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PEPTIDE TRENDS APRIL 2018

MEET US AT TIDES, MAY 7 - 10, 2018 IN BOSTON, MASSACHUSETTS

TIDES brings 1000+ global oligonucleotide and peptide leaders across Asia, Europe and North America together to present case studies, best practices and to discuss current strategies and trends to accelerate promising molecules to market. Do not miss the following presentations of the Bachem experts:

Peptides as ingredients in cosmetic products, most notably in anti-aging therapy, are of continued interest. Due to our offer for cosmeceutical-related products, our excellent service for custom peptide synthesis, and our capabilities for small to industrial scale synthesis of peptides of any complexity we are confident to be the ideal partner in the development and production of cosmetic peptides.

 

 

Pre-Conference Workshop 1:

Dr. Patrik Plattner, Group Leader Mass Spectrometry Service, Bachem AG, “LC Peak Purity Assessment Using a Novel LC-MS Data Processing Approach” May 07, 10:45am - 11:15am

 

 

Main Conference - Day 2, Peptide Chemistry, Manufacturing and Controls:

Dr. Ralph Schönleber, Vice President, Research & Development, Bachem AG, “Higher Molecular Weight (HMW) Peptide Impurities - Control Strategies and Acceptance Criteria” May 09, 8:30am - 9:00am

Dr. Stefan Eissler, Director, API Manufacturing, Bachem AG, “Highly Pure Octreotide - Development and Scale-Up of a State-of-the-art SPPS Process” May 09, 10:45am - 11:15am

 

 

Main Conference - Day 3, Peptide Chemistry, Manufacturing and Controls:

Dr. Gerhard Haas, Vice President, Quality Assurance and Regulatory Affairs, Bachem AG, “Opportunities and Challenges of the FDA Draft Guidance ANDAs for Certain Highly Purified Synthetic Drug Products That Refer to Listed Drugs of rDNA Origin” May 10, 8:50am - 9:10am

Dr. Valeska Maria Gerhardt, Specialist Regulatory Affairs, Bachem AG, “Bachem’s Experience with the Registration of Peptide APIs in Japan” May 10, 10:45am - 11:15am

 

Bachem‘s pipeline contains more than 150 customer projects in preclinical and clinical phases. Bachem’s services include pegylated peptides, lipidated peptides, various other peptide conjugates, sterile fill and finish (Clinalfa®), and selective chemical glycosylation. The glycosylation technology is applicable to large scale and has the potential to be applied to a variety of peptides, where we can pioneer the concept of improving current and future drugs. To view our webinar please click here.

 

We are excited to meet with our customers and discuss how Bachem can be a partner for their peptide API custom manufacturing needs. We invite you to visit us at our Booth #105: please contact us to schedule a meeting in advance.

 

 

 

We look forward to meeting you at TIDES 2018!

MATRIX METALLOPROTEINASES

Importance in Medical Research

Matrix metalloproteinases (MMPs) play an essential role in the homeostasis of the extracellular matrix (ECM) and are essential for developmental tissue remodeling, angio-genesis, tissue repair, and normal turnover of ECM (the female reproductive cycle, bone remodeling, nerve growth, hair follicle cycling, immune response, and apoptosis). Accordingly, their activity has been implicated in numerous clinical patterns, including severe disorders like diseases of the cardiovascular system, liver cirrhosis, Alzheimer`s and Parkinson`s disease as well as tumor growth and cancer.

 

The role of MMPs in cancer is complex. The ability of MMPs to degrade basal membranes and hence to break biological barriers is central for tumor metastasis. By cleaving several non-matrix targets, MMPs moreover regulate manifold processes and signaling pathways, which determines the entire microenvironment of growing tumors; MMPs are important mediators of the communication between tumors and the surrounding stroma.

 

Classification and Targets

MMPs constitute a multigene family of at least 28 members in vertebrates, which includes 24 members in mammals and 23 members in human. MMPs are secreted or membrane bound enzymes that degrade numerous substrates. Their targets are other proteinases, clotting factors, chemotactic molecules, latent growth factors, cell surface receptors, cell-cell adhesion molecules, and almost all structural extracellular matrix proteins. According to more recent findings, they also process proteins inside of the cells, located in the cytoplasm, mitochondria or nucleus.

 

MMPs, also known as ‘matrixins’, are a family of structurally and functionally related calcium dependent zinc-containing endopeptidases. Together with the adamlysins, astacins, pappalysins and bacterial serralysins, they form the metzincin endopeptidase superfamily. MMPs can be classified based on their substrate specificity and cellular localization. The resulting groups are the collagenases, the gelatinases, the stromelysins and the membrane-type MMPs (MT-MMPs). Since not all MMPs can be classified in this manner, an alternative classification can be conducted on basis of their domain organization (Table 1). Further data summarized for vertebrate MMPs can be found in Table 2.

Table 1 Classification and domain organisation. Domains specific for particular MMP classes are written in bold. Data on basis of Vandenbroucke and Libert, 2014.

MMP class Domain organisation Examples
Gelatinases SP/PD-Linker-CC/Fbr-Hinge-Hpx MMP-2, MMP-9
     
Archetypal MMPs    
- Collagenases SP/PD-Linker-CC-Hinge-Hpx MMP-1, MMP-8, MMP-13
- Stromelysins SP/PD-Linker-CC-Hinge-Hpx MMP-3, MMP-10
- Other SP/PD-Linker-CC-Hinge-Hpx MMP-12, MMP-19, MMP-20, MMP-27
     
Matrilysins SP/PD-Linker-CC MMP-7, MMP-26
     
Furin-activatable MMPs    
- Secreted SP/PD-Linker/PCRS-CC-Hinge-Hpx MMP-11, MMP-21, MMP-28
- Type I transmembrane P/PD-Linker/PCRS-CC-Hinge-Hpx-TM/CD MMP-14 (MT1-MMP), MMP-15 (MT2-MMP), MMP-16 (MT3-MMP), MMP-24 (MT5-MMP)
- Type II transmembrane SP/PD-Linker/PCRS-CC-CRD-IglD MMP-23
- GPI-anchored SP/PD-Linker/PCRS-CC-Hinge-Hpx-GAD MMP-17 (MT4-MMP), MMP-25 (MT6-MMP)

Abbreviations

CC: Catalytic core with bound Zn2+-ion

CC/Fbr: Catalytic core with bound Zn2+-ion and fibronectin type II motif in catalytic site

CRD: Cysteine-rich domain

GAD: Glycosylphospatidylinositol (GPI) anchoring domain

Hpx: Haemopexin-like domain

IglD: Immunoglobulin-like domain

Linker/PCRS: Linker including proprotein convertase recognition sequence

SP/PD: Signal peptide and propeptide domain

TM/CD: Transmembrane and cytoplasmic domain

Structure and Domain Organisation

MMPs are expressed as inactive zymogens (pro-MMPs), minimally consisting of a signal sequence, a prodomain of about 80 amino acids, a linker and a catalytic domain. Depending on the type of MMP, other domains accrue.

 

Gelatinases, matrilysins and archetypal MMPs are activated by extracellular convertases like plasmin or other MMPs. MMPs possessing a proprotein convertase recognition sequence are activated intracellularly by furin-type convertases.

 

MMPs are characterized by a highly conserved motif `HEXXHXXGXXH` (amino acid one-letter code, whereas `X` can be any amino acid). The three conserved histidines bind zinc (Zn2+) at the catalytic site, and the conserved glutamate acts as general base or acid during catalysis.

 

Furthermore, all metzincins identified so far contain a Met-turn, comprising an invariant methionine residue. This is a structurally and spatially conserved 1,4-turn, found directly below the zinc-binding site. Referring to crystallographic studies on wild type and mutant proteins, the Met-turn seems to link to a molecular plug, which is important for the integrity and stability of the zinc binding domain.

 

The activation of the MMPs takes place by a `cysteine-switch` mechanism, with the cysteine embedded in a conserved `PRCGXPD` motif. The latter is present in all human MMPs except MMP-23B. The cysteine of the prodomain associates in the inactive state with the Zn2+-ion of the catalytic core, which prevents binding of water to the Zn2+- ion. Proteolytic cleavage of the prodomain releases the cysteine and thus allows water to bind, which in consequence activates the MMP.

 

For a number of MMPs, the crystal structures had been solved. The catalytic core, typically consisting of about 165 residues, is divided by a shallow substrate-binding crevice into an upper and a lower subdomain, and does not contain disulfide bridges. The structures in all investigated cases revealed a rather spherical shape of the catalytic core with a diameter of about 40 Å.

 

A haemopexin-like domain found in several MMPs seems to have multiple functions, and might also be involved in triggering diverging cellular processes in a non-catalytic manner.

 

Regulation

The proteolytic activity of MMPs can be regulated at different levels and furthermore depends on the set of MMPs and their inhibitors locally expressed. The latter can differ remarkably, depending on the concerned cell type and the surrounding microenvironment.

 

A key event in the regulation is the conversion of the inactive MMP zymogen into the active protease by proteolytic removal of the prodomain. This in turn is subject to numerous and complex regulatory factors. For example, already activated MMPs of several types can degrade plasminogen, which leads to a reduced plasmin availability and hence inhibits MMP activation. Important cellular inhibitors of the MMP functions are the tissue inhibitors of metalloproteinases (TIMPs), of which four members (TIMP-1, -2, -3 and -4) had been identified in mammals. TIMPs form 1:1 stoichiometric complexes with zymogenic MMPs, by a tight, non-covalent interaction with the haemopexin-like domains. The inactivation apparently takes place by binding of the amino-terminal TIMP domains within the active site cleft of the MMPs. Moreover, MMPs are capable of an autocrine mode of up-regulation, by proteolytic degradation of physiological MMP inhibitors. This leads to reduced inactivation of MMPs, and prolongs the lifetime of the MMP enzymes.

 

Reactive oxygen species, frequently produced in large amounts by inflammatory cells in the microenvironments of tumors, can promote MMP activation due to oxidation of cysteine in the prodomain. Also formation of dimeric, trimeric or other multimeric enzyme complexes, as observed for example for proMMP-9, as well as manifold other effects, may play a role in regulation.

 

Outlook

Since MMP inhibitors exhibited significant side effects and other problems in clinical trials, they did not keep promise as new cancer therapeutics so far. While some MMPs promote pathology, others have protective functions, which raised further problems against the background of insufficient inhibitor specificity. However, MMPs remain enzymes of highest medical interest, and research unabatedly continues with the aim to get a therapeutic hold of these ubiquitous physiological players. For example, inhibitors acting outside of the highly conserved catalytic center, like on the haemopexin domain, could improve substrate specificity. In addition, diseases like inflammation, sepsis and cardiovascular disorders gained more attention in MMP research, which could lead to new medical applications beside the treatment of cancer. In any case, MMP inhibitors and substrates of high and reliable quality are required to assess the activity of MMPs experimentally, under most diverse physiological or pathological conditions. This is where our broad offering of labeled and unlabeled products is a key element for success in MMP and MMP-related research.

Table 2 The Vertebrate Matrix Metalloproteinases

MMP Common Name Mol. Wt. (latent/active) E.C.No. MEROPS ID Substrate
MMP-1 Interstitial collagenase-1, Collagenase- 1 55000/45000 3.4.24.7 M10.001 CN types I, II, III, V, VII, VIII, and X, aggrecan, gelatin, serpins
MMP-2 Gelatinase A, 72 kD Gelatinase, Type IV Collagenase 72000/66000 3.4.24.24 M10.003 CN types I, IV, V, VII, and X, gelatin, elastin, FN, LN, MMP-9, MMP-13
MMP-3 Stromelysin-1, Proteoglycanase, Transin, Procollagenase 57000/45000 3.4.24.17 M10.005 CN types II, IV, IX, X, and XI, LN, FN, proteoglycan, aggrecan, elastin, gelatin, proMMP-1, proMMP-8, proMMP-9
MMP-7 Matrilysin, Matrin, Uterine Metalloendopeptidase, Putative Metalloproteinase-1 (PUMP-1) 28000/19000 3.4.24.23 M10.008 CN type IV, elastin, proteoglycan, glycoprotein, gelatin
MMP-8 Neutrophil Collagenase, Collagenase-2 75000/58000 3.4.24.34 M10.002 CN types I, II, III, and V
MMP-9 Gelatinase B, Type V Collagenase, 92 kD Type IV Collagenase, Macrophage Gelatinase 92000/86000 3.4.24.35 M10.004 CN types IV, gelatin, aggrecan, LN
MMP-10 Stromelysin-2, Transin-2 57000/44000 3.4.24.22 M10.006 CN types I, II, III, and V
MMP-11 Stromelysin-3 51000/44000 3.4.24.B3 M10.007 LN, α1-proteinase inhibitor, α1-antitrypsin
MMP-12 Macrophage Elastase, MME, Metalloelastase 54000/45000 and 22000 3.4.24.65 M10.009 Elastin
MMP-13 Collagenase-3 60000/48000 3.4.24.B4 M10.013 CN types I, II, III, IV, V, IX, X, and XI, gelatin, LN, tenascin, aggrecan, FN
MMP-14 MT1-MMP, Membrane-type Matrix Metalloproteinase-1 66000/56000 3.4.24.80 M10.014 CN types I, II, and III, gelatin, FN, LN, VN, aggrecan, tenascin, perlecan, proMMP-2, proMMP-13, proteoglycan
MMP-17 MT4-MMP, Membrane-type Matrix Metalloproteinase-4 57000/53000 3.4.24.- M10.017 FN, fibrin, gelatin
MMP-25 MT6-MMP, Leukolysin, Membrane-type Matrix Metalloproteinase-6   3.4.24.- M10.024 proMMP-2
MMP-26 Endometase, Matrilysin-2   3.4.24.B7 M10.029 CN type IV, FN, FG
MMP-28 Epilysin   3.4.24.-   unknown

Abbreviations

CN: Collagen

FN: Fibronectin

LN: Laminin

VN: Vitronectin

FG: Fibrinogen

Explore our wide selection of MMP substrates and inhibitors or browse the full Inhibitors & Substrates index in our online shop.

 

References

Product Monograph Matrix Metalloproteinases

MATRIX METALLOPROTEINASES AND DRUGS IN CLINICAL DEVELOPMENT

Matrix Metalloproteinases (MMPs) are involved in many diseases such as autoimmune conditions, cardiovascular and neurological disorders, infectious diseases and cancer. Numerous matrix metalloproteinase inhibitors have been studied over the years. The first generation of MMP inhibitors did not distinguish between the 24 members of the MMP family resulting in disappointing clinical trials. Consequently, the design strategies for MMP inhibitors were revisited and selective inhibitors are now in the early stages of development (1). There are approximately 30 MMP inhibitors known to be in preclinical and clinical development and some of these MMP inhibitors are peptide-based (2). A few peptide-based drugs in clinical development that leverage matrix metalloproteinases are shown in Table 3.

Table 3 Matrix Metalloproteinases and Drugs in Phase I-II (2)

Product Name Active Ingredient Condition Treated Highest Phase Company
BT-1718 -- Breast Cancer; Colon Cancer; Gastric Cancer; Head And Neck Cancer; Lung Adenocarcinoma; Soft Tissue Sarcoma; Solid Tumor; Squamous Non-Small Cell Lung Cancer II Bicycle Therapeutics Ltd.
Zep-3 -- Herpes Labialis (Oral Herpes) II Shulov Innovative Science Ltd.
ZK-003 thymosin β-4 Alopecia; Corneal Ulcers; Gynecological Infections; Keratoconjunctivitis sicca (Dry Eye) II Lee's Pharmaceutical Holdings Ltd.
NL-005 thymosin β-4 Myocardial Infarction I Beijing Northland Biotech Co Ltd.

Phase II Candidates

BT-1718 is under development by Bicycle Therapeutics Ltd. for the treatment of solid tumors such as breast cancer, head and neck cancer and soft tissue carcinoma. This drug candidate consists of a constrained bicyclic peptide linked via a hindered disulfide linker to the potent anti-tubulin agent DM1 (3). The peptide targets cells expressing membrane type 1-matrix metalloproteinase-14 (MMP-14) which is overexpressed in many solid tumors. In February 2018, Cancer Research UK and Bicycle Therapeutics announced the initiation of a Phase I/IIa trial that will evaluate BT1718 in patients with advanced solid tumors (2).

 

Shulov Innovative Science Ltd. is developing a matrix metalloproteinase 9 (MMP-9) inhibitor known as Zep-3 for the treatment of Herpes Simplex Virus 1 (HSV1) cold sores. Zep-3 is a synthetic modified version of a peptide originally isolated from snake venom. In preclinical studies, Zep-3 demonstrated analgesic and anti-viral activity inhibiting the replication of HSV1 (4). In June 2015, Shulov Innovative Science registered a Phase II study to evaluate the safety, tolerability and efficacy of topical Zep-3 for the treatment of HSV1 cold sores (2).

 

Lee’s Pharmaceutical Holding Ltd. is developing ZK-003 (thymosin β-4) as a treatment for chemotherapy induced alopecia, cornea ulcers and vaginal infections. Thymosin β-4 is a 43-amino acid peptide with multiple biological activities and it plays a role in the protection, regeneration and remodeling of injured or damaged tissues. This peptide promotes the expression and secretion of matrix metalloproteinase-2 (MMP-2), which accelerates hair growth and works to reverse ophthalmic disorders. An IND application for ZK-003 is under review by the China Food and Drug Administration for the chemotherapy of induced alopecia, cornea ulcer and vaginal infection indications. The company was previously developing ZK-003 for the treatment of dry eye syndrome and the candidate was in a Phase II trial (2).

 

Phase I Candidates

NL-005 is under development by Beijing Northland Biotech Co Ltd. for the treatment of myocardial infarction. NL-005 is a thymosin β-4 receptor agonist and activation of this receptor results in an increase in synthesis of MMPs, vascular endothelial growth factor (VEGF), actin and other mediators. Beijing Northland has started a Phase I study to evaluate the tolerance and pharmacokinetics of NL-005 in healthy Chinese subjects (2).

 

Conclusion

MMPs are considered promising targets for the treatment of many diseases and the development of MMP inhibitors as therapeutic agents has been ongoing. To support researchers and organizations studying the intricate physiological and pathological functions of MMPs, Bachem offers a wide range of MMP inhibitors and substrates. In addition, Bachem offers a comprehensive custom peptide synthesis service and the production of new chemical entities to assist companies with developing peptide-based therapeutics.

 

References

(1) S. Amar and G. Fields, Potential clinical implications of recent MMP inhibitor design strategies, Expert Rev. Proteomics.  12(5), 445-447 (2015).

(2) Global Data (2018)

(3) G. Bennett et al., Development of BT1718, a novel Bicycle Drug Conjugate for the treatment of lung cancer, AACR Annual Meeting Proceedings. 77(13), 1167 (2017)

(4) Our Products, S.I.S. Shulov Innovative Science (20)

MEET BACHEM: STEFANIE DOBITZ, SALES MANAGER CUSTOM SYNTHESIS

PT: What is your official job title at Bachem?

Stefanie: Sales Manager Custom Synthesis

 

PT: How long have you been with Bachem? Where did you work before Bachem?

Stefanie: I am with Bachem for about half a year now. Prior to Bachem, I finished my doctoral thesis, which focused on the utilization of oligoproline-based multivalent ligands for tumor targeting.

 

PT: What is your academic background?

Stefanie: I have a BSc and MSc in chemistry and I recently finished my doctoral thesis at ETH Zurich.

 

PT: What do you like to do outside of work?

Stefanie: I play handball for more than 20 years and am an active club member. In addition, I enjoy running and hiking in the Alps.

 

PT: What do you like most about your job?

Stefanie: I really enjoy being in contact with our customers worldwide. To be their reliable partner and helping them to successfully carry out their experiments with custom-made peptides from Bachem is truly amazing.

 

PT: What is your preferred peptide?

Stefanie: Independent of their amino acid sequence, I really like peptides in general. Ever since I was first exposed to peptides, the principle of building numerous different peptides from a few building blocks, and their importance in nature, have always fascinated me.

 

PT: Thank you very much Stefanie.

PEPTIDE HIGHLIGHTS

Interesting news about peptides in basic research and pharmaceutical development:

Chemical attraction gives rattlesnake peptide the bite on superbugs-University of Queensland

Lab surprised to find its drug-delivery system can help even without drugs-Rice University

Peptide ‘nanodrills’ to deliver anticancer drugs into cells-In Pharmatechnologist

Personal cancer vaccines get their own boost-Harvard Paulson School

LITERATURE CITATIONS

Bachem peptides and biochemicals are widely cited in research publications. Congratulations to all our customers with recent publications!

 

D.B. Bosco et al.

A new synthetic matrix metalloproteinase inhibitor reduces human mesenchymal stem cell adipogenesis.

PLOS ONE 12, e0172925 (2017)

 

M. Sebastiano et al.

A novel mechanism regulating human platelet activation by MMP-2-mediated PAR1 biased signaling.

Blood 129, 883-895 (2017)

 

D.J. Wilkinson et al.

Matriptase induction of metalloproteinase‐dependent aggrecanolysis in vitro and in vivo: promotion of osteoarthritic cartilage damage by multiple mechanisms.

Arthritis & Rheumatology 69, 1601-1611 (2017)

 

D.H. Nam and X. Ge

Generation of highly selective MMP antibody inhibitors.

Proteases and Cancer: Methods and Protocols 307-324 (2018)