Design of Inhibitors for Human Tissue Kallikrein Using Non-Natural Aromatic and Basic Amino Acids

Abstract We explored the unique substrate specificity of the primary S1 subsite of human urinary kallikrein (hK1), which accepts both Phe or Arg synthesizing and assaying peptides derived from PhenylacetylPheSer ArgEDDnp, a previously described inhibitor with analgesic and antiinflammatory activities [Emim et al., Br. J. Pharmacol. 130 (2000), 1099 1107]. Phe was substituted by amino acids containing larger aliphatic or aromatic side chains as well as by nonnatural basic amino acids, which were designed to combine a large hydrophobic and/or aromatic group with a positivelycharged group at their side chains. In general, all peptides with basic amino acids represented better inhibitors than those with hydrophobic amino acids. Furthermore, the S1 subsite specificity proved to be much more selective than the mere distinction between Phe and Arg, for minor differences in the side chains of the nonnatural amino acids resulted in major differences in the Ki values. Finally, we present a series of peptides that were assayed as competitive inhibitors for human tissue kallikrein that may lead to the development of novel peptides, which are both more potent and selective.

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Human Tissue Kallikrein S1Subsite Recognition of Non-Natural Basic Amino Acids†

Biochemistry ◽

10.1021/bi002003u ◽

2001 ◽

Vol 40 (17) ◽

pp. 5226-5232 ◽

Cited By ~ 6

Author(s):

Robson L. Melo ◽

Roseli C. Barbosa Pozzo ◽

Daniel C. Pimenta ◽

Elisa Perissutti ◽

Giuseppe Caliendo ◽

...

Keyword(s):

Amino Acids ◽

Human Tissue ◽

Tissue Kallikrein ◽

Basic Amino Acids

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Specificity of human tissue kallikrein towards substrates containing Phe‒Phe pair of amino acids

Biochemical Journal ◽

10.1042/0264-6021:3390473 ◽

1999 ◽

Vol 339 (2) ◽

pp. 473 ◽

Cited By ~ 7

Author(s):

Daniel C. PIMENTA ◽

Julie CHAO ◽

Lee CHAO ◽

Maria A. JULIANO ◽

Luiz JULIANO

Keyword(s):

Amino Acids ◽

Human Tissue ◽

Tissue Kallikrein

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Substrate specificity of an adenohypophyseal endopeptidase capable of hydrolyzing luteinizing hormone-releasing hormone: preferential cleavage of peptide bonds involving the carboxyl terminus of hydrophobic and basic amino acids

Biochemistry ◽

10.1021/bi00534a032 ◽

1982 ◽

Vol 21 (5) ◽

pp. 1033-1036 ◽

Cited By ~ 25

Author(s):

Bernhard Horsthemke ◽

Karl Bauer

Keyword(s):

Amino Acids ◽

Luteinizing Hormone ◽

Substrate Specificity ◽

Carboxyl Terminus ◽

Peptide Bonds ◽

Luteinizing Hormone Releasing Hormone ◽

Releasing Hormone ◽

Basic Amino Acids ◽

Preferential Cleavage

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A phase transition from monoclinicC2 withZ′ = 1 to triclinicP1 withZ′ = 4 for the quasiracemateL-2-aminobutyric acid–D-methionine (1/1)

Acta Crystallographica Section C Structural Chemistry ◽

10.1107/s2053229616008858 ◽

2016 ◽

Vol 72 (7) ◽

pp. 536-543 ◽

Cited By ~ 5

Author(s):

Carl Henrik Görbitz ◽

David S. Wragg ◽

Ingrid Marie Bergh Bakke ◽

Christian Fleischer ◽

Gaute Grønnevik ◽

...

Keyword(s):

Phase Transition ◽

Amino Acids ◽

Room Temperature ◽

Aminobutyric Acid ◽

Side Chain ◽

Side Chains ◽

Significant Difference ◽

Hydrophobic Amino Acids ◽

Chain Conformations ◽

Triclinic Form

Racemates of hydrophobic amino acids with linear side chains are known to undergo a unique series of solid-state phase transitions that involve sliding of molecular bilayers upon heating or cooling. Recently, this behaviour was shown to extend also to quasiracemates of two different amino acids with opposite handedness [Görbitz & Karen (2015).J. Phys. Chem. B,119, 4975–4984]. Previous investigations are here extended to an L-2-aminobutyric acid–D-methionine (1/1) co-crystal, C4H9NO2·C5H11NO2S. The significant difference in size between the –CH2CH3and –CH2CH2SCH3side chains leads to extensive disorder at room temperature, which is essentially resolved after a phase transition at 229 K to an unprecedented triclinic form where all four D-methionine molecules in the asymmetric unit have different side-chain conformations and all three side-chain rotamers are used for the four partner L-2-aminobutyric acid molecules.

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Specificity of human tissue kallikrein towards substrates containing Phe–Phe pair of amino acids

Biochemical Journal ◽

10.1042/bj3390473 ◽

1999 ◽

Vol 339 (2) ◽

pp. 473-479 ◽

Cited By ~ 1

Author(s):

Daniel C. PIMENTA ◽

Julie CHAO ◽

Lee CHAO ◽

Maria A. JULIANO ◽

Luiz JULIANO

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Human Tissue ◽

Binding Protein ◽

General Structure ◽

Protein A ◽

Tissue Kallikrein ◽

Hybrid Peptides ◽

Loop Sequence ◽

Reactive Centre Loop

We have explored in detail the determinants of specificity for the hydrolysis by human tissue kallikrein (hK1) of substrates containing the Phe–Phe amino acid pair, after which hK1 cleaves kallistatin (human kallikrein-binding protein), a specific serpin for this protease, as well as somatostatin 1–14. Internally quenched fluorogenic peptides were synthesized with the general structure Abz-peptidyl-EDDnp [Abz, o-aminobenzoic acid; EDDnp, N-(2,4-dinitrophenyl)ethylenediamine], based on the natural reactive-centre loop sequence of kallistatin from P9 to P´13, and the kinetic parameters of their hydrolysis by hK1 were determined. All these peptides were cleaved after the Phe–Phe pair. For comparison, we have also examined peptides containing the reactive-centre loop sequences of human protein-C inhibitor (PCI) and rat kallikrein-binding protein, which were hydrolysed after Phe–Arg and Leu–Lys bonds, respectively. Hybrid peptides containing kallistatin–PCI sequences showed that the efficiency of hK1 activity on the peptides containing kallistatin and PCI sequences depended on both the nature of the P1 amino acid as well as on residues at the P- and P´-sides. Moreover, we have made systematic modifications on the hydrophobic pair Phe–Phe, and on Lys and Ile at the P3 and P4 positions according to the peptide substrate, Abz-AIKFFSRQ-EDDnp. All together, we concluded that tissue kallikrein was very effective on short substrates that are cleaved after the Phe–Arg pair; however, hydrolysis after Phe–Phe or other hydrophobic pairs of amino acids was more restrictive, requiring additional enzyme–substrate interaction and/or particular substrate conformations.

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Interaction of the neutral amino acid transporter ASCT2 with basic amino acids

Biochemical Journal ◽

10.1042/bcj20190859 ◽

2020 ◽

Vol 477 (8) ◽

pp. 1443-1457

Author(s):

Elias Ndaru ◽

Rachel-Ann A. Garibsingh ◽

Laura Zielewicz ◽

Avner Schlessinger ◽

Christof Grewer

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Binding Site ◽

Substrate Binding ◽

Ph Dependence ◽

Side Chains ◽

Substrate Binding Site ◽

Basic Amino Acids ◽

Voltage Dependent ◽

Diaminopropionic Acid

Glutamine transport across cell membranes is performed by a variety of transporters, including the alanine serine cysteine transporter 2 (ASCT2). The substrate-binding site of ASCT2 was proposed to be specific for small amino acids with neutral side chains, excluding basic substrates such as lysine. A series of competitive inhibitors of ASCT2 with low µM affinity were developed previously, on the basis of the 2,4-diaminobutyric acid (DAB) scaffold with a potential positive charge in the side chain. Therefore, we tested whether basic amino acids with side chains shorter than lysine can interact with the ASCT2 binding site. Molecular docking of L-1,3-diaminopropionic acid (L-DAP) and L-DAB suggested that these compounds bind to ASCT2. Consistent with this prediction, L-DAP and L-DAB, but not ornithine, lysine or D-DAP, elicited currents when applied to ASCT2-expressing cells. The currents were carried by anions and showed the hallmark properties of ASCT2 currents induced by transported substrates. The L-DAP response could be eliminated by a competitive ASCT2 inhibitor, suggesting that binding occurs at the substrate binding site. The KM for L-DAP was weakly voltage dependent. Furthermore, the pH dependence of the L-DAP response showed that the compound can bind in several protonation states. Together, these results suggest that the ASCT2 binding site is able to recognize L-amino acids with short, basic side chains, such as the L-DAP derivative β-N-methylamino-l-Alanine (BMAA), a well-studied neurotoxin. Our results expand the substrate specificity of ASCT2 to include amino acid substrates with positively charged side chains.

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Hydrolysis of somatostatin by human tissue kallikrein after the amino acid pair Phe-Phe

Biochemical Journal ◽

10.1042/bj3270027 ◽

1997 ◽

Vol 327 (1) ◽

pp. 27-30 ◽

Cited By ~ 8

Author(s):

Daniel C. PIMENTA ◽

Maria A. JULIANO ◽

Luiz JULIANO

Keyword(s):

Amino Acids ◽

Growth Hormone ◽

Human Tissue ◽

General Structure ◽

Tissue Kallikrein ◽

Growth Hormone Releasing Hormone ◽

Amino Acid Pair ◽

Reactive Centre Loop ◽

Hydrolysis Of ◽

Similar Kinetic

Somatostatin-(1–14) was hydrolysed by human tissue kallikrein at the Phe7-Trp8 bond, after a Phe-Phe pair of amino acids, with similar kinetic parameters to those described for human high- and low-molecular-mass kininogens. Substance P and human insulin, which also contain a Phe-Phe pair in their sequences, were both resistant. More details of this hydrolytic specificity of human tissue kallikrein were obtained by synthesizing and assaying internally quenched fluorescent peptides containing the sequence of somatostatin-(1–14), as well as the reactive-centre loop of human kallikrein-binding protein (kallistatin). We also observed that human tissue kallikrein hydrolysed growth hormone-releasing hormone at the Arg11-Lys12 bond, although this peptide contains in its structure a pair of leucines (Leu22-Leu23), in contrast with the Phe-Phe pair in somatostatin. We have also demonstrated the susceptibility to human tissue kallikrein of some chromogenic peptides with the general structure X-Phe-Phe-p-nitroanilide and of d-Pro-Phe-Phe-4-methylcoumaryl-7-amide.

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An exceptional series of phase transitions in hydrophobic amino acids with linear side chains

IUCrJ ◽

10.1107/s2052252516010472 ◽

2016 ◽

Vol 3 (5) ◽

pp. 341-353 ◽

Cited By ~ 15

Author(s):

Carl Henrik Görbitz ◽

Pavel Karen ◽

Michal Dušek ◽

Václav Petříček

Keyword(s):

Amino Acids ◽

Phase Transitions ◽

Room Temperature ◽

Side Chain ◽

Side Chains ◽

X Ray Diffraction ◽

Scanning Calorimetry ◽

Modulated Structure ◽

Hydrogen Bond Pattern ◽

Hydrophobic Amino Acids

The solid-state phase transitions and intermediate structures ofS-2-aminobutanoic acid (L-2-aminobutyric acid),S-2-aminopentanoic acid (L-norvaline),S-2-aminohexanoic acid (L-norleucine) and L-methionine between 100 and 470 K, identified by differential scanning calorimetry, have been characterized in a comprehensive single-crystal X-ray diffraction investigation. Unlike other enantiomeric amino acids investigated until now, this group featuring linear side chains displays up to five distinct phases. The multiple transitions between them involve a number of different processes: alteration of the hydrogen-bond pattern, to our knowledge the first example of this observed for an amino acid, sliding of molecular bilayers, seen previously only for racemates and quasiracemates, concerted side-chain rearrangements and abrupt as well as gradual modifications of the side-chain disorder. Ordering of L-norleucine upon cooling even proceedsviaan incommensurately modulated structure. L-Methionine has previously been described as being fully ordered at room temperature. An accurate refinement now reveals extensive disorder for both molecules in the asymmetric unit, while two previously unknown phases occur above room temperature.

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