CXCR4 ligand development: A downsizing process

Figure 3 illustrates the ligand development of CXCR4 binding peptide antagonists starting from the identification of antimicrobial peptides down to the development of small cyclic pentapeptides, which are employed as lead structures in this study. Tachyplesin I and II and polyphemusin I and II were isolated from the hemocytes of horseshoe crabs (Tachypleus tridentatus and Limulus polyphemus) due to their potent anti-HIV properties.

10 A downsizing process

Figure 3. Peptide and peptido-mimetic ligands for CXCR4. The amino acid sequence of CXCL12 (68 AS) is included with the two binding sites for CXCR4 labeled with purple (site 1) and blue (site 2) circles.

Cysteines involved in disulfide bridges are labeled green and red, respectively.

Structural modifications in the course of ligand develop-ment are indicated with blue color ^84-92.

11 These peptides contain two disulfide bridges (colored red and green, Figure 3), which stabilize the antiparallel β-sheet structure connected by a β-turn. Structure-activity relation (SAR) studies on the basis of these highly potent peptides resulted in the identification of T22 (substitution of two phenylalanine with tyrosine, and valine with lysine, starting from polyphemism II, respectively)⁸⁶, which retained the anti-parallel β-sheet structure similar to that of tachyplesin I.Importantly, the two disulfide bridges and two repeated Tyr-Arg-Lys (Y-R-K) motifs were shown to be indispensable to the anti-HIV activity of T22. With focus on molecular size reduction (T22 contains 18 AS), the outer disulfide bond (indicated in green, Figure 3) of T22, together with the two crucial Tyr-Arg-Lys (Y-R-K) sequences were retained in the novel peptide structures. In addition, turn-stabilizing motifs (D-Lys-Pro or Pro-D-Lys, indicated in blue, TW70) were introduced to the peptide sequence, resulting in the first, potent 14-residue CXCR4 antagonist TW70. TW70 maintains an antiparallel β-sheet structure even though it has only one stabilizing disulfide bridge ⁸⁷.

Further derivatization of TW70 was performed with the focus on decreasing cytotoxicity, which was believed to result from the high number of basic amino acid residues. Subsequent substitution of Arg- and Lys- residues with glutamic acid and citrulline (Cit) was conducted.

Consequently, T134 (substitution of lysine with L-Cit) and T140 (additional substitution of Trp with L-3-(2-naphthyl)alanine (Nal)) exhibited the highest CXCR4 binding affinities measured so far. Several different SAR studies, including an alanine scanning experiment of T140, revealed the pivotal role of Arg², Nal³, Tyr⁵ and Arg¹⁴ for the inhibitory activity against binding of CXCL12 to CXCR4. Subsequent approaches to decrease cytotoxicity and to increase biological stability were achieved by amidation of the C-terminus and substitution of Arg with L-Cit, which resulted in TN14003 ([Cit⁶]-T140 with a C-terminal amide). Due to the superior stability of TN14003 in human serum, a functional group was introduced to further exploit the optimized peptide. Hence, a 4-fluorobenzoyl group constituted a novel pharmacophore for T140-based CXCR4 antagonists, providing the most potent antagonist, TF14016 (4-fluorobenzoyl-TN14003, see Figure 7), with subnanomolar binding affinity ⁹³. This peptide CXCR4 antagonist was

12 further employed for ¹⁸F-or ⁶⁸Ga-based positron emission tomography (PET) imaging of CXCR4 expression in vivo, as discussed later ^94-96.

The crystal structure of a T140 peptide analogue, CVX15 clearly supported the key role of residues Arg², Nal³, Tyr⁵ and Arg¹⁴ as determined by SAR studies so far. These key residues were employed in the following molecular-size reduction approach, wherein the four amino acids were connected with a glycine linker, resulting in a cyclic pentapeptide (FC131 (R2), see Figure 3) which is equipotent to T140. The indispensable functional groups of the side chains of FC131 (R2) were illustrated in further SAR studies including alanine scanning, N-methyl amino acid scanning, optimization of amino acids and design of retro-inverso sequence peptides, which all failed to improve the binding affinity of FC131 ^97-99. A further increase of binding affinity was only accomplished by N-methylation of Arg² in FC131 (R2) (FC122, see Figure 3), which resulted in an alternative binding mode with a flipped D-Tyr¹-N(Me)Arg² peptide bond in FC122

100, 101. Within the scope of the development of molecular imaging probes for CXCR4, the N-methylation approach was also employed to enhance binding affinity, while all side chains of FC131 were tested for their feasibility of exchange. The substitution of Arg² with D-Ornithine and subsequent N-methylation of D-Orn², yielded in CPCR4 (cyclo[D-Tyr¹-N(Me)D-Orn²-Arg³ -Nal⁴-Gly⁵], (11), see Figure 3), which exhibits high binding affinity towards CXCR4 and in addition comprises an anchor point for further modification ¹⁰². Attempts to modify other side chains of FC131 resulted in a total loss of activity, however N(Me)D-Orn has been found to be a valuable attachment site for a variety of linking substituents. As expected from the massive affinity losses of residue modification in the binding scaffold of FC131, the introduction of acyl or alkyl substituents on Orn² of CPCR4 (11) reduced the binding affinity again, but unexpectedly, the attachment of a benzoic acids on Orn² retained most of the CXCR4 binding affinity. In a subsequent optimization step including more than 25 compounds, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was introduced into the molecule with an optimized linking unit to yield Pentixafor (23, see Figure 3) as the first high affinity PET tracer for CXCR4 90, 91, 103.

13 Starting from CPCR4 (11) an alternative approach was initiated to further optimize the interaction of the cyclic pentapeptide ligands with the residues of CXCR4 in the binding cavity.

NMR studies revealed, that FC122 ([N(Me)-Arg²]-FC131) despite its high affinity, exhibits two conformations in slow equilibrium, wherein only one was assumed to be the bioactive conformation ¹⁰⁰. In order to enhance stiffness of the cyclic peptides, the side chain of Orn² in CPCR4 (11) was shifted from the α-carbon to the adjacent nitrogen atom assuming that the peptide bond (cis-trans) would be frozen in its trans conformation. The resulting peptoid compound CPCR4.3 (R1) exhibited a 10-fold higher affinity compared to FC131 ⁸⁹. The binding scaffolds of R1 and CPCR4 (11) were utilized in the present work for the development of high affinity molecular imaging and endoradiotherapeutic probes targeting CXCR4. Therefore, information about the binding mode of the lead compounds can be utilized to find possible attachment sites for radiolabeling moieties.