peptidic inhibitors, as all BoNT/A LC:PLM co-crystal structures reported to date posses an Arg residue at the P19 position which provides optimal binding in the enzyme's S19 pocket [35,39,40]. This Arg side chain adopts one of two rotamers, and both conformations are stabilized by the formation of a salt bridge with the side-chain carboxylate of BoNT/A LC residue Asp 370 ( Figure 3). The conformation of the side-chain of the P19 Arg of JTH-NB72-39 closely resembles that observed for the corresponding P19 Arg residue in PLMs RRGI, RRGL, and RRATKM [39,40]. Additionally, the guanidinium group of the P19 Arg engages in a cation-p interaction with Phe 194 of the LC's substrate cleft (not shown). This is a contact that is consistently observed in other BoNT/A LC:PLM co-crystal structures, and mutations of Phe 194 have been reported to diminish the catalytic efficiency of the BoNT/A LC by ,100-fold [47]. Overall, the observed P19-S19 Arg:Asp 370/ Phe 194 interactions appear to be key for general PLM inhibitory potency.
While looking for additional contacts further down the sequence of the JTH-NB72-39 PLM, we identified an interaction never observed before for any other BoNT/A LC inhibitor. The amide nitrogen of the JTH-NB72-39 P29 Phe residue engages in a water mediated interaction with the guanidinium group of BoNT/A LC residue Arg 363 ( Figure 5A-C). In other BoNT/A LC:PLM complexes, such as RRATKM, and those of the tetrameric peptides RRGI, RRGL, RRGM, and RRGC [39,40], it is the carbonyl oxygen of the PLM's P19 that directly interacts with the enzyme's Arg 363 side-chain guanidinium group ( Figure 5A). For JTH-NB72-39, this carbonyl group is rotated 180u relative to its orientation in the tetrameric peptides ( Figure 5A). However, by virtue of this water-mediated interaction with Arg 363, the JTH-NB72-39 P29 Phe amide nitrogen replaces this direct interaction observed for other PLMs [39,40]. This is relevant, as the BoNT/A LC Arg 363 is proposed to be critical for the binding and hydrolysis of the SNAP-25 substrate, presumably by maintaining proper geometry and charge distribution around the active site; mutation of this residue results in a 80-fold decrease of the catalytic rate of SNAP25 hydrolysis by BoNT/A LC [40,43]. In addition, the side chain rotamers of Arg 363 are similar for all BoNT/A LC:PLM complexes, but differ from the rotamer observed in the unbound form of BoNT/LC, indicating that Arg 363 undergoes significant conformational changes upon PLM and substrate binding. This water-mediated contact between the JTH-NB72-39 and Arg 363 is not observed in the I1-bound BoNT/A LC complex ( Figure 5B).
Another novel interaction observed in this position of the PLM is a stabilizing, intra-molecular hydrogen bond formed between the carbonyl oxygen of the P29 Phe and the amide nitrogen of the P59 Met residues ( Figure 5D), which is not present in the I1-bound complex. There are also hydrophobic interactions between the aromatic ring of the JTH-NB72-39 P29 Phe and BoNT/A LC residues previously found to form a hydrophobic pocket for binding by the larger, indol P29 Trp moiety of I1 [35] (Figure 6). For JTH-NB72-39, the BoNT/A LC side-chains of Leu 367 and Phe 369, together with the aliphatic portions of Asn 368, contribute to the formation of this hydrophobic pocket. Additionally, the aliphatic side-chain of Leu 256 interacts with the JTH-NB72-39 P29 Phe side-chain ( Figure 6), but its electron density is weaker than observed in the BoNT/A LC:I1 complex, suggesting that the larger I1 P29 Trp is better suited than the JTH-NB72-39 P29 Phe for stabilizing this residue and forming a non-polar binding site ( Figure 6). This observation partially explains the lower potency of these three PLMs ( Figure 1) relative to I1.
A new PLM component incorporated into the design of JTH-NB72-39 also present in JTH-NB72-35 and JTH-NB72-38 is the gem-dimethyl-glycine residue, Aib, in the P39 position ( Figure 1). The rationale for incorporating this component was to stabilize the PLMs' observed 3 10 helical conformation, as inferred from the I1 binding mode [35], and to decrease the binding entropies of the new designs. In the present co-crystal structure, Aib engages in favorable intermolecular contacts (via its hydrophobic gem-dimethyl Table 1. X-ray data collection and refinement. groups) with the side-chain of BoNT/A LC residue Val 70, and an intramolecular, stabilizing, hydrogen bond with the amide nitrogen of its P69 Leu (not shown). In addition to these interactions, our structure reveals unexpected conformational restrains introduced by this Aib residue that affect neighboring residues in the PLM. The backbone atoms of the more potent I1 PLM superimpose well with corresponding atoms found in JTH-NB72-39 up to the P29 position ( Figure 6B). However, the backbone of JTH-NB72-39 abruptly contracts forming a sharp bend at the P39 position which results from the distinct conformational effects of the Aib residue. Based on this observation, we conclude that, in addition to possessing a smaller volume than the P29 Trp group of I1, and the concurrent absence of an ionic indol group, the P29 Phe of JTH-NB72-39 is hindered in its orientation by the geometrical restraints imposed on the its backbone by the adjacent P39 Aib residue. The P49 Ala residue of JTH-NB72-39 engages in intramolecular, hydrophobic interactions with the side-chain methylenes of the PLM's P1 Arg and P69 Met, as well as with the backbone amides of the PLM's P19 Arg and P39 Aib, while the P59 Met engages in intermolecular interactions with BoNT/A LC residues Glu 257, Val 258, Ser 259, and Glu 262, and intra-molecular contacts with the P1 Arg, P29 Phe, P49 Ala, and P69 Leu. The alkyl chain of the outermost residue in the inhibitor (i.e., JTH-NB72-39 P69 Leu), although solvent exposed, engages in favorable intramolecular interactions with the hydrophobic surfaces of its neighboring P29 Phe and P39 Aib PLM residues, as well as an inter-molecular interaction with the BoNT/A LC Phe 369 sidechain phenyl.

BoNT/A LC substrate binding cleft plasticity
Silvaggi et al. observed structural plasticity in the BoNT/A LC substrate cleft upon binding to three different hydroxamate derivatives [48]. This plasticity has also been documented by the distinct binding contacts identified in subsequent complexes of the BoNT/A LC with other PLM inhibitors [39,40]. Our co-crystal structure of JTH-NB72-39 bound to the BoNT/A LC further underscores this plasticity. Importantly, the complexes of BoNT/A LC with JTH-NB72-39 and with I1 (PDB reference code 3DS9, [35]), and the unbound crystal structure (PDB reference code Inhibiting interactions of JTH-NB72-39. BoNT/A LC residues are displayed as cyan sticks, and the JTH-NB72-39 backbone is shown as thin, orange sticks. Only the P19 Arg side chain of the inhibitor is shown as reference. Interactions between BoNT/A LC and JTH-NB72-39 are represented by dashed lines. The identity of the residues is indicated. The P1 amino and carbonyl groups are indicated by NH 2 and CO, respectively. The C-terminus of the inhibitor is indicated by the letter C. The Zn(II) atom is represented as a yellow sphere. doi:10.1371/journal.pone.0011378.g002 3DSE [35]) crystallized in the same space group with very similar cell dimensions. Thus, we can make comparisons of conformations between these specific crystal structures without the potential influence of crystal packing contacts.
The BoNT/A LC's overall fold is similar in the bound and unbound forms: the JTH-NB72-39-bound form superimposes to the unbound form with a 0.58 Å r.m.s.d. for all Ca atoms. In the BoNT/A LC:JTH-NB72-39 structure, the three LC residues that directly coordinate the Zn(II) ion in the active site, i.e., His 223, His 227 and Glu 262, maintain the same geometry and conformation observed in the unbound form of the LC protease. However, Glu 224 no longer associates with the Zn(II) ion via a 'catalytic water'   [39,40]. This interaction is even more pronounced in the BoNT/A LC:I1 co-crystal structure. Additionally, I1 is also in closer proximity to BoNT/A LC residue Leu 256 [35] than JTH-NB72-39 ( Figure 6). These observations reinforce the hypothesis that the P29 Trp residue in inhibitor I1 is a more favorable 'binding anchor' than the corresponding P29 Phe of JTH-NB72-39.
In the unbound form of BoNT/A LC, residues 64-70 adopt a loop conformation by packing against the b strand formed by residues 415-420. This loop is unaltered in the I1-and JTH-NB72-39-bound structures ( Figure 7A). However, for the crystal structures of the complexes with the hydroxamate derivatives and the CRATKML peptide [49], no electron density was observed for these residues ( Figure 7A). Binding of the tetrapeptides, and of the QRATKM and RRATKM PLMs results in a significant displacement of the backbone in the 70 loop away from the active site ( Figure 7B).  Figure 7C). In contrast, no electron density is observed for various residues within this loop for either the unbound form of the BoNT/A LC, or when bound to arginine hydroxamate (ArgHX) (residues 245-256), I1 (residues 250-253), or JTH-NB72-39 (residues 247-255) ( Figure 7C).
In all BoNT/A LC:inhibitor complexes described thus far, including the co-crystal described herein, there is a conformational change in the 367-372 residue segment of the 370 loop that is associated with rotamer changes in enzyme residues Phe 369 and Asp 370 ( Figure 7D). The latter engages in the salt bridge (described above) with the Arg sidechain in the P19 position of the inhibitors, a key specific interaction for binding of the SNAP-25 substrate and substrate-analog inhibitors. As for Phe 369, its side chain projects away from the binding cleft in the unbound form of the BoNT/A LC. By contrast, in the JTH-NB72-39 complex, the center of this ring moves by ,3.5 Å towards the binding cleft, facilitating the formation of a hydrophobic pocket that accommodates the Phe aromatic ring of the PLM. This conformational change for Phe 369 is even more pronounced in the BoNT/A LC:I1 complex due to this PLM's larger P29 Trp component ( Figure 7D BoNT/A LC). In all other inhibitor complexes, Phe 369 adopts a conformation that differs when compared with either the unbound form of the enzyme, the JTH-NB72-39-, or the I1bound complexes. Taken together, these conformational changes observed in the BoNT/A LC protease upon binding with different inhibitors reveal a highly 'plastic' binding cleft.

Conformational helicity and PLM inhibitors of the BoNT/ A LC
Including the present structure, there are now five co-crystal structures of PLM-based inhibitors in complex with the BoNT/A LC that are longer than four residues. A common structural feature found in all five bound PLMs is a 3 10 helical turn in the inhibitor's backbones ( Table 2). As indicated above, JTH-NB72-39 was designed to further stabilize this helical turn by introducing an Aib residue in the P39 position. Indeed, there is a 3 10 helical conformation for the backbone atoms of this PLM, although the P39 Aib residue slightly distorts the helical turn and deviates from the canonical 3 10 -helix conformation adopted by I1 ( Figure 8A), likely by virtue of the unusual geometric constrains of this residue, i.e, the gem-dimethyl effect [50]. The electron density suggests that the helical pathway also includes the Met residue in the P59 position, resembling the 3 10 helix observed for residues P29-P59 in I1.
A helical backbone conformation is also observed in the Znchelating CRATKML PLM [49], similarly encompassing positions P29 throughout P59 ( Figure 8A). Remarkably, only PLMs containing a Leu in the P69 position adopt a right-handed 3 10 a-helical conformation, in contrast to two closely related hexapeptide PLMs lacking a terminal Leu residue (Table 2) [39]. Instead, the w and y torsion angles for QRATKM and RRATKM do not correspond to a canonical right-handed helical conformation ( Figure 8B and Table 2). The backbone atoms of both peptides undergo a geometrical 'bend' which is most conspicuous along the backbone of the Thr residue in QRATKM (Table 2 and Figure 8B). Whereas inhibitors CRATKML, I1 and JTH-NB72-39 display a common right handedness (or positive chirality) of canonical a-helices, the QRATKM and RRATKM PLMs display unusual negative values in their dihedral a angles (i.e. negative helical chirality) ( Table 2). The helical segments observed for inhibitors I1, JTH-NB72-39, and CRATKML are of similar length and entail the same residue positions (i.e. P29-P59); nevertheless, the CRATKML ''helix'' is slightly shifted relative to the other two PLMs due to Zn-coordination by its thiol group in the P1 Cys. Interestingly, PLMs QRATKM and RRATKM, which do not adopt a canonical helical conformation, are less potent (K i values = 133 mM and 95 mM, respectively) than the 'helical' PLMs JTH-NB72-39, I1, and CRATKML all of     (Table S1). Hence, it is likely that the P69 Leu residue in the helical PLMs not only stabilizes their helical conformations, but also increases inhibitory potency via increased occupation of the enzyme's substrate binding cleft. Together with the favorable hydrophobic interactions of the substituents at the P29 position of JTH-NB72-35, -38, -39, and I1, the presence of a seventh residue might account for the higher potency of these PLMs over the hexa-or tetrapeptides reported by other groups (Table S1) [39,51].

Inhibition mechanism of JTH-NB72-39
The PLM JTH-NB72-39 shares the same inter-molecular contacts that facilitate the mechanism of inhibition with other PLMs (Figure 5B), in particular I1, as previously described [35]. Specifically, all PLMs possessing a free amino terminus orient this substituent such that it engages in contacts with the proton shuttle Glu 224 side-chain carboxylate ( Figure 5A-C). In this way, Glu 224 is no longer capable of ionizing the 'catalytic' water molecule and, as a consequence, it is hampered from using the protons from this water to catalyze the final cleavage of the scissile bond. In addition, all reported PLMs exhibit simultaneous, substrate-like interactions between their P1 residue backbone carbonyl oxygens and both, the side-chain hydroxyl group of Tyr 366 and the Zn(II) ion ( Figure 5A-C). Furthermore, BoNT/A LC residue Arg 363 engages in electrostatic contacts with the peptidic backbones of reported PLMs ( Figure 5A). The RRGL, RRGI, RRGM, RRGC, and RRATKM peptides [39,40] consistently contact Arg 363 via their P19 carbonyl oxygen ( Figure 5A). JTH-NB72-39, on the other hand, forms this interaction indirectly, via a bridging water molecule ( Figure 5A). This difference is most likely due to the geometric restraints imposed by the aromatic group in the P29 position of JTH-NB72-39 (which is either a Gly or an Ala in all other PLM inhibitors [39,40]).
The CRATKML inhibitor conformation deviates from those of all the other PLMs. It is likely that the Zn-coordinating geometry of the P1 Cys of CRATKML shifts the other interactions in the complex ( Figure 5C) [40]. For its first residue, P1 Cys, the terminal amino group of this PLM is not proximal to the enzyme's proton shuttle -Glu 224, but rather, this P1 Cys engages in contacts with BoNT/A LC residue Arg 363 through its P1 carbonyl oxygen, as opposed to the P19 carbonyl oxygen as observed for other PLMs [39,40] (Figure 5A and 5C). As a result, CRATKML also lacks the hydrogen bond with Tyr 366 that is detected in all other PLM cocrystal structures ( Figure 5C).

Hypotheses for improving PLM inhibitory efficacies
The PLMs described here were designed in an attempt to improve potency displayed by previously reported PLM I1 via the incorporation of a P1 position Arg residue (to engage in direct electrostatic interactions with BoNT/A LC acidic residues), and the incorporation of an Aib residue (to stabilize the inhibitors helical conformations) [35]. However, neither JTH-NB72-35 (K i = 315.5628.6 nM), JTH-NB72-38 (K i = 990.56116.9 nM), nor JTH-NB72-39 (K i = 638692.0 nM) (Figure 1) are as potent as I1 (K i = 41 nM) [35]. The co-crystal structures of JTH-NB72-39 and I1 in complex with BoNT/A LC explain this surprising result. The higher K i value for JTH-NB72-39 compared to I1 is, in part, due to fewer favorable hydrophobic contacts provided by the PLM's Phe component in the P29 position versus the larger P29 Trp of I1. Specifically, the I1 Trp component is more efficient in coalescing the non-polar side-chains of BoNT/A LC residues Leu 256 and Phe 369 in their common binding site ( Figure 6). Additionally, while the aliphatic side-chain of Leu 256 does contact the JTH-NB72-39 P29 Phe, the electron density for the side-chain of this BoNT/A LC residue is weaker than that observed in the BoNT/A LC:I1 complex [35]. Hence, the I1 P29 Trp residue is more efficient for inducing the formation of this non-polar pocket than the P29 Phe residue of JTH-NB72-39. Another unexpected result from our co-crystal structure is that the Aib residue introduces a slight deformation of the canonical 3 10 helical backbone conformation observed for I1 ( Figure 7A).
Our co-crystal structure also explains the different potencies observed for the other two PLMs (Figure 1). In particular, the most potent of the three reported PLMs (i.e., JTH-NB72-35) possesses a P29 naphthalene methylene substituent, which would more efficiently bring together, and engage in more favorable hydrophobic contacts with, the non-polar side-chains of BoNT/ A LC residues Leu 256 and Phe 369 (versus JTH-NB72-39), as well as with the aliphatic portions of the side-chain of residue Asn 368 at the enzyme binding site. Finally, it is likely that the same hypothesis applies to JTH-NB72-38; however, while the JTH-NB72-38 P29 Trp indole does provide a larger ring system for binding in the indicated BoNT/A LC hydrophobic pocket (see above), the polar pyrrole nitrogen atom is unable to engage in a favorable hydrogen bond with the backbone carbonyl of BoNT/A LC residue Glu 257, an interaction that was previously observed in the BoNT/A LC:I1 complex ( Figure 9A) [35]. Instead, modeling of a Trp side chain in the P29 position of the JTH-NB72-39 PLM revealed that none of the rotamers available for the -CH 2 -indole side chain positions it so that it engages in a hydrogen bond with any residue in the enzyme ( Figure 9B). Indeed, our analyses indicated that the polar nature of the JTH-NB72-38 pyrrole nitrogen results in unfavorable hydrophobic-polar clashes in the hydrophobic pocket indicated above. Hence, this analysis provides a rational basis for explaining the higher K i compared of this PLM versus those of JTH-NB72-35, JTH-NB72-39, and I1.
The decreased inhibitory efficacies of all three JTH PLMs compared to Il also appears to be partially due to the fact that they all incorporate an Arg residue at the P1 versus the P1 -P29 DNP-DAP component of I1. In particular, the rigidity of the DNP phenyl and the solvation of its two nitro functional groups may be necessary for stabilizing this PLM's helical structure upon binding in the enzyme's substrate cleft. This, in turn, would decrease I19s binding entropy. By comparison, the flexible side-chain of the P1 Arg of the JTH PLMs does not provide the same helix-stabilizing character. Additionally, the JTH PLMs lack the DAB component found in I1. In the BoNT/A LC:I1 co-crystal structure, this cationic component engages in a hydrogen bond with the sidechain amide of BoNT/A LC residue Gln 162 [35]. Comparatively, in the BoNT/A LC:JTH-NB72-39 co-crystal structure, the PLM's non-polar P49 Ala residue cannot engage in such a favorable H-bond. Finally, the initial weak electron density observed for the C-terminal residues of the JTH PLMs indicates that these residues may be destabilizing the overall binding modes of these inhibitors via entropic contributions, and therefore, other components that engage in more definitive hydrophobic and/or polar contacts with the enzyme cleft would be necessary for improving future PLM potencies.
Based on available structural and mechanistic data, future PLM designs will focus on optimizing the P1 -P29positions, while simultaneously introducing changes to terminal PLM residues/ components to decrease their entropic contributions. Moreover, future designs will also incorporate peptidomimetic features that will increase the drug-like character of the PLMs.

Conclusion
The design and synthesis of three new PLM inhibitors, which are pivotal for guiding the development of peptidomimetics and SMNPIs, have been presented. In order to characterize the binding modes for the PLMs, a co-crystal structure of one, JTH-NB72-39, was determined, which possesses components that have been independently reported to directly interact with the active site of the BoNT/A LC [35,39,40]. Based on comparisons between the binding mode determined for JTH-NB72-39 with other non-Zn-chelating BoNT/A LC:PLM co-crystal structures [35,39,40], a consistent inhibition mechanism has emerged [35]. Specifically, we provide a unifying PLM-based mechanism of action: in all cases the presence of a P1 amino terminal residue is key for effectively 'arresting' the proteolytic activity of the BoNT/ A LC. This discovery explains why SNAP-25 substrates Nterminally extended beyond the P1 position are cleaved by the BoNT/A LC [46]; in such peptides, due to their participation in a peptide bond with the P2 residue, the P1 amino group becomes an amide, rendering it non-competent for 'locking' the Glu 224 carboxylate group [39]. This observation emphasizes the requirement of a highly ionizable group in this position in order to strongly interact with Glu 224 -to effectively inhibit the BoNT/A LC protease and will be important for guiding the rational design of new PLMs and peptidomimetics, as well as for the discovery of new SMNPIs and the synthetic optimizations of existing SMNPIs.
The JTH-NB72-39-BoNT/A LC co-crystal structure presented here, and its comparison with all other SNAP-25-derived PLM inhibitor co-crystal structures known to date, highlights the importance of the BoNT/A LC 370 loop for substrate binding and cleavage specificity. It is this loop that contains the Asp 370 residue, which is pivotal for substrate discrimination. Also, BoNT/ A LC residue Phe 369 is located in this region, and according to the structure presented here and the co-crystal structure with I1 [35], forms part of a hydrophobic pocket that efficiently anchors non-polar groups located in the P29 position of these PLMs. Future designs will involve PLM components that can further stabilize a helical backbone orientation without interfering with binding, as well as the incorporation of bulkier non-polar components at the P29 position.

Synthesis
General. N,N-Diisopropylethylamine was sequentially distilled from ninhydrin then KOH and stored under argon. Piperidine was distilled from CaH 2 and stored under argon. Phenol was purified by dissolving the solid in diethyl ether, washing with a saturated aqueous solution of NaHCO 3 (3x), extracting with aqueous NaOH (0.1 M) (3x), acidifying the aqueous extracts with 0.1 N HCl, extracting with Et 2 O (3x), concentrating the ethereal extracts under reduced pressure, and the dry solid was stored under argon. N,N-Dimethylformamide was purchased from Alfa Aesar as anhydrous and amine free in 4 L quantities and stored in 1 L Amber bottles (dried overnight in an oven at 140uC) over activated 4 Å molecular sieves under argon. Trifluoroacetic acid (biochemical grade, 99.5+% pure) was purchased from Alfa Aesar and used as received. Methanol (HPLC grade), water (HPLC grade), and thioanisole (99% purity) were purchased from Aldrich and used as received. Triisopropylsilane (99% purity) was purchased from Acros and used as received. 1,2-ethanedithiol (.98% pure) was purchased from Fluka and used as received. All natural Fmoc-protected amino acids and 3-(diethoxy-phosphoryloxy)-3H-benzo[d] [1,2,3]triazin-4-one were purchased from either Peptides International or Advanced Automated Peptide Protein Technologies (AAPPTEC) and used as received. Unnatural Fmoc protected amino acids and Rink Amide Resin SS, 100-200 mesh, 1% DVB were purchased from Advanced Chemtech and used as received. BD Falcon BlueMax 50 mL graduated tubes and 25 mm syringe filters with a 0.45 mm nylon frit were purchased from Fischer Scientific.
The Fmoc-solid phase peptide syntheses were performed on a CEM Discover manual microwave peptide synthesizer fitted with a fiber-optic temperature probe. Solid phase peptide syntheses were performed in a 25 mL polypropylene reaction vessel. The 25 mL polypropylene reaction vessel was constructed by inserting a Teflon ring (0.4 mm height, 2.1 mm outer diameter, 1.8 mm inner diameter) into a capped 25 mL SPE reservoir purchased from Grace Davison Discovery Science (Catalogue #: 210425) containing a frit purchased from Grace Davison Discovery Science (Catalogue #: 211416) ( Figure S1).
Preparative reverse phase HPLC purifications were performed on a Gilson HPLC system with 220 and 254 nm UV detection, using a Phenomenex Luna 5m C18(2) 100 Å , AX (75630.0 mm) column at a flow rate of 10 mL/min. Unless otherwise noted, all preparative runs used linear gradients of 30-60% buffer B in A (A: water containing 0.1% TFA, B: CH 3 CN containing 0.1% TFA) over 30 min. Analytical HPLC traces of final products were performed on a Gilson HPLC system with 220 and 254 nm UV detection, using a Varian Microsorb 100-3 C18 (10064.6 mm) column at a flow rate of 0.7 mL/min. Unless otherwise noted, all analytical runs used linear gradients of 30-100% buffer B in A (A: water containing 0.1% TFA, B: MeOH) over 70 min. CD Spectra were recorded on a Jasco J-815 Circular Dichroism Spectrometer. Unless otherwise noted, all CD spectra were recorded in MeOH at a concentration of 0.5 mM, at 298 K, over a range of 300-200 nm, at a scan rate of 50 nm/min. Mass spectra were obtained using MALDI TOF/TOF with 2,5-dihydroxybenzoic acid as the matrix in the positive ion mode. Lyophilization was accomplished using a Labconco FreeZone 4.5 liter bench top freeze dry system. Centrifugation was accomplished using a Sorvall RT-7 Plus bench top centrifuge.
Proton and carbon NMR spectra were recorded using a Bruker Avance spectrometer at 600 MHz/150 MHz ( 1 H NMR/ 13 C NMR) in D 2 O (298 K), unless otherwise noted. Chemical shifts (d) are reported in parts per million (ppm) using MeOH solvent peaks as an internal reference (referenced to 3.34 ppm ( 1 H) and 49.5 ppm ( 13 C)). 1 H NMR data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublets, dt = doublet of triplets, td = triplet of doublets, qd = quartet of doublets), coupling constants (J) in Hertz (Hz), and integration. 13 C NMR spectra were obtained using a proton-decoupled pulse sequence with d1 of 6 sec, and are tabulated by observed peak.
General procedure A: solid phase peptide synthesis. To a 25 mL polypropylene reaction vessel charged with a Teflon stir bar (1063 mm) was added the Rink Amide resin (0.143 g, 0.100 mmol, loading 0.700 mmol/g, 1.00 equiv). The resin was washed with MeOH (265 mL), CH 2 Cl 2 (3610 mL) and DMF (3610 mL), suspended in CH 2 Cl 2 (5 mL) and allowed to swell at room temperature for 30 min. The resin was filtered and washed with DMF (3610 mL). The Fmoc group was cleaved by heating the resin in the Fmoc-cleavage base stock solution (1 mL) in the microwave (35 W, 78uC, 3 min). The resin was filtered and washed with DMF (3610 mL), CH 2 Cl 2 (3610 mL) and DMF (3610 mL). The first Fmoc protected amino acid was coupled to the resin by heating in a pre-mixed solution of amino acid (0.350 mmol, 3.50 equiv), DEPBT (0.105 g, 0.350 mmol, 3.50 equiv), DMF (0.80 mL), and Fmoc-coupling base stock solution (0.75 mL) in the microwave (25 W, 80uC, 5 min). The resin was filtered and washed with DMF (3610 mL), CH 2 Cl 2 (3610 mL) and DMF (3610 mL). The Fmoc group was cleaved as previously described, and the next amino acid was coupled. This process of Fmoc cleavage and amino acid coupling was repeated for each additional amino acid. After the final Fmoc cleavage, the resin was washed with DMF (30 mL) and CH 2 Cl 2 (20 mL). The protecting groups were cleaved by treatment of the dry resin with the resin cleavage cocktail stock solution (2.50 mL) for 2 h at room temperature with vigorous stirring. The resin was filtered and rinsed with the remaining resin cleavage cocktail stock solution

In vitro testing
The HPLC-based assay used to calculate PLM inhibition constants has been published extensively [46,52,53,54,55,56]. In brief, the assay utilizes an N-terminal acetylated, C-terminal aminated, synthetic peptide identical in sequence to residues 187-203 of SNAP-25. Substrate hydrolysis is determined by HPLC separation of the products from the substrate, followed by measurement of the peak areas. Assay mixtures consisted of 40 mM HEPES-0.05% Tween (pH 7.3), recombinant BoNT/A LC, peptide substrate, 0.5 mg/ml Bovine Serum Albumin, and various PLM concentrations. Assays were run at 37uC, quenched by the addition of TFA, and analyzed by reverse-phase HPLC. To eliminate Zn chelating agents, the assay is run in the presence of excess Zn (50 mM). K i values were calculated by measuring PLM mediated inhibition at different substrate concentrations and treating the kinetic data by the method of Dixon. Inhibition constants (i.e. K i values) were extracted from the slopes of Dixon plots: K i = K m /(slope x V max x S), where S is the substrate concentration. All reported values are averages of at least three independent determinations using nine PLM concentrations.

X-ray crystallography and structural analysis
BoNT/A LC: PLMs mixture preparation. Details of the bacterial expression and purification of the active form of wt BoNT/A LC used in this study have been previously described [45]. Stock solutions of wt BoNTA-LC containing 20 mM HEPES, pH 7.4 were adjusted to a final 150 mM protein concentration. Lyophilized JTH-NB72-35, -38, and -39 inhibitors were resuspended in distilled water to a final 10 mM concentration. Individual mixtures of BoNT/A LC and each of the three PLMs were prepared by mixing both stock solutions to attain a final 50 mM BoNT/A LC and 1 mM PLM concentrations.
Crystallization and data collection. Crystals were obtained by using the hanging drop vapor diffusion method at 20uC. Briefly, 3 mL of a mixture of 50 mM BoNT/A and 1 mM of each PLM inhibitor were mixed with 1.5 mL of the mother liquor containing 14% PEG MME 2000, 10 mM NiCl 2 , and 100 mM HEPES pH 8.5. A layer of a 1:1 mixture of paraffin:silicon oil was overlaid onto the mother liquor present in the well. Crystals for the BoNT/A LC:JTH-NB72-39 mixture appeared after approximately five days of incubation and they were directly transferred into a cryo-solution containing 25%(v/v) PEG600, 0.14 X PEG MME 2000, 10 mM NiCl 2 , and 100 mM HEPES pH 8.5, and then flash-frozen in liquid nitrogen. Only microcrystals were observed for the other two PLMs, and further manipulation did not result in any improvement of their size. The diffraction data were collected at beamline 11.1 of the SSRL (Stanford Synchrotron Radiation Laboratory) at a wavelength of 1 Å , and at a temperature of 100 uK. The diffracted crystals belonged to the P2 1 2 1 2 group. Integration, indexing, and scaling of the diffraction data was performed using the HKL2000 suite of programs [57].
Structure determination and refinement of the wt BoNT/ A LC:JTH-NB72-39 complex. The coordinates in the 1XTF pdb file were used as the search model to determine the structure of the wt BoNT/A LC:JTH-NB72-39 complex by molecular replacement using the PHASER module in CCP4i [58]. The initial values for the R work and R free of the generated model were 27.1% and 31.3%, respectively. The s A -weighted mF o -F c electron density map clearly indicated the presence of JTH-NB72-39 in the vicinity of the active site (Fig. 2). The coordinates of the JTH-NB72-39 inhibitor were then added to those of the BoNT/A LC in the structure of the complex using Coot [59]. Final refinement and modeling was performed using Phenix [60]. The quality of the final structure was assessed using MolProbity. Ramachandran analysis showed that the BoNT/A LC:JTH-NB72-39 structure had 97.14% residues in the favored region with no outliers. The coordinates and structure factors have been deposited in the PDB (ID 3NF3).

Analysis of the secondary structure of PLM inhibitors
In order to determine the secondary structure of all the PLM inhibitors reported to date, the pdb files for their complexes with BoNT/A LC were used as input for the DSSPcont program [61]. The output of this analysis was used to build Table 2.