The Development of a Microbial Challenge Test with Acholeplasma laidlawii To Rate Mycoplasma-Retentive Filters by Filter Manufacturers.

Mycoplasma are bacteria that can penetrate 0.2 and 0.22 µm rated sterilizing-grade filters and even some 0.1 µm rated filters. Primary applications for mycoplasma filtration include large scale mammalian and bacterial cell culture media and serum filtration. The Parenteral Drug Association recognized the absence of standard industry test parameters for testing and classifying 0.1 µm rated filters for mycoplasma clearance and formed a task force to formulate consensus test parameters. The task force established some test parameters by common agreement, based upon general industry practices, without the need for additional testing. However, the culture medium and incubation conditions, for generating test mycoplasma cells, varied from filter company to filter company and was recognized as a serious gap by the task force. Standardization of the culture medium and incubation conditions required collaborative testing in both commercial filter company laboratories and in an Independent laboratory (Table I). The use of consensus test parameters will facilitate the ultimate cross-industry goal of standardization of 0.1 µm filter claims for mycoplasma clearance. However, it is still important to recognize filter performance will depend on the actual conditions of use. Therefore end users should consider, using a risk-based approach, whether process-specific evaluation of filter performance may be warranted for their application. LAY ABSTRACT: Mycoplasma are small bacteria that have the ability to penetrate sterilizing-grade filters. Filtration of large-scale mammalian and bacterial cell culture media is an example of an industry process where effective filtration of mycoplasma is required. The Parenteral Drug Association recognized the absence of industry standard test parameters for evaluating mycoplasma clearance filters by filter manufacturers and formed a task force to formulate such a consensus among manufacturers. The use of standardized test parameters by filter manufacturers, including the preparation of the culture broth, will facilitate the end user's evaluation of the mycoplasma clearance claims provided by filter vendors. However, it is still important to recognize filter performance will depend on the actual conditions of use; therefore end users should consider, using a risk-based approach, whether process-specific evaluation of filter performance may be warranted for their application.

Synthesis and structure-activity relationships of benzophenone-bearing diketopiperazine-type anti-microtubule agents.

KPU-105 (4), a potent anti-microtubule agent that contains a benzophenone was derived from the diketopiperazine-type vascular disrupting agent (VDA) plinabulin 3, which displays colchicine-like tubulin depolymerization activity. To develop derivatives with more potent anti-microtubule and cytotoxic activities, we further modified the benzophenone moiety of 4. Accordingly, we obtained a 4-fluorobenzophenone derivative 16j that inhibited tumor cell growth in vitro with a subnanomolar IC(50) value against HT-29 cells (IC(50)=0.5 nM). Next, the effect of 16j on mitotic spindles was evaluated in HeLa cells. Treatment with 3nM of 16j partially disrupted the interphase microtubule network. By contrast, treatment with the same concentration of CA-4 barely affected the microtubule network, indicating that 16j exhibited more potent anti-mitotic effects than did CA-4.

Synthesis and structure-activity relationship study of antimicrotubule agents phenylahistin derivatives with a didehydropiperazine-2,5-dione structure.

Plinabulin (11, NPI-2358) is a potent microtubule-targeting agent derived from the natural diketopiperazine "phenylahistin" (1) with a colchicine-like tubulin depolymerization activity. Compound 11 was recently developed as VDA and is now under phase II clinical trials as an anticancer drug. To develop more potent antimicrotubule and cytotoxic derivatives based on the didehydro-DKP skeleton, we performed further modification on the tert-butyl or phenyl groups of 11, and evaluated their cytotoxic and tubulin-binding activities. In the SAR study, we developed more potent derivatives 33 with 2,5-difluorophenyl and 50 with a benzophenone in place of the phenyl group. The anti-HuVEC activity of 33 and 50 exhibited a lowest effective concentration of 2 and 1 nM for microtubule depolymerization, respectively. The values of 33 and 50 were 5 and 10 times more potent than that of CA-4, respectively. These derivatives could be a valuable second-generation derivative with both vascular disrupting and cytotoxic activities.

Trypanocidal activity of ß-lactone-γ-lactam proteasome inhibitors.

Four ß-lactone- γ-lactam proteasome inhibitors of natural origin were tested for their trypanocidal activities in vitro using culture-adapted bloodstream forms of Trypanosoma brucei. All four compounds displayed activities in the nanomolar range. The most trypanocidal compounds with 50% growth inhibition (GI(50)) values of around 3 nM were the bromine and iodine analogues of salinosporamide A, a potent proteasome inhibitor produced by the marine actinomycete Salinispora tropica. In general, trypanosomes were more susceptible to the compounds than were human HL-60 cells. The data support the potential of ß-lactone- γ-lactam proteasome inhibitors for rational anti-trypanosomal drug development.

Specific and prolonged proteasome inhibition dictates apoptosis induction by marizomib and its analogs.

Marizomib (NPI-0052) is a naturally derived irreversible proteasome inhibitor that potently induces apoptosis via a caspase-8 and ROS-dependent mechanism in leukemia cells. We aim to understand the relationship between the irreversible inhibition of the proteasome and induction of cell death in leukemia cells by using analogs of marizomib that display reversible and irreversible properties. We highlight the importance of sustained inhibition of at least two proteasome activities as being key permissive events for the induction of the apoptotic process in leukemia cells. These data provide the basis for the development of new approaches to generate more effective anti-proteasome therapies.

Proteasome structure, function, and lessons learned from beta-lactone inhibitors.

The 26S proteasome is the enzymatic core engine of the ubiquitin and proteasome dependent proteolytic system (UPS), the major eukaryotic pathway for regulated protein degradation. The UPS plays a pivotal role in cellular protein turnover, protein quality control, antigen processing, signal transduction, cell cycle regulation, cell differentiation and apoptosis, inspiring in-depth studies of proteasome structure and function and the search for selective inhibitors. Structural studies revealed that the 26S proteasome comprises up to two 19S regulatory caps flanking a cylindrical 20S core particle, which houses the proteolytic subunits and is present in all kingdoms of life. This review highlights current understanding of 20S architecture, maturation and assembly, the mechanism for selective degradation of protein substrates targeted for destruction, and relationships to other proteases. This knowledge base has benefited from structurally diverse proteasome inhibitors discovered from unique sources, including terrestrial and marine actinomycetes that produce the ß-lactone-γ- lactam superfamily of inhibitors, including omuralide, salinosporamide A (marizomib; NPI-0052) and the cinnabaramides. These "minimalist inhibitors" utilize dense functionality to maximum efficiency for potent and selective proteasome inhibition and have advanced from biochemical tools to potential agrochemicals and anticancer agents. In this review, lessons learned from the ß-lactone-γ-lactam superfamily are presented, with an emphasis on their unique binding mechanisms elucidated through structural biology in concert with medicinal chemistry. Distinctions between slowly reversible and irreversible inhibitors are discussed, together with the relationship of irreversible binding at the molecular level to prolonged duration proteasome inhibition in tumor cells, and in vitro and in vivo efficacy.

Proteasome regulator marizomib (NPI-0052) exhibits prolonged inhibition, attenuated efflux, and greater cytotoxicity than its reversible analogs.

The present study was undertaken to compare the cellular transport characteristics of [(3)H]NPI-0052 (1R,4R,5S)-4-(2-chloroethyl)-1-((S)-((S)-cyclohex-2-enyl)(hydroxy)methyl)-5-methyl-6-oxa-2-azabicyclo[3.2.0]heptane-3,7-dione (marizomib; salinosporamide A) and [(3)H]NPI-0047 (1R,4R, 5S)-1-((S)-((S)-cyclohex-2-enyl)(hydroxy)methyl)-4-ethyl-5-methyl-6-oxa-2-azabicyclo[3.2.0]heptane-3,7-dione in RPMI 8226 multiple myeloma and PC-3 prostate adenocarcinoma cells to determine whether these properties explain differences in the cytotoxic potencies of these chemical analogs. The results indicate that marizomib, which possesses a chemical-leaving group, is more cytotoxic to both cell lines and inhibits proteasome activity more completely at lower concentrations than NPI-0047, a nonleaving-group analog. Moreover, it was found that both compounds accumulate in these cells by simple diffusion and the same carrier-mediated transport system. Although the rate of uptake is similar, the cellular efflux, which does not seem to be mediated by a major ATP-binding cassette (ABC)-efflux transporter, is more rapid for NPI-0047 than for marizomib. Experiments revealed that the irreversible binding of marizomib to the proteasome is responsible for its slower efflux, longer duration of action, and greater cytotoxicity compared with NPI-0047. The discovery that major ABC transporters of the multidrug resistance-associated protein family do not seem to be involved in the accumulation or removal of these agents suggests they may not be affected by multidrug resistance mechanisms during prolonged administration.

Generating a generation of proteasome inhibitors: from microbial fermentation to total synthesis of salinosporamide a (marizomib) and other salinosporamides.

The salinosporamides are potent proteasome inhibitors among which the parent marine-derived natural product salinosporamide A (marizomib; NPI-0052; 1) is currently in clinical trials for the treatment of various cancers. Methods to generate this class of compounds include fermentation and natural products chemistry, precursor-directed biosynthesis, mutasynthesis, semi-synthesis, and total synthesis. The end products range from biochemical tools for probing mechanism of action to clinical trials materials; in turn, the considerable efforts to produce the target molecules have expanded the technologies used to generate them. Here, the full complement of methods is reviewed, reflecting remarkable contributions from scientists of various disciplines over a period of 7 years since the first publication of the structure of 1.

Concise formal synthesis of (-)-salinosporamide A (marizomib) using a regio- and stereoselective epoxidation and reductive oxirane ring-opening strategy.

Expedient access to a highly functionalized 2-pyrrolidinone (8), the gamma-lactam core of 20S proteasome inhibitor (-)-salinosporamide A (marizomib; NPI-0052; 1), using a regio- and stereoselective epoxide formation/reductive oxirane ring-opening strategy is presented. Notably, the sequential construction of the C-4, C-3, and C-2 stereocenters of 1 in a completely stereocontrolled fashion is a key feature of streamlining the synthesis of intermediate 12. A related strategy is also discussed.

The odyssey of marine pharmaceuticals: a current pipeline perspective.

The global marine pharmaceutical pipeline consists of three Food and Drug Administration (FDA) approved drugs, one EU registered drug, 13 natural products (or derivatives thereof) in different phases of the clinical pipeline and a large number of marine chemicals in the preclinical pipeline. In the United States there are three FDA approved marine-derived drugs, namely cytarabine (Cytosar-U((R)), Depocyt((R))), vidarabine (Vira-A((R))) and ziconotide (Prialt((R))). The current clinical pipeline includes 13 marine-derived compounds that are either in Phase I, Phase II or Phase III clinical trials. Several key Phase III studies are ongoing and there are seven marine-derived compounds now in Phase II trials. The preclinical pipeline continues to supply several hundred novel marine compounds every year and those continue to feed the clinical pipeline with potentially valuable compounds. From a global perspective the marine pharmaceutical pipeline remains very active, and now has sufficient momentum to deliver several additional compounds to the marketplace in the near future; this review provides a current view of the pipeline.

Pharmacodynamic and efficacy studies of the novel proteasome inhibitor NPI-0052 (marizomib) in a human plasmacytoma xenograft murine model.

Our previous study showed that the novel proteasome inhibitor NPI-0052 induces apoptosis in multiple myeloma (MM) cells resistant to conventional and bortezomib (Velcade, Takeda, Boston, MA, USA) therapies. In vivo studies using human MM-xenografts demonstrated that NPI-0052 is well tolerated, prolongs survival, and reduces tumour recurrence. These preclinical studies provided the basis for an ongoing phase-1 clinical trial of NPI-0052 in relapsed/refractory MM patients. Here we performed pharmacodynamic (PD) studies of NPI-0052 using human MM xenograft murine model. Our results showed that NPI-0052: (i) rapidly left the vascular compartment in an active form after intravenous (i.v.) administration, (ii) inhibited 20S proteasome chymotrypsin-like (CT-L, beta5), trypsin-like (T-L, beta2), and caspase-like (C-L, beta1) activities in extra-vascular tumours, packed whole blood (PWB), lung, liver, spleen, and kidney, but not brain and (iii) triggered a more sustained (>24 h) proteasome inhibition in tumours and PWB than in other organs (<24 h). Tissue distribution analysis of radiolabeled compound (3H-NPI-0052) in mice demonstrated that NPI-0052 left the vascular space and entered organs as the parent compound. Importantly, treatment of MM.1S-bearing mice with NPI-0052 showed reduced tumour growth without significant toxicity, which was associated with prolonged inhibition of proteasome activity in tumours and PWB but not normal tissues.

Snapshots of the fluorosalinosporamide/20S complex offer mechanistic insights for fine tuning proteasome inhibition.

Many marketed drugs contain fluorine, reflecting its ability to modulate a variety of biological responses. The unique 20S proteasome inhibition profile of fluorosalinosporamide compared to chlorinated anticancer agent salinosporamide A (NPI-0052) is exemplary and relates to each halogen's leaving group potential. Crystal structures of fluoro-, hydroxy-, and bromosalinosporamide in complex with the yeast 20S proteasome core particle (CP) provide mechanistic insights into ligand binding and leaving group elimination and the ability to fine-tune the duration of proteasome inhibition. Fluorosalinosporamide/CP crystal structures determined over time offer striking snapshots of the ligand trapped with an intact fluoroethyl group in anticipation of fluoride elimination, followed by complete nucleophilic displacement of fluoride to give the highly stabilized cyclic ether found for salinosporamide A and bromosalinosporamide. This two-step reaction pathway is consistent with a mechanism for partially reversible proteasome inhibition by fluorosalinosporamide. Proteasome catalyzed fluoride displacement provides preliminary insights into the active site Thr1N pK(a).

Antiprotealide is a natural product.

Large-scale fermentation of the marine actinomycete Salinispora tropica for production of salinosporamide A (NPI-0052; 1) clinical trials materials provided crude extracts containing minor secondary metabolites, including salinosporamide B (2) and a new congener, 3. Spectroscopic characterization revealed that 3 is identical to antiprotealide, a molecular hybrid of 20S proteasome inhibitors 1 and omuralide (4) not previously described as a natural product. Analysis of crude extracts from shake flask cultures of three wild-type S. tropica strains confirmed the production of antiprotealide at 1.1, 0.8, and 3.0 mg/L. Thus, antiprotealide is a natural product metabolite of S. tropica.

Discovery and development of the anticancer agent salinosporamide A (NPI-0052).

The discovery of the anticancer agent salinosporamide A (NPI-0052) resulted from the exploration of new marine environments and a commitment to the potential of the ocean to yield new natural products for drug discovery and development. Driving the success of this process was the linkage of academic research together with the ability and commitment of industry to undertake drug development and provide the resources and expertise to advance the entry of salinosporamide A (NPI-0052) into human clinical trials. This paper offers a chronicle of the important events that facilitated the rapid clinical development of this exciting molecule.

Tubulin photoaffinity labeling with biotin-tagged derivatives of potent diketopiperazine antimicrotubule agents.

NPI-2358 (1) is a potent antimicrotubule agent that was developed from a natural diketopiperazine, phenylahistin, which is currently in Phase I clinical trials as an anticancer drug. To understand the precise recognition mechanism of tubulin by this agent, we focused on its potent derivative, KPU-244 (2), which has been modified with a photoreactive benzophenone structure, and biotin-tagged KPU-244 derivatives (3 and 4), which were designed and synthesized for tubulin photoaffinity labeling. Introduction of the biotin structure at the p'-position of the benzophenone ring in 2 exhibited reduced, but significant biological activities with tubulin binding, tubulin depolymerization and cytotoxicity in comparison to the parent KPU-244. Therefore, tubulin photoaffinity labeling studies of biotin-derivatives 3 and 4 were performed by using Western blotting analysis after photoirradiation with 365 nm UV light. The results indicated that tubulin was covalently labeled by these biotin-tagged photoprobes. The labeling of compound 4 was competitively inhibited by the addition of diketopiperazine 1 or colchicine, and weakly inhibited by the addition of vinblastine. The results suggest that photoaffinity probe 4 specifically recognizes tubulin at the same binding site as anticancer drug candidate 1, and this leads to the disruption of microtubules. Probe 4 serves well as a useful chemical probe for potent antimicrotubule diketopiperazines, much like phenylahistin, and it also competes for the colchicine-binding site.

Leaving groups prolong the duration of 20S proteasome inhibition and enhance the potency of salinosporamides.

Salinosporamide A ( 1 (NPI-0052)) is a potent, monochlorinated 20S proteasome inhibitor in clinical trials for the treatment of cancer. To elucidate the role of the chlorine leaving group (LG), we synthesized analogues with a range of LG potentials and determined their IC 50 values for inhibition of chymotrypsin-like (CT-L), trypsin-like (T-L), and caspase-like (C-L) activities of 20S proteasomes. Proteasome activity was also determined before and after attempted removal of the inhibitors by dialysis. Analogues bearing substituents with good LG potential exhibited the greatest potency and prolonged duration of proteasome inhibition, with no recovery after 24 h of dialysis. In contrast, activity was restored after