COMBINED IN SILICO APPROACHES FOR DRUG DESIGN AND PHARMACOKINETIC OPTIMIZATION OF A SET OF CARNOSINE ANALOGUES AS POTENT AND SELECTIVE CARBONYL QUENCHERS

Vistoli G., Pedretti A., Marconi C., Aldini G.

Dipartimento di Scienze Farmaceutiche “Pietro Pratesi”, Università degli Studi di Milano, Via Mangiagalli 25, I-20133 Milano, Italia.

References

1. Aldini G, Dalle-donne I, Maffei Facino R, Milzani A, Carini M. 2007 Med. Res. Rev. 6, 817-68

2. Vistoli G, Orioli M, Pedretti A, Regazzoni L, Canevotti R, Negrisoli G, Carini M, Aldini G. 2009 Chemmedchem. 4, 967-75

3. Vistoli G, Pedretti A, Cattaneo M, Aldini G, Testa B. 2006 J Med Chem. 49, 3269-77

4. Pedretti A, De Luca L, Marconi C, Negrisoli G, Aldini G, Vistoli G. 2008. Chemmedchem., 3, 1913-21

5. Vistoli G, Pedretti A, Mazzolari A, Testa B. 2010. Bioorg Med Chem., 18, 320-9

Predicting the active absorption: the homology model for hPepT1

Background� Reactive carbonyl species (RCS) are cytotoxic mediators generated by lipidoxidation of PUFAs, leading to

alteration of cellular functions and inducing irreversible structural modifications to biomolecules.

� RCS and the corresponding adducts with proteins (that is, carbonylated proteins) are widely used as

biomarkers of lipidperoxidation and, in general, of oxidative stress.

� Moreover, there are several convincing evidences supporting a pathogenic role for RCS, such as in the

case of diabetic-related diseases, age-dependent tissue dysfunction, and metabolic distress syndrome.

� Consequently, RCS, in addition to being a predictive biomarker, also represents a biological target for drug

discovery.

• We recently found that the endogenous dipeptide carnosine (β-alanyl-L-histidine) is a specific quencher of

α,β-unsaturated aldehydes [1]. Although carnosine is actively absorbed by intestinal transporter hPepT1,

its therapeutic use is limited since it is unstable in human plasma due to the serum carnosinase activity (5).

Moreover, the reactivity of carnosine towards RCS is markedly lower compared to other known quenchers.

• Hence, the rational design of new carnosine analogues should (1) increase the quenching activity of

carnosine, maintaining its selectivity (2) confer plasma stability against human serum carnosinase, and (3)

conserve an optimal recognition by hPepT1.

• Accordingly, in silico approaches can support the design of carnosine derivatives by (1) parameterizing the

factors which govern the quenching activity, (2) predicting the effects of serum carnosinase and (3)

modeling the recognition by hPepT1.

• When the modifications were so significant to prevent the active transport, the marked hydrophilicity of

carnosine analogues was modulated by designing prodrugs whose hydrolysis was predicted in silico by

docking simulations with the major human carboxylesterases.

NH3 CO

SC1

NH

COO

SC2

+ _Glu23Glu26Tyr588

Phe297Leu296Ala295

Asn22Glu23

Ile331Trp294

Phe293Trp294

Glu291Thr327

NH3 CO

SC1

NH

COO

SC2

+ _Glu23Glu26Tyr588

Phe297Leu296Ala295

Asn22Glu23

Ile331Trp294

Phe293Trp294

Glu291Thr327

The ammonium head probably plays the most critical role since it realizes a reinforced H-bond with Tyr588 as well as ion-pairs with Glu23and/or Glu26. Notably, the contact between Tyr588 and ammonium head characterizes the most affinitive ligands.

The carboxy terminus appears less involved in ligand recognition, since it stabilizes only H-bonds with the backbone of Ala295, Leu296, and Phe297without forming strong ionic interactions

The residues which interact with the side chains are heterogeneous, justifying the ability of hPepT1 to interact with structurally diverse substrates. It is possible to recognize a set of residues involved in the interaction with the N-terminal side chain (SC1) such as Asn22, Glu23, and Phe293, while the C-terminal side chain (SC2) contacts Trp294, Ile331, Glu291 and Thr327.

The central peptide bond can stabilize H-bonds with backbone atoms of Phe293 and Trp294.

pKi = 0.235 ∆∆∆∆distance – 7.912 10-3 Zapbind +1.534 Int_Tyr588 - 0.480 n = 50; r2 = 0.85; s = 0.45; F = 89.92

Docking results suggest that the most affinitive compounds have a distance between charged termini about equal to 6 Å. ∆∆∆∆distance defines the difference between the distance value of a given ligand and the optimal distance (6.02 Å) as evidenced by the most affinitive ligand.

Zapbind accounts for the ionic interactions. Despite the known predilection of hPepT1 for hydrophobic ligands, the role of Zapbind score emphasizes the relevance of the polar interactions mostly realized by the ligand’s charged groups.

Given the beneficial role of the interaction between Tyr588 and ammonium head, we introduced a binary descriptor (Int_Tyr588), which is equal to 1 for substrates which realize such a reinforced H-bond and 0 otherwise.

The hPepT1 structure was modeled by fragments based on the resolved structure of lactose permease, LacY. Docking analysesinvolved a set of 50 known substrates and allowed the identification of a common pattern of interactions [3].

Docking results confirmed the key role of chiral centers in hPepT1 recognition, thus explaining why D-car derivatives are not actively transported. Docking results also allowed to derive a reliable correlative equation which was used to predict the affinity of carnosine analogues. The figure shows the putative complex carnosine-hPepT1. The predicted affinity is in line with the experimental value: 1.41 mM (pred) vs. 2.48 mM (exp).

Trp294

Glu23

Tyr588

Ala295

Leu296

Phe297

Predicting the plasma stability: the serum carnosinase

LID DOMAIN

CATALYTIC DOMAIN

N

O OH

N

O

N

O N NH3

+

O

NH

N

OO

H

- N

O

O

O

-

-

N

OO

O

Asp 116

Gly 115

Glu 451

Thr 424

Leu 254

Zn2+ Zn2+

N

O OH

N

O

N

O N NH3

+

O

NH

N

OO

H

-

N NH3

+

O

NH

N

OO

H

-- N

O

O

O

-

N

O

O

O

--

-

N

OO

O--

N

OO

O

Asp 116

Gly 115

Glu 451

Thr 424

Leu 254

Zn2+ Zn2+

D-carnosine

L-carnosine

The human serum carnosinase was modeled using the structure of β-alaninesynthase as template. Docking analyses allowed to identify the ligand moieties which are critically involved in enzyme recognition, emphasizing the key role of the contacts with metal ions [4].Furthermore, docking studies can be used to predict the plasma stability of novel analogues. For instance, they can well explain why D-carnosine is not recognized by serum carnosinase and is stable in human plasma.

Predicting the prodrug hydrolysis: the carboxylresterase 1

Leu255

Leu144

Glu225

Ser226Phe

177Tyr170

His140

Ile139Trp

138

Gly224

Gly223

Ala222

Ser221

Glu220

Leu358

Tyr152

Val146

Leu472

Phe101

Leu97

Asp470

Phe476

Alkyl/Aryl group

Acyl group

C-O=O1

2

3

4

5

6

7

1 2 3 4 5 6 7

Exp pKm

Pred pKi

training settest set

pKm = – 1.66MLPInS

– 0.381DistSer221

+ 4.18

n = 40; r2 = 0.85; q2 = 0.73; SE = 0.49; F = 64.06; p < 0.0001

His468

Ser221

Leu358

Phe303

Leu304

Met145

Leu255

Leu144

Leu97

Phe101

The design of suitable prodrugs for D-carnosine was supported by docking analyses involving the human carboxylesterase-1. Such studies firstly involved a set ok known substrates and allowed the identification of key residues involved in complex stabilization as well as the development of a robust predictive equations [5].

The obtained results were then exploited to rationalize the stability of a set of D-carnosine prodrugs also predicting the more labile function for the bi-protected derivatives.

Globally, the performed simulations lead to the design of octyl-D-carnosine as most promising prodrug for clinical studies.

Conclusions: summarizing the obtained SARs

NNH

NH

OH

NH2

O

O

Mandatory for quenchingIt can be replaced by other moieties able to yield Michael adducts Important for CN2 but not for PepT1

It can be substituted by aromatic but not aliphatic groups

It can be largely modified. Required by CN2 but not byPepT1

Irrelevant for quenching but crucial for PepT1

It can be largely modified. Required by CN2 but not byPepT1

Mandatory for quenching.It can be replaced by hydrazines, hydrazides and diamines lacking in selectivity. Important for bothCN2 and PepT1

Based on obtained SARs and performed simulations, the rational design of new carnosine analogues followed such a pathway:

L-carnosine

Active, absorbed but unstable

D-carnosine

Active, stable but not absorbed

Octyl-D-carnosine

Active, stable, absorbed but toxic

FL-927-A

Active, stable and absorbed

Enhancing the quenching activity

The modifications aimed to enhance the reactivity of the amino group are not largely exploitable since they would

mine the specificity quenching also physiological aldehydes. Conversely, the specific Michael adduction can be

optimized, by (1) modifying the imidazole ring to increase its nucleophilicity and (2) modulating the conformational

profile of the Schiff base intermediate in order to favor a close conformation in which the imidazole ring approaches

the reactive C3 to form the corresponding Michael adduct.

The key role of conformational profile: the case of Schiff base carnosine-HNE

3.8 Å3.8 Å

13.2 Å13.2 Å13.2 Å

NH

N

3.8 Å3.8 Å

13.2 Å13.2 Å13.2 Å

NH

N

FL-926-A-010

0.0 0.5 1.0 1.5 2.0 2.5 3.00

102030

405060

708090

Quenching activity (carnosine units)

Fo

lded

con

form

atio

n(%

) carnosine

r2= 0.886

FL-926-A-010

0.0 0.5 1.0 1.5 2.0 2.5 3.00

102030

405060

708090

Quenching activity (carnosine units)

Fo

lded

con

form

atio

n(%

) carnosine

r2= 0.886

0.0 0.5 1.0 1.5 2.0 2.5 3.00

102030

405060

708090

Quenching activity (carnosine units)

Fo

lded

con

form

atio

n(%

) carnosine

r2= 0.886

Open and unreactive

conformation

~ 50%~ 50%

Folded and reactive

conformationNoticeably, suitable modifications in aryl-analogues enhances the folded percentage up to 75% with a corresponding increase of quenching activity as seen in 4-metoxybenzyl derivative [2].

The Michael adduction can be parameterized calculating the nucleophilicity of new analogues using suitable indices

mainly based on ab initio simulations. Preliminary analyses confirmed the significant reactivity of imidazole ring and

unveiled the promising activity of furan ring.

N

NH

NH

OH

NH2 O

O

MeO

(S)

+ NH

O OH

N

N

NO OH

NH

O OH

NH

N

NH2O

Carnosine HNE

OOH

NH

O OH

NH

N

NOOH

Schiff Base Michael adduct