Co-reporter:Manishabrata Bhowmick, Dorota Tokmina-Roszyk, Lillian Onwuha-Ekpete, Kelli Harmon, Trista Robichaud, Rita Fuerst, Roma Stawikowska, Bjorn Steffensen, William Roush, Hector R. Wong, and Gregg B. Fields
Journal of Medicinal Chemistry May 11, 2017 Volume 60(Issue 9) pp:3814-3814
Publication Date(Web):April 10, 2017
DOI:10.1021/acs.jmedchem.7b00018
The design of selective matrix metalloproteinase (MMP) inhibitors that also possess favorable solubility properties has proved to be especially challenging. A prior approach using collagen-model templates combined with transition state analogs produced a first generation of triple-helical peptide inhibitors (THPIs) that were effective in vitro against discrete members of the MMP family. These THPI constructs were also highly water-soluble. The present study sought improvements in the first generation THPIs by enhancing thermal stability and selectivity. A THPI selective for MMP-2 and MMP-9 was redesigned to incorporate non-native amino acids (Flp and mep), resulting in an increase of 18 °C in thermal stability. This THPI was effective in vivo in a mouse model of multiple sclerosis, reducing clinical severity and weight loss. Two other THPIs were developed to be more selective within the collagenolytic members of the MMP family. One of these THPIs was serendipitously more effective against MMP-8 than MT1-MMP and was utilized successfully in a mouse model of sepsis. The THPI targeting MMP-8 minimized lung damage, increased production of the anti-inflammatory cytokine IL-10, and vastly improved mouse survival.
Co-reporter:Jun Yong Choi, Rita Fuerst, Anna M. Knapinska, Alexander B. Taylor, Lyndsay Smith, Xiaohang Cao, P. John Hart, Gregg B. Fields, and William R. Roush
Journal of Medicinal Chemistry July 13, 2017 Volume 60(Issue 13) pp:5816-5816
Publication Date(Web):June 27, 2017
DOI:10.1021/acs.jmedchem.7b00514
We describe the use of comparative structural analysis and structure-guided molecular design to develop potent and selective inhibitors (10d and (S)-17b) of matrix metalloproteinase 13 (MMP-13). We applied a three-step process, starting with a comparative analysis of the X-ray crystallographic structure of compound 5 in complex with MMP-13 with published structures of known MMP-13·inhibitor complexes followed by molecular design and synthesis of potent but nonselective zinc-chelating MMP inhibitors (e.g., 10a and 10b). After demonstrating that the pharmacophores of the chelating inhibitors (S)-10a, (R)-10a, and 10b were binding within the MMP-13 active site, the Zn2+ chelating unit was replaced with nonchelating polar residues that bridged over the Zn2+ binding site and reached into a solvent accessible area. After two rounds of structural optimization, these design approaches led to small molecule MMP-13 inhibitors 10d and (S)-17b, which bind within the substrate-binding site of MMP-13 and surround the catalytically active Zn2+ ion without chelating to the metal. These compounds exhibit at least 500-fold selectivity versus other MMPs.
Co-reporter:Maciej J. Stawikowski, Roma Stawikowska, and Gregg B. Fields
Biochemistry 2015 Volume 54(Issue 19) pp:3110-3121
Publication Date(Web):April 21, 2015
DOI:10.1021/acs.biochem.5b00110
Although collagenolytic matrix metalloproteinases (MMPs) possess common domain organizations, there are subtle differences in their processing of collagenous triple-helical substrates. In this study, we have incorporated peptoid residues into collagen model triple-helical peptides and examined MMP activities toward these peptomeric chimeras. Several different peptoid residues were incorporated into triple-helical substrates at subsites P3, P1, P1′, and P10′ individually or in combination, and the effects of the peptoid residues were evaluated on the activities of full-length MMP-1, MMP-8, MMP-13, and MMP-14/MT1-MMP. Most peptomers showed little discrimination between MMPs. However, a peptomer containing N-methyl Gly (sarcosine) in the P1′ subsite and N-isobutyl Gly (NLeu) in the P10′ subsite was hydrolyzed efficiently only by MMP-13 [nomenclature relative to the α1(I)772–786 sequence]. Cleavage site analysis showed hydrolysis at the Gly–Gln bond, indicating a shifted binding of the triple helix compared to the parent sequence. Favorable hydrolysis by MMP-13 was not due to sequence specificity or instability of the substrate triple helix but rather was based on the specific interactions of the P7′ peptoid residue with the MMP-13 hemopexin-like domain. A fluorescence resonance energy transfer triple-helical peptomer was constructed and found to be readily processed by MMP-13, not cleaved by MMP-1 and MMP-8, and weakly hydrolyzed by MT1-MMP. The influence of the triple-helical structure containing peptoid residues on the interaction between MMP subsites and individual substrate residues may provide additional information about the mechanism of collagenolysis, the understanding of collagen specificity, and the design of selective MMP probes.
Co-reporter:Dr. Manishabrata Bhowmick;Roma Stawikowska;Dorota Tokmina-Roszyk; Gregg B. Fields
ChemBioChem 2015 Volume 16( Issue 7) pp:1084-1092
Publication Date(Web):
DOI:10.1002/cbic.201402716
Abstract
Matrix metalloproteinases (MMPs) have been implicated in numerous pathologies. An overall lack of selectivity has rendered active-site-targeted MMP inhibitors problematic. The present study describes MMP inhibitors that function by binding both secondary binding sites (exosites) and the active site. Heterotrimeric triple-helical peptide transition-state analogue inhibitors (THPIs) were assembled utilizing click chemistry. Three different heterotrimers were constructed, allowing for the inhibitory phosphinate moiety to be present uniquely in the leading, middle, or trailing strand of the triple helix. All heterotrimeric constructs had sufficient thermally stability to warrant analysis as inhibitors. The heterotrimeric THPIs were effective against MMP-13 and MT1-MMP, with Ki values spanning 100–400 nM. Unlike homotrimeric THPIs, the heterotrimeric THPIs offered complete selectivity between MT1-MMP and MMP-1. Exosite-based approaches such as this provide inhibitors with desired MMP selectivities.
Co-reporter:Janelle L. Lauer-Fields, Dmitriy Minond, Peter S. Chase, Pierre E. Baillargeon, S. Adrian Saldanha, Roma Stawikowska, Peter Hodder, Gregg B. Fields
Bioorganic & Medicinal Chemistry 2009 Volume 17(Issue 3) pp:990-1005
Publication Date(Web):1 February 2009
DOI:10.1016/j.bmc.2008.03.004
The major components of the cartilage extracellular matrix are type II collagen and aggrecan. Matrix metalloproteinase 13 (MMP-13) has been implicated as the protease responsible for collagen degradation in cartilage during osteoarthritis (OA). In the present study, a triple-helical FRET substrate has been utilized for high throughput screening (HTS) of MMP-13 with the MLSCN compound library (n ∼ 65,000). Thirty-four compounds from the HTS produced pharmacological dose–response curves. A secondary screen using RP-HPLC validated 25 compounds as MMP-13 inhibitors. Twelve of these compounds were selected for counter-screening with 6 representative MMP family members. Five compounds were found to be broad-spectrum MMP inhibitors, 3 inhibited MMP-13 and one other MMP, and 4 were selective for MMP-13. One of the selective inhibitors was more active against MMP-13 triple-helical peptidase activity compared with single-stranded peptidase activity. Since the THP FRET substrate has distinct conformational features that may interact with MMP secondary binding sites (exosites), novel non-active site-binding inhibitors may be identified via HTS protocols utilizing such assays.
Co-reporter:David R. Khan;Evonne M. Rezler;Janelle Lauer-Fields ;Gregg B. Fields
Chemical Biology & Drug Design 2008 Volume 71( Issue 1) pp:3-7
Publication Date(Web):
DOI:10.1111/j.1747-0285.2007.00610.x
A major obstacle in drug delivery is the inability to effectively deliver drugs to their intended biological target without deleterious side-effects. Delivery vehicles such as liposomes can minimize toxic side-effects by shielding the drug from reaction with unintended targets while in systemic circulation. Liposomes have the ability to accommodate both hydrophilic and hydrophobic drugs, either in the internal aqueous core or the lipid bilayer, respectively. In the present study, fluorescein and rhodamine have been used to model hydrophilic and hydrophobic drugs, respectively. We have compared the stabilities of liposomes encapsulating these fluorophores as a function of lipid content, time, and temperature. At 25 and 37 °C, liposomes containing distearoyl phosphatidylcholine as the major phospholipid component were found to be more stable over time than those containing dipalmitoyl phosphatidylcholine, regardless of the fluorophore encapsulated. Liposomes loaded with fluorescein were found to be more stable than those with rhodamine. Dipalmitoyl phosphatidylcholine liposomes that encapsulated rhodamine were the least stable. The results indicate that the physical properties of the drug cargo play a role in the stability, and hence drug delivery kinetics, of liposomal delivery systems, and desired drug release times can be achieved by adjusting/fine-tuning the lipid compositions.
Co-reporter:Janelle L. Lauer-Fields, Timothy P. Spicer, Peter S. Chase, Mare Cudic, Gayle D. Burstein, Hideaki Nagase, Peter Hodder, Gregg B. Fields
Analytical Biochemistry 2008 Volume 373(Issue 1) pp:43-51
Publication Date(Web):1 February 2008
DOI:10.1016/j.ab.2007.09.014
The major components of the cartilage extracellular matrix are type II collagen and aggrecan. Type II collagen provides cartilage with its tensile strength, whereas the water-binding capacity of aggrecan provides compressibility and elasticity. Aggrecan breakdown leads to an increase in proteolytic susceptibility of articular collagen; hence, aggrecan may also have a protective effect on type II collagen. Given their role in aggrecan degradation and differing substrate specificity profiles, the pursuit of inhibitors for both aggrecanase 1 (a disintegrin and metalloproteinase with thrombospondin motifs-4 [ADAMTS-4]) and aggrecanase 2 (ADAMTS-5) is desirable. We previously described collagen model fluorescence resonance energy transfer (FRET) substrates for aggrecan-degrading members of the ADAMTS family. These FRET substrate assays are also fully compatible with multiwell formats. In the current study, a collagen model FRET substrate was examined for inhibitor screening of ADAMTS-4. ADAMTS-4 was screened against a small compound library (n = 960) with known pharmacological activity. Five compounds that inhibited ADAMTS-4 > 60% at a concentration of 1 μM were identified. A secondary screen using reversed-phase high-performance liquid chromatography (RP–HPLC) was developed and performed for verification of the five potential inhibitors. Ultimately, piceatannol was confirmed as a novel inhibitor of ADAMTS-4, with an IC50 value of 1 μM. Because the collagen model FRET substrates have distinct conformational features that may interact with protease secondary substrate sites (exosites), nonactive site-binding inhibitors can be identified via this approach. Selective inhibitors for ADAMTS-4 would allow a more definitive evaluation of this protease in osteoarthritis and also represent a potential next generation in metalloproteinase therapeutics.
Co-reporter:Orsolya Giricz, Janelle L. Lauer-Fields, Gregg B. Fields
Analytical Biochemistry 2008 Volume 380(Issue 1) pp:137-139
Publication Date(Web):1 September 2008
DOI:10.1016/j.ab.2008.05.024
We examined a panel of 26 melanoma and fibroblast samples (tissues and cultured cells) to evaluate the suitability of two commonly used housekeeping genes, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 18S ribosomal RNA (rRNA), for quantitative real-time PCR. Both genes showed significant variations within the individual cell line and tissue groups. Although no overall trends were observed in the expression of the 18S rRNA, GAPDH was up-regulated in melanoma tissue and cultured cells compared with the corresponding normal samples. In melanoma and fibroblast cell lines and tissues, absolute quantification appears to be more appropriate than normalizing messenger RNA (mRNA) expression via GAPDH or 18S rRNA housekeeping genes.
Co-reporter:Janelle L Lauer-Fields, Péter Kele, Guodong Sui, Hideaki Nagase, Roger M Leblanc, Gregg B Fields
Analytical Biochemistry 2003 Volume 321(Issue 1) pp:105-115
Publication Date(Web):1 October 2003
DOI:10.1016/S0003-2697(03)00460-3
The consequences of improper regulation of collagen turnover include diseases such as tumor cell metastasis and arthritis. Several fluorogenic triple-helical peptide (fTHP) substrates have been constructed presently to examine collagenolytic behavior. These substrates incorporate l- or d-2-amino-3-(7-methoxy-4-coumaryl)propionic acid (Amp) or l- or d-2-amino-3-(6,7-dimethoxy-4-coumaryl)propionic acid (Adp) as the fluorophore and N-2,4-dinitrophenyl (Dnp) as the quencher. The desired sequences were C6-(Gly-Pro-Hyp)5-Gly-Pro-[Amp/Adp]-Gly-Pro-Gln-Gly∼Leu-Arg-Gly-Gln-Lys(Dnp)-Gly-Val-Arg-(Gly-Pro-Hyp)5-NH2. All four fTHPs formed stable triple-helices. Matrix metalloproteinase-2 (MMP-2) rates of hydrolysis for all fTHPs were considerably more rapid than corresponding MMP-1 rates. Evaluation of individual kinetic parameters indicated that MMP-2 bound to the fTHPs more efficiently than MMP-1. Comparison to a triple-helical substrate incorporating the same sequence but with a different fluorophore [Lys((7-methoxycoumarin-4-yl)acetyl); Lys(Mca)] demonstrated that the shorter side chain of Amp or Adp was better tolerated by MMP-1 and MMP-2. Adp may well be the fluorophore of choice for fTHPs, as (a) fTHPs incorporating Adp were obtained in significantly higher yields than the Amp-containing fTHPs, (b) Adp has a larger Stokes shift than either Amp or Lys(Mca) and thus has less chance of self-quenching, (c) Adp has a relatively high quantum yield, (d) the Adp/Dnp pair is compatible with multiwell plate reader formats, and (e) MMPs better tolerate Adp than Lys(Mca).
Co-reporter:Janelle L Lauer-Fields, Hideaki Nagase, Gregg B Fields
Journal of Chromatography A 2000 Volume 890(Issue 1) pp:117-125
Publication Date(Web):18 August 2000
DOI:10.1016/S0021-9673(00)00396-4
The matrix metalloproteinase (MMP) family has been implicated in the process of a variety of diseases such as arthritis, atherosclerosis, and tumor cell metastasis. We have been designing single-stranded peptides (SSPs) and triple-helical peptides (THPs) as potential discriminatory MMP substrates. Edman degradation sequence and matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analyses of proteolytic activity have been utilized to aid in further substrate design. THP models of the α1(I)772–786 sequence from type I collagen were synthesized to examine the triple-helical substrate specificity of MMP family members. Sequence and MALDI-MS analyses were used in conjunction with a fluorometric assay to determine the exact point of cleavage by each MMP. MMP-1 (interstitial collagenase) cleaved the substrates at a single Gly–Ile bond, analogous to the cleavage site in type I collagen. MMP-2 (Mr 72 000 type IV collagenase; gelatinase A) was found to cleave the substrates at two sites, a Gly–Ile bond and a Gly–Gln bond. MMP-3 (stromelysin 1) was found to cleave only one of the substrates after reaction for 48 h. Ultimately, sequence and MALDI-MS analyses allowed us to detect an additional cleavage site for MMP-2 in comparison to MMP-1, while MMP-3 was found to cleave a substrate after an extended time period. The second cleavage site would cause the kinetic parameters for MMP-2 to be overestimated by the fluorometric assay. Further design variations for these substrates need to consider the presence of more stable triple-helical conformation (to eliminate MMP-3 binding) and the removal of Gly–Gln bonds that may be susceptible to MMP-2.
Co-reporter:Pilar Forns;Janelle L. Lauer-Fields;Su Gao;Gregg B. Fields
Biopolymers 2000 Volume 54(Issue 7) pp:
Publication Date(Web):31 AUG 2000
DOI:10.1002/1097-0282(200012)54:7<531::AID-BIP60>3.0.CO;2-X
Numerous approaches have been described for creating relatively small folded biomolecular structures. “Peptide-amphiphiles,” whereby monoalkyl or dialkyl hydrocarbon chains are covalently linked to peptide sequences, have been shown previously to form specific molecular architecture of enhanced stability. The present study has examined the use of monoalkyl hydrocarbon chains as a more general method for inducing protein-like structures. Peptide and peptide-amphiphiles have been characterized by CD and one- and two-dimensional nmr spectroscopic techniques. We have examined two structural elements: α-helices and collagen-like triple helices. The α-helical propensity of a 16-residue peptide either unmodified or acylated with a C6 or C16 monoalkyl hydrocarbon chain has been examined initially. The 16-residue peptide alone does not form a distinct structure in solution, whereas the 16-residue peptide adopts predominantly an α-helical structure in solution when a C6 or C16 monoalkyl hydrocarbon chain is N-terminally acylated. The thermal stability of the α-helix is greater upon addition of the C16 compared with the C6 chain, which correlates to the extent of aggregation induced by the respective hydrocarbon chains. Very similar results are seen using a 39-residue triple-helical model peptide, in that structural thermal stability (a) is increasingly enhanced as alkyl chain length is increased and (b) correlates to the extent of peptide-amphiphile aggregation. Overall, structures as diverse as α-helices, triple helices, and turns/loops have been shown to be induced and/or stabilized by alkyl chains. Increasing alkyl chain length enhances stability of the structural element and induces aggregates of defined sizes. Hydrocarbon chains may be useful as general tools for protein-like structure initiation and stabilization as well as biomaterial modification. © 2000 John Wiley & Sons, Inc. Biopoly 54: 531–546, 2000
Co-reporter:Anna M. Knapinska, Sabrina Amar, Zhong He, Sandro Matosevic, Claudia Zylberberg, Gregg B. Fields
Enzyme and Microbial Technology (November 2016) Volumes 93–94() pp:29-43
Publication Date(Web):1 November 2016
DOI:10.1016/j.enzmictec.2016.07.009
Recombinant MMPs were compared to bacterial collagenase for mesenchymal stem cell isolation.MMP-12 isolated cells as efficiently as bacterial collagenase with a purer population of cells.A single activation cocktail can be used for MMPs prior to stem cell isolation.Cell isolation methods for therapeutic purposes have seen little advancement over the years. The original methods of stem cell and islet isolation using bacterial collagenases were developed in the early 1980s and are still used today. Bacterial collagenases are subject to autodegradation, and isolates obtained with these enzymes may be contaminated with endotoxins, reducing cell viability and contributing to toxicity in downstream applications. Here we describe a novel method for isolation of mesenchymal stem cells from adipose tissue (ADSC) utilizing recombinantly produced matrix metalloproteases (MMPs). The ADSCs isolated by MMPs displayed essentially identical morphological and phenotypical characteristics to cells isolated by bacterially-derived collagenase I and Liberase. Samples isolated with MMPs and Liberase had comparable levels of CD73, CD90, and CD105. The adipogenic and osteogenic potential of the ADSCs isolated by MMPs was retained as compared to cells isolated with Liberase. However, ADSCs isolated by Liberase displayed 6% contamination with other cells as per negative markers revealed by PE staining, as opposed to <1% for all MMP-treated samples. MMP-based cell isolation may contribute to optimization of transplantation technology.
Co-reporter:Gregg B. Fields
Matrix Biology (May–July 2015) Volumes 44–46() pp:239-246
Publication Date(Web):1 May 2015
DOI:10.1016/j.matbio.2015.01.002
•Matrix metalloproteinase (MMP) inhibitor design has considered secondary binding sites (exosites) to improve specificity.•Small molecules and peptides have been developed that bind exosites in MMP-2, MMP-9, MMP-13, and MT1-MMP.•Antibody-based approaches have resulted in selective inhibitors for MMP-9 and MT1-MMP.•Biomolecules designed for specific exosite targeting have provided selective MMP probes.The development of matrix metalloproteinase (MMP) inhibitors has often been frustrated by a lack of specificity and subsequent off-target effects. More recently, inhibitor design has considered secondary binding sites (exosites) to improve specificity. Small molecules and peptides have been developed that bind exosites in the catalytic (CAT) domain of MMP-13, the CAT or hemopexin-like (HPX) domain of MT1-MMP, and the collagen binding domain (CBD) of MMP-2 and MMP-9. Antibody-based approaches have resulted in selective inhibitors for MMP-9 and MT1-MMP that target CAT domain exosites. Triple-helical “mini-proteins” have taken advantage of collagen binding exosites, producing a family of novel probes. A variety of non-traditional approaches that incorporate exosite binding into the design process has yielded inhibitors with desirable selectivities within the MMP family.
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