Batimastat Induces Cytotoxic and Cytostatic Effects in In Vitro Models of Hematological Tumors.

The role of metalloproteinases (MMPs) in hematological malignancies, like acute myeloid leukemia (AML), myelodysplastic neoplasms (MDS), and multiple myeloma (MM), is well-documented, and these pathologies remain with poor outcomes despite treatment advancements. In this study, we investigated the effects of batimastat (BB-94), an MMP inhibitor (MMPi), in single-administration and daily administration schemes in AML, MDS, and MM cell lines. We used four hematologic neoplasia cell lines: the HL-60 and NB-4 cells as AML models, the F36-P cells as an MDS model, and the H929 cells as a model of MM. We also tested batimastat toxicity in a normal human lymphocyte cell line (IMC cells). BB-94 decreases cell viability and density in a dose-, time-, administration-scheme-, and cell-line-dependent manner, with the AML cells displaying higher responses. The efficacy in inducing apoptosis and cell cycle arrests is dependent on the cell line (higher effects in AML cells), especially with lower daily doses, which may mitigate treatment toxicity. Furthermore, BB-94 activated apoptosis via caspases and ERK1/2 pathways. These findings highlight batimastat's therapeutic potential in hematological malignancies, with daily dosing emerging as a strategy to minimize adverse effects.

Neutrophil Infiltration and Matrix Metalloproteinase-9 in Lacunar Infarction.

We use the modified pial vessel disruption rat model to elucidate the cellular and molecular mechanisms of cavitation as it plays a role in lacunar infarction. Here we discuss the similarities between the genesis of pulmonary cavitation in various animal models and lacunar infarction in the cerebral cortex of rats. Both pathological processes involve the creation of a cavity surrounded by fibroblasts or reactive astrocytes. A crucial step in both, the lung and the cerebral cortex, appears to be the migration of neutrophils across the endothelial barrier into the parenchyma. In the lung and cerebral cortex this involves release of matrix metalloproteinase-9 (MMP-9). Inside the parenchyma neutrophils continue to release MMP-9. In both situations batimastat (BB-94) and minocycline reduce release of MMP-9 and prevent cavitation. In the cerebral cortex MMP-9 release by resident microglia plays an additional role. We therefore advance the hypothesis that cavitation in both tissues is driven by MMP-9 originating from invading neutrophils. Therapeutic intervention has to focus on these blood-borne intruder cells and specific MMP actions. Batimastat and its derivatives (marimastat, BB-1101, mCGS-27023-A, ilomastat, GM6001, CTK8G1150) are already in clinical or experimental use in humans for anti-cancer treatment, and these clinically relevant drugs could be repurposed to act as anti-inflammatory to counter neutrophil contribution to lung or cerebral cortex cavitation.

Prevention of abdominal aortic aneurysm progression by targeted inhibition of matrix metalloproteinase activity with batimastat-loaded nanoparticles.

RATIONALE: Matrix metalloproteinases (MMPs)-mediated extracellular matrix destruction is the major cause of development and progression of abdominal aortic aneurysms. Systemic treatments of MMP inhibitors have shown effectiveness in animal models, but it did not translate to clinical success either because of low doses used or systemic side effects of MMP inhibitors. We propose a targeted nanoparticle (NP)-based delivery of MMP inhibitor at low doses to the abdominal aortic aneurysms site. Such therapy will be an attractive option for preventing expansion of aneurysms in patients without systemic side effects. OBJECTIVE: Our previous study showed that poly(d,l-lactide) NPs conjugated with an antielastin antibody could be targeted to the site of an aneurysm in a rat model of abdominal aortic aneurysms. In the study reported here, we tested whether such targeted NPs could deliver the MMP inhibitor batimastat (BB-94) to the site of an aneurysm and prevent aneurysmal growth. METHODS AND RESULTS: Poly(d,l-lactide) NPs were loaded with BB-94 and conjugated with an elastin antibody. Intravenous injections of elastin antibody-conjugated BB-94-loaded NPs targeted the site of aneurysms and delivered BB-94 in a calcium chloride injury-induced abdominal aortic aneurysms in rats. Such targeted delivery inhibited MMP activity, elastin degradation, calcification, and aneurysmal development in the aorta (269% expansion in control versus 40% elastin antibody-conjugated BB-94-loaded NPs) at a low dose of BB-94. The systemic administration of BB-94 alone at the same dose was ineffective in producing MMP inhibition. CONCLUSIONS: Targeted delivery of MMP inhibitors using NPs may be an attractive strategy to inhibit aneurysmal progression.

Vaccinia virus G1 protein: absence of autocatalytic self-processing.

Vaccinia virus relies on a series of proteolytic cleavage events involving two viral proteins, I7 and G1, to complete its life cycle. Furthermore, G1 itself is cleaved during vaccinia virus infection. However, convincing evidence is lacking to show whether G1 participates in autoproteolysis or is a substrate of another protease. We employed both biochemical and cell-based approaches to investigate G1 cleavage. G1, when expressed in bacteria, rabbit reticulocyte lysates or HeLa cells, was not processed. Moreover, G1 was cleaved in infected cells, but only in the presence of virus late gene expression; cleavage was strongly inhibited by proteasome inhibitors. Thus, these results imply a more complex G1 cleavage reaction than previously envisaged.

Inhibition of HIV-1 release by ADAM metalloproteinase inhibitors.

HIV-1 gp120 glycan binding to C-type lectin adhesion receptor L-selectin/CD62L on CD4 T cells facilitates viral attachment and entry. Paradoxically, the adhesion receptor impedes HIV-1 budding from infected T cells and the viral release requires the shedding of CD62L. To systematically investigate CD62L-shedding mediated viral release and its potential inhibition, we screened compounds specific for serine-, cysteine-, aspartyl-, and Zn-dependent proteases for CD62L shedding inhibition and found that a subclass of Zn-metalloproteinase inhibitors, including BB-94, TAPI, prinomastat, GM6001, and GI25423X, suppressed CD62L shedding. Their inhibition of HIV-1 infections correlated with enzymatic suppression of both ADAM10 and 17 activities and expressions of these ADAMs were transiently induced during the viral infection. These metalloproteinase inhibitors are distinct from the current antiretroviral drug compounds. Using immunogold labeling of CD62L, we observed association between budding HIV-1 virions and CD62L by transmission electron microscope, and the extent of CD62L-tethering of budding virions increased when the receptor shedding is inhibited. Finally, these CD62L shedding inhibitors suppressed the release of HIV-1 virions by CD4 T cells of infected individuals and their virion release inhibitions correlated with their CD62L shedding inhibitions. Our finding reveals a new therapeutic approach targeted at HIV-1 viral release.

The Natural Ligand for Metalloproteinase-A Multifaceted Drug Target.

Metalloproteinase is one of the key components of Russell viper venom and it is the root cause of edema, blood coagulation, local tissue damage, hemorrhage, and inflammation during snakebite envenoming. Hence, finding a suitable metalloproteinase inhibitor from natural source will be of great biological importance in mitigating pathological effects. In this current study, we employed computational analysis to examine the inhibition of metalloproteinase by phytochemicals present in Andrographis paniculata. Molecular docking studies revealed interaction of A. paniculata phytochemicals with the catalytic M domain's active site amino acid residues, namely ASN203, ARG293, PHE203, LEU206, LYS199, and ALA122, similar to that of the reference compound Batimastat. 14-acetylandrographolide, 14-deoxy-11,12 didehydroandrographolide, Andrograpanin, Isoandrographolide, and 14-deoxy-11-oxoandrographolide displayed high binding energy and inhibition against the metalloproteinase. Molecular dynamic simulation analysis revealed less root mean square fluctuation of amino acid residues of metalloproteinase-14-acetylandrographolide complex than metalloproteinase-Batimastat complex indicating the high stability for metalloproteinase with the phytochemical. In silico analysis of parameters like ADME properties and drug-likeness of the phytochemicals exhibited good pharmacokinetic properties. Ligand-based virtual screening of phytochemicals to identify similarity to FDA-approved drugs and identification of their possible targets were also performed. The outcome of the current study strengthens the significance of these phytochemicals as promising lead candidates for the treatment of snakebite envenomation. Moreover, the study also encourages the in vivo and in vitro evaluation of the phytochemicals to validate the computational findings.

Computational insights into the identification of a potent matrix metalloproteinase inhibitor from Indigofera aspalathoides to control cancer metastasis.

Matrix metalloproteinases (MMPs) are the major proteolytic enzymes which assist in regulating the metastatic process by degrading the extracellular matrix proteins. In this study, we have investigated the anti-metastatic potential of major bioactive compounds in the medicinal plant Indigofera aspalathoides targeting matrix metalloproteinases (MMP2 & MMP9) and it's in silico pharmacokinetic profiles using computational studies. Indigofera aspalathoides (Sivanar vembu in Tamil) is a renowned medicinal herb in traditional Indian medicine which contains indigocarpan, mucronulatol, indigocarpan diacetate, erythroxydiol X and erythroxydiol Y as the major constituents. The 3-dimensional structure of MMP2 and MMP9 was designed by using I-tasser and Modeller and it was validated by PROCHECK. The structures of mucronulatol and indigocarpan have been retrieved from PubChem and indigocarpan diacetate, erythroxydiol X & Y were drawn by using Chemdraw Ultra 6.0. Batimastat was used as a positive control. Molecular docking was performed by using AutoDock 4.2 tools and AutoDock vina, an open-source program which signifies an effective interaction between the phytoligands and MMP2 & MMP9. From the results, AutoDock 4.2 have showed that indigocarpan possesses strong binding energy (ΔG) of - 7.68 kcal/mol towards MMP2 and - 6.35 kcal/mol towards MMP9, whereas batimastat showed binding energy (ΔG) of - 6.34 kcal/mol for MMP2 and - 5.66 kcal/mol for MMP9, meanwhile the results from AutoDock vina indicates that indigocarpan possesses strong binding energy (ΔG) of - 8.0 kcal/mol towards MMP2 and - 8.2 kcal/mol towards MMP9, whereas batimastat showed binding energy (ΔG) of - 7.2 kcal/mol for MMP2 and - 7.6 kcal/mol for MMP9. Also, the ADME and toxicity results suggest that the indigocarpan compound possesses a druggable pharmacokinetic potentiality and does not have carcinogenicity and Ames mutagenesis compared with other phytoligands. Hence, it is evident from our results that both AutoDock platforms strongly revealed that the phytoligand, indigocarpan possesses strong inhibitory activity against MMP2 and MMP9 to control cancer metastasis. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02731-w.

Repurposing Cancer Drugs Batimastat and Marimastat to Inhibit the Activity of a Group I Metalloprotease from the Venom of the Western Diamondback Rattlesnake, Crotalus atrox.

Snakebite envenomation causes over 140,000 deaths every year, predominantly in developing countries. As a result, it is one of the most lethal neglected tropical diseases. It is associated with incredibly complex pathophysiology due to the vast number of unique toxins/proteins present in the venoms of diverse snake species found worldwide. Here, we report the purification and functional characteristics of a Group I (PI) metalloprotease (CAMP-2) from the venom of the western diamondback rattlesnake, Crotalus atrox. Its sensitivity to matrix metalloprotease inhibitors (batimastat and marimastat) was established using specific in vitro experiments and in silico molecular docking analysis. CAMP-2 shows high sequence homology to atroxase from the venom of Crotalus atrox and exhibits collagenolytic, fibrinogenolytic and mild haemolytic activities. It exerts a mild inhibitory effect on agonist-induced platelet aggregation in the absence of plasma proteins. Its collagenolytic activity is completely inhibited by batimastat and marimastat. Zinc chloride also inhibits the collagenolytic activity of CAMP-2 by around 75% at 50 µM, while it is partially potentiated by calcium chloride. Molecular docking studies have demonstrated that batimastat and marimastat are able to bind strongly to the active site residues of CAMP-2. This study demonstrates the impact of matrix metalloprotease inhibitors in the modulation of a purified, Group I metalloprotease activities in comparison to the whole venom. By improving our understanding of snake venom metalloproteases and their sensitivity to small molecule inhibitors, we can begin to develop novel and improved treatment strategies for snakebites.

Peptidomimetic hydroxamate metalloproteinase inhibitors abrogate local and systemic toxicity induced by Echis ocellatus (saw-scaled) snake venom.

The ability of two peptidomimetic hydroxamate metalloproteinase inhibitors, Batimastat and Marimastat, to abrogate toxic and proteinase activities of the venom of Echis ocellatus from Cameroon and Ghana was assessed. Since this venom largely relies for its toxicity on the action of zinc-dependent metalloproteinases (SVMPs), the hypothesis was raised that toxicity could be largely eliminated by using SVMP inhibitors. Both hydroxamate molecules inhibited local and pulmonary hemorrhagic, in vitro coagulant, defibrinogenating, and proteinase activities of the venoms in conditions in which venom and inhibitors were incubated prior to the test. In addition, the inhibitors prolonged the time of death of mice receiving 4 LD50s of venom by the intravenous route. Lower values of IC50 were observed for in vitro and local hemorrhagic activities than for systemic effects. When experiments were performed in conditions that simulated the actual circumstances of snakebite, i.e. by administering the inhibitor after envenoming, Batimastat completely abrogated local hemorrhage if injected immediately after venom. Moreover, it was also effective at inhibiting lethality and defibrinogenation when venom and inhibitor were injected by the intraperitoneal route. Results suggest that these, and possibly other, metalloproteinase inhibitors may become an effective adjunct therapy in envenomings by E. ocellatus when administered at the anatomic site of venom injection rapidly after the bite.

Protective effects of batimastat against hemorrhagic injuries in delayed jellyfish envenomation syndrome models.

Previously, we established delayed jellyfish envenomation syndrome (DJES) models and proposed that the hemorrhagic toxins in jellyfish tentacle extracts (TE) play a significant role in the liver and kidney injuries of the experimental model. Further, we also demonstrated that metalloproteinases are the central toxic components of the jellyfish Cyanea capillata (C. capillata), which may be responsible for the hemorrhagic effects. Thus, metalloproteinase inhibitors appear to be a promising therapeutic alternative for the treatment of hemorrhagic injuries in DJES. In this study, we examined the metalloproteinase activity of TE from the jellyfish C. capillata using zymography analyses. Our results confirmed that TE possessed a metalloproteinase activity, which was also sensitive to heat. Then, we tested the effect of metalloproteinase inhibitor batimastat (BB-94) on TE-induced hemorrhagic injuries in DJES models. Firstly, using SR-based X-ray microangiography, we found that BB-94 significantly improved TE-induced hepatic and renal microvasculature alterations in DJES mouse model. Secondly, under synchrotron radiation micro-computed tomography (SR-µCT), we also confirmed that BB-94 reduced TE-induced hepatic and renal microvasculature changes in DJES rat model. In addition, being consistent with the imaging results, histopathological and terminal deoxynucleotidyl transferase-mediated UTP end labeling (TUNEL)-like staining observations also clearly corroborated this hypothesis, as BB-94 was highly effective in neutralizing TE-induced extensive hemorrhage and necrosis in DJES rat model. Although it may require further clinical studies in the near future, the current study opens up the possibilities for the use of the metalloproteinase inhibitor, BB-94, in the treatment of multiple organ hemorrhagic injuries in DJES.

An MMP-inhibitor modified adhesive primer enhances bond durability to carious dentin.

OBJECTIVES: To evaluate the effect of adding a matrix metalloproteinase (MMP) inhibitor (BB94, Batimastat) to the primer of a three-step etch and rinse adhesive system on caries-affected dentin (CaD) MMP activity, and to assess the effect of such an inclusion on the chemical content of the CaD-adhesive interface. METHODS: Caries-infected dentin (CiD) was excavated selectively from freshly extracted human carious teeth using a chemo-mechanical agent. Each tooth was sectioned into three slabs through the CaD retained cavity. These were treated with either Optibond FL "OB" (Kerr, Orange, USA) without MMP inhibitor, or with 500 µM BB94 prior to the application of OB primer and bond, or with OB primer that contained 5 µM BB94. In situ zymography and Raman micro-spectroscopy were used to investigate MMP activity and the changes in the chemical content at the CaD/adhesive interface, respectively. RESULTS: Data showed the use of OB adhesive with BB94 resulted in immediate interfacial MMP inhibition, by direct application (93.3%) and by means of a drug delivery system (80%), as demonstrated by in situ zymography. Raman imaging revealed 33% higher resin infiltration into MMP-inhibited adhesive interfaces (SE 3.88). SIGNIFICANCE: Through competitive inhibition by batimastat (BB94), a proportion of the MMPs found in CaD were inhibited immediately and irreversibly. Such a competitive mechanism brings the adhesive primer close to the collagen matrix and enhances the dental adhesive wettability, which is a proposed mechanism to explain the presence of more resin within the hybrid layer.

Chemotherapy stimulates syndecan-1 shedding: a potentially negative effect of treatment that may promote tumor relapse.

In patients with multiple myeloma, the heparan sulfate proteoglycan syndecan-1 (CD138) is shed from the surface of tumor cells and accumulates in the serum and within the extracellular matrix of the bone marrow where it promotes tumor growth and metastasis. In the present study we discovered that commonly used anti-myeloma drugs stimulate syndecan-1 shedding both in vitro and in animals bearing myeloma tumors. Enhanced shedding is accompanied by increased syndecan-1 synthesis prior to drug induced tumor cell death. Addition of a caspase inhibitor blocks the drug-induced shedding of syndecan-1 in vitro indicating that shedding is linked to the onset of apoptosis. ADAM inhibitors or siRNA targeting ADAMs blocked drug-induced shedding suggesting that upregulation or activation of ADAMs is responsible for cleaving syndecan-1 from the tumor cell surface. These results reveal that myeloma chemotherapy stimulates synthesis and shedding of syndecan-1, a potentially negative side effect that may lead to the accumulation of high levels of syndecan-1 to establish a microenvironment that nurtures relapse and promotes tumor progression. Interestingly, we also found that chemotherapeutic drugs stimulated syndecan-1 shedding from pancreatic cancer cells as well, indicating that drug-induced shedding of syndecan-1 may occur in many cancer types. Overall, our results indicate that the use of metalloproteinase inhibitors (to inhibit syndecan-1 shedding) in combination with chemotherapy may represent a novel therapeutic strategy to prevent re-establishment of a microenvironment conducive for tumor relapse.

The phosphoinositide-dependent protein kinase 1 inhibitor, UCN-01, induces fragmentation: possible role of metalloproteinases.

Phosphoinositide-dependent protein kinase 1 (PDK1) is a key enzyme, master regulator of cellular proliferation and metabolism; it is considered a key target for pharmacological intervention. Using membranes obtained from DDT1 MF-2 cells, phospho-PDK1 was identified by Western blotting, as two major protein bands of Mr 58-68 kDa. Cell incubation with the PDK1 inhibitor, UCN-01, induced a time- and concentration-dependent decrease in the amount of phospho-PDK1 with a concomitant appearance of a ≈42 kDa phosphorylated fragment. Knocking down PDK1 diminished the amount of phospho-PDK1 detected in membranes, accompanied by similarly decreased fragment generation. UCN-01-induced fragment generation was also observed in membranes from cells stably expressing a myc-tagged PDK1 construct. Other PDK1 inhibitors were also tested: OSU-03012 induced a clear decrease in phospho-PDK1 and increased the presence of the phosphorylated fragment in membrane preparations; in contrast, GSK2334470 and staurosporine induced only marginal increases in the amount of PDK1 fragment. Galardin and batimastat, two metalloproteinase inhibitors, markedly attenuated inhibitor-induced PDK1 fragment generation. Metalloproteinases 2, 3, and 9 co-immunoprecipitated with myc-PDK1 under baseline conditions and this interaction was stimulated by UCN-01; batimastat also markedly diminished this effect of the PDK1 inhibitor. Our results indicate that a series of protein kinase inhibitors, namely UCN-01 and OSU-03012 and to a lesser extent GSK2334470 and staurosporine induce PDK1 fragmentation and suggest that metalloproteinases could participate in this effect.