Descripción textual de los Casos de Uso de Negocio

In a recent work,³⁰ using solute tempering replica exchange (REM) molecular dynamics simulations, Procacci and all. have shown that the molecular recognition of known FKBP12 proline-mimetic drugs such as (1R)-1,3-diphenyl-1-propyl-(2S)-1-(3,3-dimethyl-1,2-dioxopenty1)-2-piperidinecarboxylate (sb3, Figure 3.7 left) resides in the correct 3D orientation and in the stability of the two binding carbonyls of the ligand acting like a sort of “pipecolic clamp” in bulk water solution.³¹Such a feature is favored by persistent through-space intraligand hydrophobic interactions that significantly reduce the entropy loss upon binding by imparting to the drug a pseudocyclic structure (like Rapamycin and FK506) with solvent exposure of the highly polar pipecolic clamp.

Figure 3.7. Left: sb3; right: Elte421

The activity of sb3 is related to the mean mutual orientation of the two carbonyl units (C1-O2 and C8-O3) in bulk solution that is in turn controlled by the mutual arrangement of the dihedral involving the C2 and N7 substituents, that is, angle ω (C9C8N7C2) and angle ψ (O1C1C2N7). When the former torsional angle is in the trans and the latter in the cis configuration, the orientation of the carbonyl units and the O2-O3 distance (around 4.5 Å) is optimal for binding in the active site with Tyr86 and Ile56, the two most conserved residues across the entire FKBP family.³

In this work, it was further shown that in the right-handed stereoisomer (15R)-sb3, this optimal trans-cis arrangement is stabilized in bulk solution by a stereospecific intraligand hydrophobic interaction of the 1,1-dimethyl propyl group of the N2 substituent with one phenyl ring of the C2 substituent. These hydrophobic interactions are not observed in the (15S)-sb3 epimer, consequently reducing the probability of potential binding conformation in bulk solution, with a remarkable impact on the observed potency of the compound (30-60 times less effective than the (15R) counterpart).

Using this guidance, starting from sb3, a new FKBP12 inhibitor, 1-phenylhexan-3-yl-1-(2-oxo-2-phenylacetyl)piperidine-2-carboxylate, a 1:1 disteroisomeric mixture named Elte421 (1) (Figure 3.7, right), was in silico designed, replacing the isopropyl moiety at carbon 9 of sb3 with a phenyl ring and the phenyl ring at the stereocenter C15 with a propyl chain (Figure 3.8).

193 Figure 3.8. Schematic diagram of FKBP12−Elte421 interactions. The diagram has been obtained

running the LigPLot program31 on a snapshot configuration taken from an MD trajectory of the ligand-protein complex at 300 K.

18 ns REM simulations with solute tempering³² of (15R)-Elte and (15S)-Elte in water solution at T = 300 K and P = 1 atm, using the ORAC program,³³ were performed to evaluate at the atomistic level the free-energy surface (FES) with respect to ω and ψ angles, characterizing the conformation of the binding carbonyl groups. The results show that both the Elte421 diastereoisomers exhibit a significant conformational population in the FKBP12 binding ω-ψ (gridded quadrant, Figure 3.9) comparable to that of the potent (15R)-sb3 ligand, confirming the optimal redislocation of the hydrophobic groups in sb3 (phenyl and alkyl chains) so as to stabilize at the same time via π-π stacking interactions the 15R stereoisomer and, as in (15R)-sb3, via phenyl-alkyl hydrophobic interactions the 15S partner.³⁰ Starting from these promising data, Elte421 (1) was synthesized in our laboratory, confirming the prediction based on the above described FES analysis, the (15R)-Elte and (15S)-Elte 1:1 mixture therefore was three times as potent as (15R)-sb3.

Figure 3.9. Free-energy surface with respect to the dihedral angles ω and ψ of Elte421 and sb3 in water at T = 300 K and P = 1 atm calculated via solute tempering REM.

194 Moreover, as correctly inferred from calculation, 15R-Elte421 and 15S-Elte421 have both Kd of nanomolar order with S-Elte having a slightly higher affinity. This behavior can be rationalized by assessing the exposure of the two carbonyls in the PKA as derived from the FES in the ω, ψ space calculated in a REM simulation of the drug in a water environment in standard conditions. The S-Elte421 compound is characterized by a deep minimum in the FES close to the optimal ω, ψ values.

Such a minimum is less pronounced in the R-Elte compound and somewhat shifted toward the ω, ψ trans−trans rotameric state. Correspondingly, the measured binding affinity of S-Elte421 is larger than that of the R-compound. We also notice that the R-Elte421 epimer has a less populated ω-cis rotamer with respect to S-Elte. The incidence of ω-cis conformation in R-Elte421 has a negative impact on the FKBP affinity since only the ω-trans rotamer is able to form the two conserved H-bonds in the PPI domain. Therefore, the low population of the cis conformer at equilibrium in bulk water solution partly compensates, in R-Elte421, the nonoptimal exposure of the two binding carbonyls. As a consequence and in contrast to the sb3 parent compound where the S and R diastereoisomer affinities differ by 2 orders of magnitude, in Elte421 the stereocenter at C15 has a marginal role: the two Elte421 diastereoisomers have both nanomolar binding affinity versus FKBP12. The full confirmation derived from the enantioselective synthesis of the two diasteroisomers that, again, showed both nanomolar affinity for FKBP12.³⁴

Encouraged by the promising results, the chemical-physical determinants of the peptidomimetic FKBP ligands was further characterized.³⁴ In order to stabilize cis−trans configuration of the ω, ψ dihedral angles, we tried to substitute the carboxylate group in Elte421 with an amide group, arriving at (15R/S,2S)-1-(2- oxo-2-phenylacetyl)-N-(1-phenylhexan-3-yl)piperidine-2-carboxamide, named ElteN420 (4). The N1-H moiety, by interacting via H-bond with O3 thus mimicking a tight turn of δ type, should force at the same time the trans conformation for ω dihedral and the cis conformation for ψ dihedral. The potency of the 1:1 diasteroisomeric mixture ElteN420 disgruntledly turned out to be lower than that of the Elte421 mixture (1). An accurate inspection of the highly populated binding cluster structures reported in the ω, ψ trans-cis region revealed that the propyl moiety points in both cases outward, in the same direction of the pipecolic clamp, possibly interfering, at least in one epimer, with FKBP binding. Hence we decided to eliminate altogether the propyl group along with the stereocenter C15, finally landing on the compound (2S)-1-(2-oxo-2-phenylacetyl)-N-(3-phenylpropyl)-piperidine-2-carboxamide, named ElteN378 (5). From REM configuration analysis showed that the population of the most populated binding clusters is characterized by a parallel displaced stacking of the two phenyl rings with one of these rings additionally stacked right above the planar α-keto amide moiety (Figure 3.10, left). More precisely, the extensive stacking system involving the two phenyl and the planar amide moiety is stabilized by solvophobic effects due to the free energy gain (notably of the intrasolvent enthalpy) promoted by the minimization of the solvent- ElteN378 interface upon intraligand stacking.

195 Figure 3.10. Left: Free energy surface (FES) with respect to the dihedral angles ω and ψ (in degrees)

of ElteN378 calculated via solute tempering REM in water bulk solution. Right: Contour map of the O2−O3 distance (in Å) as a function of ω and ψ (in degrees) in ElteN378

A 3D interpolation of the resulting surface using the pm3d algorithm³⁵ revealed that basically the entire O2-O3 distance spread in the case of ElteN378 (5) is restricted to the binding range 4.2-5.0 found in the experimental structures of ligand-FKBP complexes (Figure 3.10, right), imparting to the molecule an extraordinary rigidity. In this case, in silico calculations have been so predictive that drove us to the synthesis of ElteN378 (5), the most powerful synthetic inhibitor of FKBP12 ever described in literature.