Design and implementation of a particle concentration fluorescence method for the detection of HIV-1 protease inhibitors

Manetta, Joseph; Lai, Mei‐Huei T.; Osborne, Harold E.; Dee, Albert; Margolin, Nara; Sportsman, J. Richard; Vlahos, Chris J.; Heath, William F.

doi:10.1016/0003-2697(92)90198-g

Cited by 16 publications

(5 citation statements)

References 21 publications

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“…A number of the thiadiazoles listed in Table 3 were evaluated against stromelysin using a continuous fluorescence inhibition assay. In addition, a particle concentration fluorescent assay (PCFA) was developed based on that reported by Manndetta 26 for screening HIV protease inhibitors. This assay was used for the evaluation of the analogues reported in Tables 4 and 5.…”

Section: Resultsmentioning

confidence: 99%

“…Particle Concentration Fluorescent Assay. This assay, based on that described by Mandetta, 26 was used to assess inhibitory properties of compounds against three MMPs: collagenase, gelatinase A, and stromelysin-1. The same polypeptide substrate, 34 labeled at the amino end with fluorescein and biotin at the carboxyl end, was used with all three enzymes.…”

Section: N-[[(45-dihydro-5-thioxo-134-thiadiazol-2-yl)amino]carbonyl]...mentioning

confidence: 99%

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Synthesis of a Series of Stromelysin-Selective Thiadiazole Urea Matrix Metalloproteinase Inhibitors

Jacobsen¹,

Mitchell²,

Hendges³

et al. 1999

J. Med. Chem.

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The synthesis and enzyme inhibition data for a series of thiadiazole urea matrix metalloproteinase (MMP) inhibitors are described. A broad screening effort was utilized to identify several thiadiazoles which were weak inhibitors of stromelysin. Optimization of the thiadiazole leads to include an alpha-amino acid side chain with variable terminal amide substituents provided a series of ureas which were moderately effective stromelysin inhibitors, with Ki's between 0.3 and 1.0 microM. The most effective analogues utilized an L-phenylalanine as the amino acid component. In particular, unsubstituted 46 had a Ki of 710 nM, while the p-fluoro analogue 52 displayed increased potency (100 nM). Stromelysin inhibition was further improved using a pentafluorophenylalanine substituent which resulted in 70, a 14 nM inhibitor. While gelatinase inhibition was generally poor, the use of 1-(2-pyridyl)piperazine as the amide component usually provided for enhanced activity, with 71 inhibiting gelatinase with a Ki of 770 nM. The combination of this heterocycle with a p-fluorophenylalanine substituent provided the only analogue, 69, with collagenase activity (13 microM). The SAR for analogues described within this series can be rationalized through consideration of the X-ray structure recently attained for70 complexed to stromelysin. Uniquely, this structure showed the inhibitor to be completely orientated on the left side of the enzyme cleft. These results suggest that thiadiazole urea heterocycles which incorporate a substituted phenylalanine can provide selective inhibitors of stromelysin. Careful selection of the amide substituent can also provide for analogues with modest gelatinase inhibition.

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Section: Resultsmentioning

confidence: 99%

Section: N-[[(45-dihydro-5-thioxo-134-thiadiazol-2-yl)amino]carbonyl]...mentioning

confidence: 99%

Synthesis of a Series of Stromelysin-Selective Thiadiazole Urea Matrix Metalloproteinase Inhibitors

Jacobsen¹,

Mitchell²,

Hendges³

et al. 1999

J. Med. Chem.

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“…Biological Methods. Protease inhibition assays 17 and whole cell antiviral testing 18 have been described previously.…”

Section: Methodsmentioning

confidence: 99%

Potent Human Immunodeficiency Virus Type 1 Protease Inhibitors That Utilize Noncoded d-Amino Acids as P₂/P₃ Ligands

et al. 1996

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Noncoded D-amino acids have been designed to replace the quinaldic amide-asparaginyl moiety (P2/P3 ligand) found in several potent human immunodeficiency virus (HIV) protease inhibitors such as LY289612. The substituted nitrogen, optimally an N-methanesulfonyl moiety, served as a CH2CONH2 (asparagine side chain mimic), while the amino acid side chain became the backbone and P3 ligand of these novel inhibitors. Compounds derived from S-aryl-D-cysteine proved to be potent HIV protease inhibitors which also exhibited potent whole cell antiviral activity. Oxidation of the cysteines to the sulfoxide or sulfone oxidation states resulted in significant improvements in potency. For example, the compound derived from N-(methyl-sulfonyl)-2-S-naphthylcysteine sulfone, 17c, was a 3.5 nM inhibitor of HIV protease which inhibited the spread of virus in MT4 cells with an IC50 = 4.3 nM. Compounds 17c,g,i were found to be orally bioavailable in a rat model.

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“…Kinetic readings were taken at 10 minute intervals for 180 minutes. Figure 3D Fluorescence polarization technology is well suited to high-throughput screening since inhibition of macromolecular binding can be done rapidly, in solution, without any washing steps [73][74][75]. The availability of adequate supplies of well-characterized probe and target protein molecules is fundamental to development of HTS assays.…”

Section: Cell-free Hts Assaysmentioning

confidence: 99%

Application of High-Throughput, Molecular-Targeted Screening to Anticancer Drug Discovery

Shoemaker

Scudiero²,

Melillo³

et al. 2002

CTMC

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Increasing insight into the genetics and molecular biology of cancer has resulted in the identification of an increasing number of potential molecular targets for anti-cancer drug discovery and development. These targets can be approached through exploitation of emerging structural biology, "rational" drug design, screening of chemical libraries, or a combination of these methods. In this article we discuss the application of high-throughput screening to anti-cancer drug discovery, with special reference to approaches used at the U.S. National Cancer Institute.

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Design and implementation of a particle concentration fluorescence method for the detection of HIV-1 protease inhibitors

Cited by 16 publications

References 21 publications

Synthesis of a Series of Stromelysin-Selective Thiadiazole Urea Matrix Metalloproteinase Inhibitors

Synthesis of a Series of Stromelysin-Selective Thiadiazole Urea Matrix Metalloproteinase Inhibitors

Potent Human Immunodeficiency Virus Type 1 Protease Inhibitors That Utilize Noncoded d-Amino Acids as P₂/P₃ Ligands

Application of High-Throughput, Molecular-Targeted Screening to Anticancer Drug Discovery

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Design and implementation of a particle concentration fluorescence method for the detection of HIV-1 protease inhibitors

Cited by 16 publications

References 21 publications

Synthesis of a Series of Stromelysin-Selective Thiadiazole Urea Matrix Metalloproteinase Inhibitors

Synthesis of a Series of Stromelysin-Selective Thiadiazole Urea Matrix Metalloproteinase Inhibitors

Potent Human Immunodeficiency Virus Type 1 Protease Inhibitors That Utilize Noncoded d-Amino Acids as P2/P3 Ligands

Application of High-Throughput, Molecular-Targeted Screening to Anticancer Drug Discovery

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Potent Human Immunodeficiency Virus Type 1 Protease Inhibitors That Utilize Noncoded d-Amino Acids as P₂/P₃ Ligands