Peptides having tetrahedral mimicking groups as inhibitors of rhomboid proteases

Abstract

The present invention describes rhomboid protease inhibitors having high specificity and inhibition characteristics providing novel antibiotics, anti-malarial pharmaceutical agents, and provides a strategy for designing RiBns (rhomboid-inhibiting boronates) to target rhomboid selectively in unrelated organisms.

Claims

1. A compound comprising a tetrahedral mimicking group attached to a first end of a rhomboid protease substrate, wherein the rhomboid protease substrate comprises either KRFRSMQYSA (SEQ ID NO: 3) or KRFRSNQYSA (SEQ ID NO: 4), and wherein the rhomboid protease is PfROM4.

2. The compound of claim 1, wherein the rhomboid protease substrate is selected from the group consisting of: Ac-KRFRSMQYSA (SEQ ID NO: 3) and Ac-KRFRSNQYSA (SEQ ID NO: 4), wherein Ac is an N-terminal acetyl moiety, and wherein the tetrahedral mimicking group is attached to the C-terminal end of the rhomboid protease substrate.

3. The compound of claim 1, wherein the tetrahedral mimicking group is B(OH).sub.2, or trifluoromethylketone.

4. The compound of claim 1, wherein the rhomboid protease substrate has an acetyl group attached to a second end.

5. A method for inhibiting PfROM4 comprising the steps of providing a compound of claim 1 and applying it to PfROM4.

6. A method of inhibiting PfROM4 in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of a compound of claim 1 and inhibiting PfROM4.

7. The method of claim 6, wherein the subject is a human.

8. A method of treating malaria in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of a compound of claim 1.

9. The method of claim 8, wherein the subject is a human.

10. The method of claim 8, wherein the malaria is antibiotic resistant.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A-1E illustrates differential substrate specificity of PfROM4 is mediated by steric exclusion not a new recognition sequence. A. Diagram of GFP-tagged chimeras of EBA175 (EBA), AMA1 (AMA), and Spitz (Spi), with the membrane depicted as 2 horizontal lines (cytoplasm is down). Here and below, panels are western analyses of media and cell fractions from co-transfected HEK293 cells probed with the indicated antibodies. Substrate cleavage releases products into media. ‘mut’ denotes the inactive H578A mutant of PfROM4. Substrate cleavage was verified in at least two independent experiments. Figure discloses “MPYY,” “Kill” and “MLEK” as SEQ ID NOs: 9-11, respectively. B. Western analysis of cleavage by PfROM4 of GFP-EBA175 P4-P2 mutants is shown. Arrowheads illustrate that a P4 isoleucine alone is sufficient to block processing to the same extent as a triple isoleucine stretch (that naturally occurs in AMA1). ‘FL’ denotes full-length substrate. All constructs were analyzed in at least two independent experiments. Figure discloses SEQ ID NOs: 12-20, respectively, in order of appearance. C. Processing of GFP-EBA175 harboring all 20 amino acids in the P4 position by PfROM4. Arrowheads denote substitutions that hindered PfROM4 cleavage. Lower levels of DNA encoding PfROM4 were transfected in the right panel to facilitate quantification of increased mutant substrate processing. Cleavage of all mutants (except C and M) were tested in three independent experiments. D. P4 mutants of GFP-EBA175 that blocked PfROM4 cleavage activated cleavage by PfROM1. Shown are western analyses of cleavage products released into culture media. E. Left, alignment of a short stretch of L1 loop residues bound by the universally conserved R and H residues. Right, processing of a panel of P4 (P, I, R, E) and P2 mutants (A, I) of GFP-EBA175 by wildtype or indicated L1 loop mutants of PfROM1 or PfROM4. Shown are western analyses of proteolytic products released into cell culture media. Arrowheads denote substrates acquired by L1 loop mutants (while the endogenous profile remained intact). Constructs in D and E were tested in at least two, and often three, independent experiments. Figure discloses SEQ ID NOs: 21-23, respectively, in order of appearance.

(2) FIG. 2A-2D illustrates engineering a ‘super-substrate’ for PfROM4 by manually guided evolution of EBA175. A. Western analysis revealed substituting a serine at the P2 position of GFP-EBA175 enhanced proteolysis of P4 R, D, and Q mutants by ˜5-fold. Shown here and in c are western analyses of media and cell fractions from co-transfected HEK293 cells probed with the indicated antibodies. Substrate cleavage releases products into media. ‘FL’ denotes full-length GFP-EBA175. B. Mass spectra of C-terminal proteolytic fragments immuno-affinity isolated (anti-Flag) from HEK293 cells co-transfected with GFP-EBA175-Flag and PfROM4. Cleavage sites, based on experimentally measured molecular masses (in the spectra), are indicated with corresponding predicted masses on the left. Note a mass of 5487 corresponds to a cleavage site shift. Figure discloses SEQ ID NO: 24. C. Western analysis showing that substituting K, R and R and P7, P9, and P10, respectively (diagram at right), enhanced PfROM4-catalyzed processing of the quintuple GFP-EBA175 mutant by ˜20-fold (last lane). Note that PYY (first lane) is the natural P4-2 sequence. Figure discloses SEQ ID NOs: 25-30, respectively, in order of appearance. D. Mass spectrum the quintuple GFP-EBA175 mutant that was processed 20-fold better by PfROM4 revealed cleavage occurred at only the natural site. Data in a/c are representative of at least two independent biological experiments, while the mass spectra in b/d are representative of multiple spectral acquisitions. Figure discloses SEQ ID NO: 31.

(3) FIG. 3A-3F illustrates atomic structures of E. coli rhomboid GlpG in catalytic complex with peptide aldehyde (CHO), chlorometylketone (CMK), and boronate (B (OH)2) inhibitors. A. Overall structure of GlpG (left, in ribbon) in catalytic complex with Ac-VRMA-B(OH)2 (SEQ ID NO: 2) (electron density map 2Fo-Fc at 2s is shown in mesh). Note that in all cases electron density is continuous between the catalytic serine 5201 and the inhibitors, indicating that catalysis had taken place. Right, ball-and-stick overlay of all three inhibiting Ac-VRMA (SEQ ID NO: 2) peptides with different warheads reveals that all adopted nearly indistinguishable conformations in the GlpG active site. B. Active site comparison of the CMK singly reacted with the catalytic serine S201, versus CMK doubly reacted with the catalytic serine S201 and histidine H254 (PDB=4QO2). Note the dramatic shifts in position (denoted by arrows) of the substrate oxyanion, which has left the oxyanion hole, and catalytic histidine base (H254). C.-E. Atomic structures of tetrapeptide inhibitors in the active site of GlpG. The experimental electron density maps (2Fo-Fc at 2s) are shown in outline for the catalytic residues, and for the Ac-VRMA-CMK (SEQ ID NO: 2) in C, for the Ac-VRMA-CHO (SEQ ID NO: 2) (PDB=5F5B) in D, and for the Ac-VRMA-B(OH)2 (SEQ ID NO: 2) in E. In all cases, distances are in Å, and oxyanion-stabilizing interactions are the dashed lines. F. Inhibition of endogenous GlpG activity in living E. coli cells grown in liquid culture. Δ is the negative control in which GlpG was deleted from the E. coli genome. Peptide inhibitors were added at the indicated concentrations directly to growing cultures. Shown are quantitative infrared anti-flag westerns, with the top band being the TatA-Flag uncut substrate, and the lower band being the cut product. Quantification of cleavage was used to calculate the EC50 concentrations (graph on right, mean±s.e.m.). Inhibition was analyzed in three independent biological experiments, each with a full spectrum of concentrations per compound. Figure discloses SEQ ID NO: 2.

(4) FIG. 4A-F illustrates RiBn is a non-toxic, selective inhibitor of only PfROM4. A. Chemical structure of one embodiment of RiBn, a peptide of 10 aa with boronate warhead (SEQ ID NO: 3). Ac denotes an N-terminal acetyl moiety, while the C-terminal boronate warhead is on the right. B. Western blot analysis of EBA175 and AMA1 cleavage by PfROM4 in a heterologous cleavage assay. Co-transfected HEK293 cells were conditioned in the presence of absence of the indicated concentrations of RiBn. Cells transfected with AMA1 were conditioned in the presence or absence of the metalloprotease inhibitor (WI) BB-94 to reduce background cleavage by endogenous cellular proteases. N-terminal cleavage products were released into media (anti-GFP, top panels). Lysates were probed for expression of substrate (anti-GFP, middle panel) and enzyme (anti-HA, bottom panel). Quantification of PfROM4 inhibition by RiBn (right panel, mean±s.e.m.). Data was fitted with a 4-parameter logistic regression model, accounting for baselines, slope and the EC50. Shading represents the 95% confidence interval of the model fit. Inhibition was analyzed in three independent biological experiments. C. Fluorescence microscopy of HEK293 cells cultured in the presence or absence of RiBn for ˜24 hours (left panels) and stained with calcein-AM (live cells) and ethidium homodimer-1 (dead cells). Flow cytometry analysis (calcein AM fluorescence) of the same cells (right panels). D. Cell cycle analysis of HEK293 cells cultured in the presence or absence of RiBn for 24 hours was conducted by flow cytometry (propidium iodine fluorescence). Populations of cells in G1, S, and G2/M are indicated with gates. For data in D and E, toxicity analyses were conducted in three independent biological experiments (once for each assay readout). E. Western analysis of EBA175 cleavage by PfROM4 (anti-GFP, conditioned media). Co-transfected HEK293 cells were conditioned in the presence or absence of the indicated concentrations of either the peptide-aldehyde (top panel) or RiBn (bottom panel). The dose-response of RiBn was conducted in three independent biological experiments (at various concentrations). F. Western analysis (anti-GFP, conditioned media) of RiBn inhibition of all known cell surface rhomboid enzymes and their substrates. Inhibition was verified in three independent biological experiments.

(5) FIG. 5A-5F illustrates RiBn inhibits P. falciparum malaria growth on human erythrocytes. A. Quantification of parasitemia inhibition by RiBn (mean±s.e.m.). Absolute parsitemia was determined by flow cytometry (using acridine orange staining) of parasitized erythrocytes cultured in the presence of various concentrations of RiBn. Inhibitor potency was verified in three independent biological experiments. B. Bright-field microscopy (top panel) and flow cytometry analysis (Forward Scatter vs Side Scatter, bottom panel) of erythrocytes cultured in the presence or absence of RiBn for ˜18 hours. Quantification was conducted once. C. Quantification of parasitemia in parasitized erythrocytes cultured in the presence of the indicated concentrations of pepA (peptide-aldehyde, left-hand side of each concentration) or RiBn (right-hand side of each concentration) for ˜80 hours. D. Blood cultures of well synchronized schizont-stage parasites were treated with the indicated concentrations of RiBn for ˜24 hours and probed for the presence of ring stage parasites by flow cytometry of parasitized (DNA+) and uninfected (DNA−) erythrocytes. E. Fluorescent microscopy images of acridine orange stained parasitized erythrocytes cultured in the presence or absence of RiBn for ˜24 hours. F. Flow cytometry (acridine orange staining) at the indicated time points of RiBn treated or untreated parasitized erythrocytes. Parasite invasion and growth defects in d-f are representative of three independent biological experiments.

(6) FIG. 6A-6C illustrates RiBn cures blood stage cultures of malaria by iteratively inhibiting parasite invasion and retarding growth. A. Blood stage cultures containing 1% well synchronized schizont stage parasitized erythrocytes were cultured in the presence or absence of 0.3 mM RiB, as indicated, and analyzed at various time points via flow cytometry (acridine orange, top panels) and fluorescent microscopy (acridine orange staining, bottom panels). After 18 hours of culturing, RiBn was washed out of one replicate of the RiBn treated cultures and monitored as above, as indicated. B. Dose-response of RiBn (mean±s.e.m.) at various timepoints in the above experiment revealed iterative inhibition of parasite invasion. C. Quantification of absolute parasitemia at various time points in blood stage parasite cultures treated with the indicated concentrations of RiBn. In a-c, growth defect was confirmed in three independent biological experiments, while the parallel washout was conducted once and is shown.

(7) FIG. 7 illustrates compositions with a tetrahedral mimicking group (W) of the present invention kill artemisinin drug-resistant parasites including C580Y.

DETAILED DESCRIPTION OF THE INVENTION

(8) Intramembrane proteases are diverse enzymes that form a protease active site from their membrane-spanning segments within the plane of the membrane. Although these ancient enzymes fall into four evolutionarily and chemically distinct families, only the rhomboid and site-2 protease families are conserved in nearly all known bacterial, fungal, and protozoan pathogens. This broad distribution, coupled with multiple functions in pathogenesis by unrelated microbes, suggests understanding and ultimately inhibiting these enzymes could be an exciting pan-antimicrobial strategy.

(9) Site-2 proteases are zinc metalloenzymes that play central roles in the virulence of many bacterial pathogens; they usually activate latent transcription factors by releasing them from the bacterial inner membrane. These deceptively simple ‘triggered release’ circuits regulate a wide variety of processes central to pathogenesis, including protective alginate production in Pseudomonas aeruginosa, outer membrane composition in Mycobacterium tuberculosis, toxin production in Vibrio cholerae, and conjugation and/or nosocomial antibiotic resistance in Enterococcus faecalis. More recent work has implicated site-2 proteolysis in virulence of the fungus Cryptococcus neoformans by facilitating deep-tissue dissemination.

(10) Although rhomboid proteases likely originated in bacteria, the function of these unusual serine proteases have been studied predominantly in protozoan parasites. Early work identified adhesin molecules of apicomplexan parasites as probable substrates, including those from Plasmodium falciparum, the agent of malaria, and Toxoplasma gondii, a related parasite that infects nearly a third of the world's population. These obligate intracellular parasites must iteratively invade host cells, and rely upon an extensive arsenal of transmembrane adhesin molecules to attach to and invade cells. Ultimately shedding adhesins from the parasite surface was postulated to be important for completing invasion. Recent work has further broadened possible roles for rhomboid proteolysis to non-invasive, cytolytic parasites Entamoeba histolytica and Trichomonas vaginalis. Rhomboid cleavage of adhesins in these parasites is proposed to regulate parasite adherence, contact-dependent host-cell killing, and/or immune evasion, all of which are central to the virulence of these extracellular parasites.

(11) Despite the great promise that rhomboid and site-2 proteases hold as therapeutic targets for treating multiple and diverse infectious diseases, three challenges have nearly quelled progress towards this goal. First, neither the rhomboid nor site-2 protease family has ever been successfully targeted pharmacologically in any microbe despite over a decade of effort. In fact, all but one recent class of rhomboid inhibitors act irreversibly and require pre-incubation, sometimes for hours, to be effective. This limitation has raised concerns whether such membrane-immersed active sites and/or unusual chemical environments should be considered readily accessible to conventional small-molecule inhibition. The rapid nature of apicomplexan invasion, which can take only seconds, magnifies the challenge of whether there is even sufficient time to target rhomboid proteolysis prior to parasites completing invasion and taking refuge in a host cell. A second major complication is the conservation of both site-2 and rhomboid protease families in humans, which necessitates developing parasite-selective inhibitors.

(12) The third challenge stems from an incomplete understanding of rhomboid function: although the processes in which rhomboid enzymes participate are essential, it remains uncertain whether rhomboid proteolysis is itself essential. Understanding the precise role played by the principal cell surface rhomboid, PfROM4, has been hindered by the inability to isolate malaria parasites lacking PfROM4. However, recent rhomboid knockouts in the related apicomplexan Toxoplasma gondii revealed surprisingly non-essential roles for rhomboid proteolysis; absence of rhomboid cleavage led to elevated and non-polarized adhesin distribution on the parasite surface, but many parasites nevertheless invaded successfully. These first glimpses call into question whether rhomboid proteases, even if successfully inhibited, are targets worthy of continued effort.

(13) An Exclusion Mechanism Underlies Differential Substrate Selectivity of PfROM4

(14) PfROM4 is able to process over a dozen malaria adhesins, but was the first active rhomboid protease found that failed to cleave the canonical rhomboid substrate Drosophila Spitz. Conversely, animal rhomboid proteases cannot cleave malaria adhesins, implying that PfROM4 evolved distinct substrate specificity. In fact, P. falciparum contains at least one rhomboid protease, PfROM1, the specificity of which resembles Spitz-cleaving animal rhomboid proteases, and an adhesin, apical membrane antigen 1 (AMA1), which is cleaved by canonical rhomboid proteases including PfROM1 but not by PfROM4.

(15) We focused on P. falciparum erythrocyte-binding antigen 175 (EBA175) and AMA1 to map the substrate requirements underlying PfROM4's atypical substrate specificity, because these are the best-characterized malaria adhesins; they were the earliest to be identified, are essential, had their crystal structures solved, and their cleavage sites have been mapped as they are shed from parasites. We examined rhomboid cleavage in a standardized cell-based assay by co-transfecting human HEK293 cells with GFP-tagged adhesin constructs and HA-tagged PfROM1 and 4.

(16) AMA1 and EBA175 are large, type I transmembrane proteins of 622 and 1502 residues, respectively. To map the region of EBA175 responsible for targeting by PfROM4, we first created a chimeric AMA1 adhesin containing transmembrane and juxtamembrane residues from EBA175, with the junction being 5 residues N-terminal to their cleavage sites (termed P5 in protease nomenclature). Despite only having 40 residues from EBA175, this chimera converted AMA1 into a strong substrate for PfROM4 (FIG. 1a). Conversely, a chimera that contained the extracellular region of EBA175 and the analogous juxtamembrane AMA1 residues (from P5 onwards) failed to be cleaved, yet one that contained AMA1 residues from P1 was cleaved efficiently, implying that PfROM4 recognizes EBA175 by the four residues P5 through P2. Accordingly, transplanting just these four residues of EBA175 (MPYY (SEQ ID NO: 9)) was sufficient to convert AMA1 into a strong PfROM4 substrate. Reciprocally, mutating just the PYY residues (P4-P2) of EBA175 to III (P4-P2 of AMA1) completely abrogated EBA175 cleavage. Finally, we tested single mutants of EBA175 and found that mutating just the P4 proline residue to isoleucine was sufficient to block EBA175 cleavage, while isoleucine or alanine substituted at either the P2 or P3 positions supported proteolysis (FIG. 1b). These mapping experiments therefore highlighted the P4 proline of EBA175 as key for conferring cleavage by PfROM4.

(17) To decipher the mechanistic basis of this effect, we installed each of the 20 naturally occurring amino acids at the P4 position of EBA175 and evaluated cleavage by PfROM4 (FIG. 1c). Surprisingly, all but an isoleucine and valine supported cleavage, and many changes even enhanced EBA175 proteolysis. These changes included residues that were small and non-polar or polar (alanine, glycine, serine), large and hydrophobic (leucine, phenylalanine), positively charged (arginine, lysine), and negatively changed (aspartate, glutamate). The disparity in chemical characteristics and size suggested that, surprisingly, beta-branched residues are simply not tolerated at the P4 position, while all others are accommodated with ease or even preferred. This proved to be precisely reciprocal for conferring cleavage by PfROM1; only P4 mutants harboring beta-branched residues converted EBA175 into a PfROM1 substrate (FIG. 1c). Finally, although neither AMA1 nor Spitz are substrates for PfROM4, a chimera with the extracellular region from AMA1 and remaining residues from Spitz (from its P5 residue onwards) was cleaved efficiently by PfROM4 (FIG. 1a).

(18) These experiments suggested that PfROM1 and 4 do not recognize separate sequences directly, but instead that they cannot cleave certain substrates because of residues that block cleavage. This model predicts that steric clashes with rhomboid explain the differences in substrate preferences. Although no structural information exists for malaria rhomboid enzymes, we could align PfROM4 and PfROM1 to Escherichia coli GlpG, whose high-resolution structure has been solved with bound substrate peptides, in a short region of the L1 loop between a universally conserved arginine residue that supports L1 loop architecture, and an oxyanion-stabilizing histidine near the start of transmembrane segment 2 (FIG. 1d). This region of GlpG has been shown structurally to interact with the P4 residue of substrates. We therefore mutated the corresponding A144 and N145 in PfROM1 to glycine, which naturally occur in PfROM4, and found that the mutant PfROM1 gained the ability to cleave wildtype EBA175 with a P4 proline, or large and charged P4 residues (FIG. 1d), while retaining its ability to process natural substrates. Reciprocally, mutating the corresponding glycines in PfROM4 to AN, as occur naturally in PfROM1, still allowed PfROM4 to cleave its natural substrates, but now also allowed it to cleave substrates with the previously non-tolerated beta-branched isoleucine in the P4 position (FIG. 1d). The change is selectivity was specific, because neither the PfROM1 nor the PfROM4 L1 loop mutants changed the ability or inability of either enzyme to process P2 substrate mutants (FIG. 1d).

(19) Taken together, these mapping and mutagenesis studies indicate that the mechanistic basis underlying the differential selectivity of PfROM4 and PfROM1 for substrates is governed by negatively acting steric clashes that hinder cleavage, rather than presence of a unique, positively acting targeting sequence.

(20) An Alternative Strategy: Engineering a Super-Substrate by Manually Directed Evolution

(21) The steric exclusion mechanism that defines differential substrate specificity of PfROM4 precluded identifying a sequence that we could use for inhibitor design. However, we recently discovered using a structural approach with E. coli GlpG that fostering ‘extra’ interactions in substrates at otherwise non-essential positions can enhance natural substrate processing. In fact, we found a variant that increased substrate processing by a modest 2-fold actually increased inhibitor potency by nearly 10-fold. We therefore focused on evolving an optimized targeting sequence for PfROM4 by engineering EBA175 mutations that enhanced natural substrate processing.

(22) As a starting point, we subjected three diverse P4 mutants (Q, R, D) that enhanced processing of EBA175 to a second round of mutagenesis, and isolated an additional serine substitution at the P2 residue that enhanced proteolysis by 5-fold (FIG. 2a). Rhomboid proteases do not normally bind a target sequence in substrates, and accordingly mutations often induce shifts in the site of cleavage. Since such shifts would undermine our attempt to evolve a true binding sequence, we used mass spectrometry to map the cleavage site of every enhanced mutant that we isolated. Although the presence of R at P4 shifted cleavage site, the natural cleavage site was maintained in the Q and D P4 mutants (FIG. 2b). We thus subjected the enhanced double mutant substrates to a third round of mutagenesis, and ultimately discovered that incorporating positively charged residues at the P7, P9 and P10 positions further enhanced processing (FIG. 2c). The best quintuple mutant substrate that we engineered enhanced processing by ˜20-fold over wildtype EBA175, and fully maintained cleavage at the natural site (FIG. 2d). This sequence was therefore an ideal candidate for inhibitor design.

(23) Peptide Boronates are >100-Fold More Potent Rhomboid Inhibitors

(24) Our next goal was to install a chemical warhead for PfROM4 inhibition. We recently discovered that, contrary to expectation, peptides bearing C-terminal aldehydes that act as reversible inhibitors are more effective at inhibiting E. coli rhomboid proteolysis than peptides harboring an irreversible chloromethylketone warhead. Since this was a new and unexpected observation, we sought to test whether other reversible warheads might prove to be even more potent rhomboid inhibitors.

(25) E. coli and P. falciparum rhomboid proteases are grossly dissimilar, sharing only ˜9% sequence identity overall. However, the local constellation of residues performing catalysis in their active sites is likely to be conserved, thereby potentially allowing structural analysis of GlpG to inform our attempt at PfROM4 inhibitor development. We therefore solved and compared the tetrahedral complex structures between E. coli GlpG and tetrapeptides harboring C-terminal chloromethylketone, aldehyde, or boronate warheads at 2.3 Å resolution.

(26) In all cases, the position of the substrate peptide in the active site was similar, being dictated by the peptide sequence (FIG. 3a). Interestingly, we were able to isolate the singly-linked tetrahedral complex with even the peptide chlorometylketone (FIG. 3b, c), in contrast to the available structures that contain only trapped, doubly-linked covalent adducts. A key difference is the oxyanion being dramatically pushed outwards by 1.64 Å with the doubly-reacted chloromethylketone (FIG. 3b), but clearly in the oxyanion hole with both the singly reacted peptide aldehyde (FIG. 3d) and chloromethylketone (FIG. 3c). Interestingly, the boronate also adopted the ideal substrate position, but the second hydroxyl group of the boronate made an additional strong hydrogen bond to the catalytic H254 base of GlpG at a distance of 2.61 Å (FIG. 3e). In fact, H254 moved closer to the substrate as a whole. This was an encouraging observation, because such a strong, extra hydrogen bond could result in inhibitors of considerably higher affinity.

(27) We next tested the effect of the boronate on the inhibition of GlpG in the natural membrane of living E. coli cells (FIG. 3f). Remarkably, just the presence of the boronate moiety itself increased potency of the same tetrapeptide by >400-fold relative to the chloromethylketone, and >100-fold relative to the aldehyde in a direct, side-by-side comparison. In fact, the peptide boronate, with a sub-micro molar EC.sub.50, became the most potent inhibitor of the endogenous GlpG in E. coli cells ever discovered and without needing preincubation, suggesting that a boronate might provide an excellent warhead for PfROM4 inhibition.

(28) Parasite-Selective Rhomboid Inhibition by a Designed Peptide Boronate

(29) We combined our 20-fold enhanced EBA175 substrate sequence with a C-terminal boronate warhead to engineer a rhomboid-inhibiting boronate, or RiBn (pronounced ‘ribbon’), as a candidate compound for the selective inhibition of PfROM4 (FIG. 4a). Treating transfected HEK293 cells with the RiBn inhibited EBA175 processing by PfROM4 in a dose-dependent manner (FIG. 4b), and had no apparent effect on cell viability or function (FIG. 4b, c). In fact, even at the highest concentrations, a fluorescence-based live/dead cell assay on living cells revealed no change in cell viability (FIG. 4b). We also used flow cytometry to quantify cell viability, and found 92.4% viability in cells treated with the highest RiBn dose, relative to 93.8% in untreated controls analyzed in parallel (FIG. 4c). Moreover, flow cytometry also revealed no apparent change in the adherence, growth, or cell cycle distribution of treated versus untreated cells (FIG. 4d). Finally, testing the same peptide sequence but replacing the boronate with an aldehyde moiety completely abolished PfROM4 inhibition, again highlighting the specificity of the designed RiBn (FIG. 4e).

(30) More exciting was the fact that the RiBn had no inhibitory effect on any of the other five rhomboid proteases known to reside on the cell surface, including the human rhomboid enzyme, analyzed in parallel in the same transfection-based assay (FIG. 4f). In fact, even PfROM1, which is also a P. falciparum rhomboid but with distinct substrate specificity, proved completely unaffected even at the highest RiBn concentration. Under all of these conditions, treated cells translated, glycosylated, trafficked, and secreted several different unrelated substrates indistinguishably from cells treated with the highest RiBn concentrations, again revealing no apparent effect on cell viability or function (FIG. 4f). These observations indicate that we succeeded in designing a PfROM4-selective inhibitor that has no discernible effect on other rhomboid enzymes, or on cell viability or function.

(31) PfROM4 Inhibition Blocks Host-Cell Invasion by Malaria Parasites

(32) Although several different species of Plasmodium naturally infect humans, P. falciparum causes the key lethal form of malaria worldwide. As such, we evaluated the effect of our RiBn on P. falciparum merozoites iteratively growing on human red blood cells, which constitutes the symptomatic and persistent stage of the parasite lifecycle, and thus the desired window for therapeutic intervention. Treating malaria cultures with the RiBn resulted in dose-dependent inhibition of parasite growth (FIG. 5a). Toxicity analysis with red blood cells again verified that the compound was not affecting host cell viability (FIG. 5b), and substitution of the boronate with an aldehyde abolished inhibition (FIG. 5c). We next sought to characterize the basis of the inhibition.

(33) Treating synchronized cultures with the RiBn revealed dose-dependent inhibition of merozoite invasion of red blood cells (FIG. 5d). Interestingly, the maximal effect was ˜50% inhibition in the first round of infection as quantified by flow cytometry. Fluorescence microscopy revealed that many of the parasites that were able to invade red blood cells nevertheless were ‘stuck’ to the inner cell periphery (FIG. 5e), implying they could not get fully internalized or free themselves from the plasma membrane. Subsequent time points revealed that these parasites grew considerably slower than vehicle-treated controls (FIG. 5f). Ultimately the delayed parasites progressed through all of the lifecycle stages and released merozoites from schizonts, indicating that other parasite stages including egress itself were minimally or not affected by the RiBn. This was significant because egress is an intricate process that can be quite sensitive to indirect perturbations.

(34) Finally, we examined the effect of prolonged culturing parasites in the presence or absence of the peptide boronate (FIG. 6a). Washing out of the RiBn after the first round of invasion rescued parasite growth, which became evident in the second round of invasion. Conversely, single application of the RiBn had an even stronger effect on the second and subsequent rounds of invasion (FIG. 6a,b), such that red blood cell cultures could be entirely cured of parasitemia within 2 to 3 invasive cycles without applying any additional RiBn (FIG. 6c). These observations reveal an essential role for PfROM4 in host-cell invasion, and suggest that targeting its enzymatic activity selectively is both pharmacologically feasible and potentially curative.

(35) The present invention combined substrate profiling in living cells, manually directed evolution, X-ray crystallography, and parasitology to design and evaluate the first parasite-selective rhomboid proteolysis inhibitor. Chemical genetic dissection of P. falciparum infection revealed an essential and druggable role for PfROM4 in the invasion of human red blood cells. Remarkably, blood-stage cultures were cured of malaria with a single dose of the RiBn, even without the aid of an immune system that would likely be enhanced in its ability to attack parasites displaying accumulated cell-surface antigens and/or incompletely invaded and/or slow-growing parasites. These encouraging observations now compel initiating development of anti-PfROM4 drugs after the decade-long gap since rhomboid proteases were identified as adhesin-processing enzymes.

(36) The present invention also establishes a rational and specific strategy for generating selective RiBns for a variety of rhomboid proteases. In fact, many rhomboid proteases are thought to be involved in a diversity of parasitic diseases for which treatments are rudimentary or even ineffective, but their importance and/or targetability remain speculative. These include trichomonasis, the most common non-viral sexually-transmitted disease, and non-viral dysentery, which has recently surpassed malaria as a leading cause of childhood death worldwide. Other targets of rhomboid inhibitors of the present invention include but not limited to Cryptosporidium spp., Toxoplasma gondii, Pseudomonas aeruginosa, pathogenic E. coli, Streptococcus spp, Vibrio cholerae.

(37) Although it also seems unlikely that any parasitic rhomboid proteases naturally evolved high-affinity binding of recognition motifs within their substrates, transfection-based assays have already been established for over a dozen parasitic rhomboid enzymes. Coupled with our approach described here, this core set of standardized assays should allow a directed-evolution approach to engineering ‘super substrates’ and subsequently selective RiBns. Mounting chemical genetic screens will identify which cell surface rhomboid proteases are themselves important for pathogenic functions, which has been the bottleneck for the past decade. Identifying those rhomboid proteases that play essential and druggable roles will allow prioritizing them for subsequent drug development to start realizing the much-anticipated therapeutic promise of rhomboid proteases.

(38) Pharmaceutical Composition

(39) Embodiments of the disclosure concern methods and/or compositions for treating and/or preventing a disease such as malaria, trichomonaisis, cryptosporidiosis, toxoplasmosis, and non-viral dysentery, in which inhibition of one or more rhomboid intramembrane protease is directly or indirectly related. In certain embodiments, individuals with such a disease are treated with an inhibitor of a rhomboid protease, and in specific embodiments an individual with malaria is provided an inhibitor of rhomboid protease, such as an inhibitor of PfROM4.

(40) In certain embodiments, the level to which an inhibitor of rhomboid proteases decreases rhomboid protease activity may be any level so long as it provides amelioration of at least one symptom of a disease, including malaria, trichomonaisis, cryptosporidiosis, toxoplasmosis, and non-viral dysentery. The level of activity may decrease by at least 2, 3, 4, 5, 10, 25, 50, 100, 200, 300, 400, 500, 1000, or more fold activity compared to the level of expression in a standard or reference, in at least some cases. An individual may monitor activity levels of rhomboid protease using standard methods in the art, such as western assays, for example.

(41) An individual known to have a disease such as malaria, trichomonaisis, cryptosporidiosis, toxoplasmosis, or non-viral dysentery, suspected of having such a disease, or at risk for having such a disease, may be provided an effective amount of an inhibitor of rhomboid protease, including Compound I of the present invention, preferably the compound of FIG. 4 a.

(42) In particular embodiments of the disclosure, an individual is given an agent for therapy in addition to the one or more inhibitors of rhomboid proteases. When combination therapy is employed with one or more inhibitors of rhomboid protease, the additional therapy may be given prior to, at the same time as, and/or subsequent to the inhibitor of rhomboid protease.

(43) Pharmaceutical Preparations

(44) Pharmaceutical compositions of the present invention comprise an effective amount of one or more inhibitors of rhomboid protease such as the compound of FIG. 4 a, dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases “pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that comprises at least one inhibitor of a rhomboid protease or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington: The Science and Practice of Pharmacy, 21.sup.st Ed. Lippincott Williams and Wilkins, 2005, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.

(45) The inhibitor of activity of a rhomboid protease may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The present compositions can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

(46) The inhibitor of rhomboid protease activity may be formulated into a composition in a free base, neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as formulated for parenteral administrations such as injectable solutions, or aerosols for delivery to the lungs, or formulated for alimentary administrations such as drug release capsules and the like.

(47) Further in accordance with the present disclosure, the composition of the present invention suitable for administration is provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should be assimilable and includes liquid, semi-solid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of a composition contained therein, its use in administrable composition for use in practicing the methods of the present invention is appropriate. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof. The composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof. In accordance with the present invention, the composition is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are routine for those skilled in the art.

(48) In a specific embodiment of the present invention, the composition is combined or mixed thoroughly with a semi-solid or solid carrier. The mixing can be carried out in any convenient manner such as grinding. Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers for use in an the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.

(49) In further embodiments, the present invention may concern the use of a pharmaceutical lipid vehicle compositions that include inhibitors of the activity of rhomboid protease, one or more lipids, and an aqueous solvent. As used herein, the term “lipid” will be defined to include any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds are well known to those of skill in the art, and as the term “lipid” is used herein, it is not limited to any particular structure. Examples include compounds which contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic (i.e., designed or produced by man). However, a lipid is usually a biological substance. Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that are understood by one of skill in the art as lipids are also encompassed by the compositions and methods of the present invention.

(50) One of ordinary skill in the art would be familiar with the range of techniques that can be employed for dispersing a composition in a lipid vehicle. For example, the inhibitor of the activity of rhomboid protease may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art. The dispersion may or may not result in the formation of liposomes.

(51) The actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

(52) In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

(53) In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.

(54) Kits of the Disclosure

(55) Any of the compositions described herein may be comprised in a kit. In a non-limiting example, an inhibitor of activity of rhomboid protease (for example the compound of FIG. 4a) may be comprised in a kit.

(56) The kits may comprise a suitably aliquoted inhibitor of rhomboid protease activity and, in some cases, one or more additional agents. The component(s) of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the inhibitor of rhomboid protease activity and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

(57) When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. The inhibitor of rhomboid protease activity composition(s) may be formulated into a syringeable composition. In which case, the container means may itself be a syringe, pipette, and/or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, and/or even applied to and/or mixed with the other components of the kit.

(58) However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.

EXAMPLES/METHODS

(59) The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The following Examples are offered by way of illustration and not by way of limitation.

(60) DNA Constructs

(61) All open reading frames were cloned into the pcDNA3.1 vector (Invitrogen) as N-terminal GFP-tagged/C-terminal Flag-tagged substrates or N-terminal 3×HA-tagged rhomboid proteases for expression from the pCMV promoter, exactly as described previously.sup.10. Substrate and enzyme mutants were generated by QuikChange Site-Directed Mutagenesis (Agilent Genomics) or an inverse PCR strategy. All constructs were confirmed by DNA sequencing (Genewiz).

(62) Heterologous Cell-Based Rhomboid Cleavage Assay

(63) Cleavage of substrates and the activity of enzymes was determined in a slightly modified heterologous cleavage assay. Briefly, HEK293T (ATCC CRL-11268) cells were seeded into 12-well plates, and ˜80% confluent cells were co-transfected with the appropriate substrates and/or enzymes with X-tremeGENE-HP (Roche). To identify a ‘super-substrate’, cells were transfected with reduced levels of plasmid encoding rhomboid enzyme: substrate:enzyme ˜250:1. 18-hours post transfection, cells were washed once and conditioned in serum-free DMEM (Sigma) containing the appropriate chemical compound where indicated. For cells transfected with AMA1, the metalloprotease inhibitor BB-94 (20 μM) was included to reduce spontaneous ectodomain shedding. Media containing the GFP-tagged N-terminal cleavage product and their respective cells were harvested ˜16-20-h later, re-suspension in reducing Laemmli buffer, resolved on 4-12% gradient Bis-Tris gels in MES running buffer (Life Technologies), and transferred onto nitrocellulose membranes with a Trans-Blot (BioRad) semi-dry system. Membranes were probed with anti-GFP (AbCam), anti-HA (Roche) primary antibodies, anti-rabbit/rat secondary antibodies conjugated to infrared fluorophores (Li-COR Biosciences) and imaged on an Odyssey infrared laser scanner (Li-COR Biosciences). Two-color quantitative westerns were converted to greyscale.

(64) Cleavage Site Determination

(65) To determine the cleavage site of rhomboid substrates, HEK293T cells were transfected as described above, lysed in RIPA buffer, subjected to anti-FLAG immunopurification (Sigma). Eluent was spotted onto a sinapinic acid matrix analyzed by MALDI-TOF mass spectrometry on a standards calibrated Voyager DE Instrument (AB SCIEX) as previously described. Resultant spectra were analyzed and plotted in the R environment with aid of the Maldiquant package.

(66) Crystallization, Data Collection and Refinement

(67) Crystals of E. coli GlpG consisting of residues 87-276 (DN-GlpG) were prepared as described previously. Briefly, purified ΔN-GlpG was concentrated to 5 mg/ml in a buffer of 25 mM Tris (pH 8.0), 250 mM NaCl, and 0.2% (w/v) nonyl-glycoside, and crystallized by hanging-drop method over a reservoir buffer of 0.1M Tris pH 8.5, 3M NaNO.sub.3, and 15% glycerol at room temperature. For soaking experiments with Ac-VRMA-CMK (SEQ ID NO: 2) and Ac-VRMA-B(OH).sub.2 (SEQ ID NO: 2), harvested crystals were transferred twice to a fresh drop consisting of 25 mM Tris (pH 7.0), 2.5 M NaCl, 0.2% nonyl-glycoside, and 15% glycerol for an hour. Peptide aldehydes and boronates were commercially custom synthesized using solid-phase chemistry, purified to >90% purity by reverse-phase high-performance liquid chromatography, and verified by ESI mass spectrometry. The peptide inhibitors were dissolved in 2.5 M NaCl to a concentration of 20 mM and added to the crystals to a final soaking concentration of 5 mM. After 5 hrs of soaking, crystals were flash-frozen in a nitrogen stream and diffraction data were collected on beamline F1 of the Cornell High Energy Synchrotron Source. Data was processed with iMosflm 7.1.1 and structures were determined by molecular replacement with Molrep in CCP4 using an apo-structure (PDB ID 2IC8) as the model. The solutions of molecular replacement clearly showed inhibitors directly connected with catalytic S201 in electron density maps, and were modeled as the peptide-CMK and peptide-boronate after several iterative rounds of model building using COOT and refinement using Refmac5. Structures were further refined using refmac5 and PHENIX, and R/R.sub.free values of the final models were 0.217/0.258 and 0.215/0.242 at 2.3 Å with good geometry. Crystallographic data and refinement statistics are summarized in Supplementary Table 1.

(68) Fitting and Statistical Analysis of Dose-Response Curves

(69) Dose-response curves were analyzed in the R environment. Curve fitting and graphing was performed with the DRC package using data appropriate models. EC.sub.50 values are depicted along with the standard error of the fit.

(70) P. falciparum Culture and Invasion Assays

(71) Asexual P. falciparum 3d7 parasites (kind gift of Dr David Sullivan, Johns Hopkins School of Public Health) were cultured in human erythrocytes (O-donor, purchased from Interstate Blood Bank, Inc.) under standard conditions. Sorbitol synchronization was performed ˜1/week or as needed for experiments. For invasion assays, schizont-stage parasites were magnetically purified as previously described (MACS Miltenyi Biotec) and incubated at the appropriate parasitemia (0.2% for dose-response curves) in a 96-well plate in the presence or absence of the peptide-warhead inhibitors at 1.5% hematocrit. Flow cytometry analysis for absolute parasitemia determination and growth characterization was performed by removing a small volume of culture, washing cells in 1×PBS, resuspending them in 1×PBS containing 0.1 μg/mL acridine orange and monitoring FL1/FL3 fluorescence on a FACSCalibur instrument (BD Biosciences). Methanol-fixed, acridine orange stained parasitized erythrocytes were imaged on an inverted fluorescence microscope (Nikon) with bandpass filters. Images were overlaid in Photoshop CC (Adobe). All flow cytometry data were analyzed in FlowJo.

(72) Analysis of RiBn Effects on Human Cells

(73) Cell viability of RiBn-treated HEK293 cells was determined with a LIVE/DEAD Viability/Cytotoxicity Kit (Molecular Probes). Calcein AM and ethidium homodimer-1 stained cells were imaged on an inverted fluorescence microscope (Nikon) with the appropriate bandpass filters. Flow cytometry on the same samples was performed with a FACSCalibur instrument (BD Biosciences). For cell cycle distribution determination, RiBn+/− treated HEK293 cells growing in serum were washed in 1×PBS, fixed in ice-cold 70% ethanol, RNase A treated and stained with 50 μg/mL propidium iodine. Flow cytometry was performed as above by monitoring the FL2/FL3 channels.

(74) All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

(75) The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

(76) Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.