Thrombostatin FM compounds: direct thrombin inhibitors – mechanism of action in vitro and in vivo

Nieman, Marvin T.; Burke, F.; Warnock, Mark; Zhou, Yihua; Sweigart, J.; Chen, A.; Ricketts, D.; Lucchesi, Benedict R.; Chen, Z.; Di, Enrico; Hilfinger, John M.; Kim, J. S.; Mosberg, Henry I.

doi:10.1111/j.1538-7836.2008.02937.x

Cited by 11 publications

(14 citation statements)

References 24 publications

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“…More recent approaches include the study of several synthetic analogs of the angiotensin converting enzyme breakdown product of bradykinin (RPPGF) [17], [18], [19], [20] as well as 1,3,5-trisubstituted benzenes [21] as potent thrombin inhibitors.…”

Section: Introductionmentioning

confidence: 99%

Rational Design and Characterization of D-Phe-Pro-D-Arg-Derived Direct Thrombin Inhibitors

et al. 2012

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The tremendous social and economic impact of thrombotic disorders, together with the considerable risks associated to the currently available therapies, prompt for the development of more efficient and safer anticoagulants. Novel peptide-based thrombin inhibitors were identified using in silico structure-based design and further validated in vitro. The best candidate compounds contained both l- and d-amino acids, with the general sequence d-Phe(P3)-Pro(P2)-d-Arg(P1)-P1′-CONH2. The P1′ position was scanned with l- and d-isomers of natural or unnatural amino acids, covering the major chemical classes. The most potent non-covalent and proteolysis-resistant inhibitors contain small hydrophobic or polar amino acids (Gly, Ala, Ser, Cys, Thr) at the P1′ position. The lead tetrapeptide, d-Phe-Pro-d-Arg-d-Thr-CONH2, competitively inhibits α-thrombin's cleavage of the S2238 chromogenic substrate with a Ki of 0.92 µM. In order to understand the molecular details of their inhibitory action, the three-dimensional structure of three peptides (with P1′ l-isoleucine (fPrI), l-cysteine (fPrC) or d-threonine (fPrt)) in complex with human α-thrombin were determined by X-ray crystallography. All the inhibitors bind in a substrate-like orientation to the active site of the enzyme. The contacts established between the d-Arg residue in position P1 and thrombin are similar to those observed for the l-isomer in other substrates and inhibitors. However, fPrC and fPrt disrupt the active site His57-Ser195 hydrogen bond, while the combination of a P1 d-Arg and a bulkier P1′ residue in fPrI induce an unfavorable geometry for the nucleophilic attack of the scissile bond by the catalytic serine. The experimental models explain the observed relative potency of the inhibitors, as well as their stability to proteolysis. Moreover, the newly identified direct thrombin inhibitors provide a novel pharmacophore platform for developing antithrombotic agents by exploring the conformational constrains imposed by the d-stereochemistry of the residues at positions P1 and P1′.

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Section: Introductionmentioning

confidence: 99%

Rational Design and Characterization of D-Phe-Pro-D-Arg-Derived Direct Thrombin Inhibitors

et al. 2012

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“…The difficulty to date in considering thrombin inhibition for cancer treatment is that all presently approved agents are parenteral and potent, putting the cancer patient at bleeding risk. We have developed an oral, reversible direct thrombin inhibitors based the angiotensin converting enzyme (ACE) breakdown product of bradykinin, BK1-5 or RPPGF (18)(19)(20)(21). FM19 consists of D-isomers and unusual amino acid substitutions to increase stability and allow for oral activity (20).…”

Section: Introductionmentioning

confidence: 99%

“…FM19 is a direct thrombin inhibitor with a K i of 4.4 ± 2.4 μM that inhibits the threshold of α-thrombin-induced human platelet aggregation at 8.4 ± 4.7 μM (21). On crystallography, FM19 is a retrobinder in thrombin's active site (21). When administered in drinking water at 5 mg/ml, but not at 3 mg/ml, it delays induced arterial thrombosis (21).…”

Section: Introductionmentioning

confidence: 99%

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Oral thrombostatin FM19 inhibits prostate cancer

Nieman¹,

LaRusch²,

Fang³

et al. 2010

Thromb Haemost

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Thrombin stimulates proliferation, invasion and metastasis by cleaving PAR1 on a human prostate cancer cells. Current direct thrombin inhibitors pose risks for bleeding in the cancer patients. We have developed an oral reversible direct thrombin inhibitor called FM19. FM19 inhibits thrombin-induced calcium mobilization of PC3 cells with an IC50 of 15 μM with a 95% confidence interval of 7.3–31.6 μM. Thrombin stimulation increases PC3 cell invasion 3-fold from 27.1 ± 11.4 to 66 ± 11.6. FM19 or bivalirudin reduces cell invasion at ≥ 0.1 μM (p≤0.02). After inoculation with PC3 cells, nude mice were treated with oral FM19 at 3 mg/ml in the drinking water. The treated mice do not have long bleeding times and only a 1.4-fold increase in their thrombin clotting time. However, with treatment, the mice have a reduced rate of tumor growth 0.26 ± 0.17 fold change/day versus 0.55 ± 0.35 for untreated (p = 0.038), reduced fold change in tumor size 5.3 ± 0.47 to 8.9 ± 1.8 (untreated) (p=0.048), and reduced overall tumor weight 0.5 ± 0.31 g versus 0.82 ± 0.32 g (untreated) (p=0.04). On microscopic examination, FM19 treatment reduces the number of large vessels in the tumors from 4.6±2.1 per high-powered field in untreated samples to 1.4±1.4 in treated samples (p≤0.04). These studies show FM19 reduces prostate tumor growth in vivo at a concentration below that needed for anticoagulation. These data suggest novel opportunities for oral direct thrombin inhibitors in cancer therapy.

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“…SAR studies were carried out to follow-up on RPPGF antithrombotic activity and culminated in the development of lead compound, FM 19, with the sequence D-Arg-Oic-Pro-D-Ala-Phe(p-Me)-NH 2 , where Oic is ((2S,3aS,7aS)-octahydroindole-2-carboxylic acid) ( Figure 1) (9). Although FM 19 is more potent than RPPGF (K i = 4.4 lM versus 1.75 mM), FM 19 is still only a modest inhibitor of thrombin activity (10). The crystal structure of FM 19 in thrombin's active site has recently been determined, showing that, like RPPGF, FM 19 is a retrobinder (Figure 2) (10).…”

mentioning

confidence: 99%

Structure‐Based Design of Residue 1 Analogs of the Direct Thrombin Inhibitor Pentapeptide FM 19

Girnys

Sobczyk-Kojiro

Mosberg³

2009

Chem Biol Drug Des

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Myocardial ischemia and other acute coronary syndromes are leading causes of death worldwide, and often result from a thrombus that blocks an atherosclerotic coronary artery. A key enzyme in thrombus formation is the serine protease thrombin, which is responsible for both the conversion of soluble fibrinogen into insoluble fibrin, as well as the activation of the GPCRs, PAR1 and PAR4, which stimulate platelet aggregation. Thus, thrombin is an attractive target for anticoagulant and antithrombotic therapy. Previous studies in our laboratory led to the development of lead compound FM 19 (D-Arg-Oic-Pro-D-Ala-Phe(p-Me)-NH 2 ), which shows modest potency as a thrombin inhibitor. The recently determined X-ray structure of FM 19 in the active site of thrombin has revealed potential sites for modification to improve potency. This study reports replacements to the first residue (D-Arg 1 ) of FM 19, which seek to improve potency by removing the N-terminal amine to eliminate an adverse electrostatic interaction, and alterations to the length of the side chain to eliminate an unfavorable eclipsed conformation observed in the X-ray structure. This study produced two compounds, 1 and 9, with improved a-thrombin inhibition (IC 50 values of 0.66 ± 0.20 lM and 0.57 ± 0.12 lM, respectively).

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Thrombostatin FM compounds: direct thrombin inhibitors – mechanism of action in vitro and in vivo

Cited by 11 publications

References 24 publications

Rational Design and Characterization of D-Phe-Pro-D-Arg-Derived Direct Thrombin Inhibitors

Rational Design and Characterization of D-Phe-Pro-D-Arg-Derived Direct Thrombin Inhibitors

Oral thrombostatin FM19 inhibits prostate cancer

Structure‐Based Design of Residue 1 Analogs of the Direct Thrombin Inhibitor Pentapeptide FM 19

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