Comparative chemical genomics in Babesia species identifies the alkaline phosphatase PhoD as a determinant of antiparasitic resistance.

Babesiosis is an emerging zoonosis and widely distributed veterinary infection caused by 100+ species of Babesia parasites. The diversity of Babesia parasites and the lack of specific drugs necessitate the discovery of broadly effective antibabesials. Here, we describe a comparative chemogenomics (CCG) pipeline for the identification of conserved targets. CCG relies on parallel in vitro evolution of resistance in independent populations of Babesia spp. (B. bovis and B. divergens). We identified a potent antibabesial, MMV019266, from the Malaria Box, and selected for resistance in two species of Babesia. After sequencing of multiple independently derived lines in the two species, we identified mutations in a membrane-bound metallodependent phosphatase (phoD). In both species, the mutations were found in the phoD-like phosphatase domain. Using reverse genetics, we validated that mutations in bdphoD confer resistance to MMV019266 in B. divergens. We have also demonstrated that BdPhoD localizes to the endomembrane system and partially with the apicoplast. Finally, conditional knockdown and constitutive overexpression of BdPhoD alter the sensitivity to MMV019266 in the parasite. Overexpression of BdPhoD results in increased sensitivity to the compound, while knockdown increases resistance, suggesting BdPhoD is a pro-susceptibility factor. Together, we have generated a robust pipeline for identification of resistance loci and identified BdPhoD as a resistance mechanism in Babesia species.

The anti-cancer efficacy of a novel phenothiazine derivative is independent of dopamine and serotonin receptor inhibition.

Introduction: An attractive, yet unrealized, goal in cancer therapy is repurposing psychiatric drugs that can readily penetrate the blood-brain barrier for the treatment of primary brain tumors and brain metastases. Phenothiazines (PTZs) have demonstrated anti-cancer properties through a variety of mechanisms. However, it remains unclear whether these effects are entirely separate from their activity as dopamine and serotonin receptor (DR/5-HTR) antagonists. Methods: In this study, we evaluated the anti-cancer efficacy of a novel PTZ analog, CWHM-974, that was shown to be 100-1000-fold less potent against DR/5-HTR than its analog fluphenazine (FLU). Results: CWHM-974 was more potent than FLU against a panel of cancer cell lines, thus clearly demonstrating that its anti-cancer effects were independent of DR/5-HTR signaling. Our results further suggested that calmodulin (CaM) binding may be necessary, but not sufficient, to explain the anti-cancer effects of CWHM-974. While both FLU and CWHM-974 induced apoptosis, they induced distinct effects on the cell cycle (G0/G1 and mitotic arrest respectively) suggesting that they may have differential effects on CaM-binding proteins involved in cell cycle regulation. Discussion: Altogether, our findings indicated that the anti-cancer efficacy of the CWHM-974 is separable from DR/5-HTR antagonism. Thus, reducing the toxicity associated with phenothiazines related to DR/5-HTR antagonism may improve the potential to repurpose this class of drugs to treat brain tumors and/or brain metastasis.

CWHM-974 is a fluphenazine derivative with improved antifungal activity against Candida albicans due to reduced susceptibility to multidrug transporter-mediated resistance mechanisms.

Multidrug resistance (MDR) transporters such as ATP-Binding Cassette (ABC) and Major Facilitator Superfamily proteins are important mediators of antifungal drug resistance, particularly with respect to azole class drugs. Consequently, identifying molecules that are not susceptible to this mechanism of resistance is an important goal for new antifungal drug discovery. As part of a project to optimize the antifungal activity of clinically used phenothiazines, we synthesized a fluphenazine derivative (CWHM-974) with 8-fold higher activity against Candida spp. compared to the fluphenazine and with activity against Candida spp. with reduced fluconazole susceptibility due to increased MDR transporters. Here, we show that the improved C. albicans activity is because fluphenazine induces its own resistance by triggering expression of Candida drug resistance (CDR) transporters while CWHM-974 induces expression but does not appear to be a substrate for the transporters or is insensitive to their effects through other mechanisms. We also found that fluphenazine and CWHM-974 are antagonistic with fluconazole in C. albicans but not in C. glabrata, despite inducing CDR1 expression to high levels. Overall, CWHM-974 is one of the few examples of a molecule in which relatively small structural modifications significantly reduced susceptibility to multidrug transporter-mediated resistance.

Comparative chemical genomics in Babesia species identifies the alkaline phosphatase phoD as a novel determinant of resistance.

Babesiosis is an emerging zoonosis and widely distributed veterinary infection caused by 100+ species of Babesia parasites. The diversity of Babesia parasites, coupled with the lack of potent inhibitors necessitates the discovery of novel conserved druggable targets for the generation of broadly effective antibabesials. Here, we describe a comparative chemogenomics (CCG) pipeline for the identification of novel and conserved targets. CCG relies on parallel in vitro evolution of resistance in independent populations of evolutionarily-related Babesia spp. ( B. bovis and B. divergens ). We identified a potent antibabesial inhibitor from the Malaria Box, MMV019266. We were able to select for resistance to this compound in two species of Babesia, achieving 10-fold or greater resistance after ten weeks of intermittent selection. After sequencing of multiple independently derived lines in the two species, we identified mutations in a single conserved gene in both species: a membrane-bound metallodependent phosphatase (putatively named PhoD). In both species, the mutations were found in the phoD-like phosphatase domain, proximal to the predicted ligand binding site. Using reverse genetics, we validated that mutations in PhoD confer resistance to MMV019266. We have also demonstrated that PhoD localizes to the endomembrane system and partially with the apicoplast. Finally, conditional knockdown and constitutive overexpression of PhoD alter the sensitivity to MMV019266 in the parasite: overexpression of PhoD results in increased sensitivity to the compound, while knockdown increases resistance, suggesting PhoD is a resistance mechanism. Together, we have generated a robust pipeline for identification of resistance loci, and identified PhoD as a novel determinant of resistance in Babesia species. Highlights: Use of two species for in vitro evolution identifies a high confidence locus associated with resistance Resistance mutation in phoD was validated using reverse genetics in B. divergens Perturbation of phoD using function genetics results in changes in the level of resistance to MMV019266Epitope tagging reveals localization to the ER/apicoplast, a conserved localization with a similar protein in diatoms Together, phoD is a novel resistance determinant in multiple Babesia spp .

Structure-Activity Relationship Studies of the Aryl Acetamide Triazolopyridazines against Cryptosporidium Reveals Remarkable Role of Fluorine.

Our previous work identified compound 1 (SLU-2633) as a potent lead compound toward the identification of a novel treatment for cryptosporidiosis, caused by the parasite Cryptosporidium (EC50 = 0.17 µM). While this compound is potent and orally efficacious, the mechanism of action and biological target(s) of this series are currently unknown. In this study, we synthesized 70 compounds to develop phenotypic structure-activity relationships around the aryl "tail" group. In this process, we found that 2-substituted compounds are inactive, confirmed that electron withdrawing groups are preferred over electron donating groups, and that fluorine plays a remarkable role in the potency of these compounds. The most potent compound resulting from this work is SLU-10482 (52, EC50 = 0.07 µΜ), which was found to be orally efficacious with an ED90 < 5 mg/kg BID in a Cryptosporidium-infection mouse model, superior to SLU-2633.

MEPicides: α,ß-unsaturated Fosmidomycin N-Acyl Analogs as Efficient Inhibitors of Plasmodium falciparum 1-Deoxy-d-xylulose-5-phosphate reductoisomerase.

Malaria, a mosquito-borne disease caused by several parasites of the Plasmodium genus, remains a huge threat to global public health. There are an estimated 0.5 million malaria deaths each year, mostly among African children. Unlike humans, Plasmodium parasites and a number of important pathogenic bacteria employ the methyl erythritol phosphate (MEP) pathway for isoprenoid synthesis. Thus, the MEP pathway represents a promising set of drug targets for antimalarial and antibacterial compounds. Here, we present new unsaturated MEPicide inhibitors of 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), the second enzyme of the MEP pathway. A number of these compounds have demonstrated robust inhibition of Plasmodium falciparum DXR, potent antiparasitic activity, and low cytotoxicity against HepG2 cells. Parasites treated with active compounds are rescued by isopentenyl pyrophosphate, the product of the MEP pathway. With higher levels of DXR substrate, parasites acquire resistance to active compounds. These results further confirm the on-target inhibition of DXR in parasites by the inhibitors. Stability in mouse liver microsomes is high for the phosphonate salts, but remains a challenge for the prodrugs. Taken together, the potent activity and on-target mechanism of action of this series further validate DXR as an antimalarial drug target and the α,ß-unsaturation moiety as an important structural component.

Structure-Activity relationships of replacements for the triazolopyridazine of Anti-Cryptosporidium lead SLU-2633.

Cryptosporidiosis is a diarrheal disease particularly harmful to children and immunocompromised people. Infection is caused by the parasite Cryptosporidium and leads to dehydration, malnutrition, and death in severe cases. Nitazoxanide is the only FDA approved drug but is only modestly effective in children and ineffective in immunocompromised patients. To address this unmet medical need, we previously identified triazolopyridazine SLU-2633 as potent against Cryptosporidium parvum, with an EC50 of 0.17 µM. In the present study, we develop structure-activity relationships (SAR) for the replacement of the triazolopyridazine head group by exploring different heteroaryl groups with the aim of maintaining potency while reducing affinity for the hERG channel. 64 new analogs of SLU-2633 were synthesized and assayed for potency versus C. parvum. The most potent compound, 7,8-dihydro-[1,2,4]triazolo[4,3-b]pyridazine 17a, was found to have a Cp EC50 of 1.2 µM, 7-fold less potent than SLU-2633 but has an improved lipophilic efficiency (LipE) score. 17a was found to decrease inhibition in an hERG patch-clamp assay by about two-fold relative to SLU-2633 at 10 µM despite having similar inhibition in a [3H]-dofetilide competitive binding assay. While most other heterocycles were significantly less potent than the lead, some analogs such as azabenzothiazole 31b, have promising potency in the low micromolar range, similar to the drug nitazoxanide, and represent potential new leads for optimization. Overall, this work highlights the important role of the terminal heterocyclic head group and represents a significant extension of the understanding of the SAR for this class of anti-Cryptosporidium compounds.

CWHM-974 is a fluphenazine derivative with improved antifungal activity against Candida albicans due to reduced susceptibility to multidrug transporter-mediated resistance mechanisms.

Multidrug resistance (MDR) transporters such as ATP Binding Cassette (ABC) and Major Facilitator Superfamily (MFS) proteins are important mediators of antifungal drug resistance, particularly with respect to azole class drugs. Consequently, identifying molecules that are not susceptible to this mechanism of resistance is an important goal for new antifungal drug discovery. As part of a project to optimize the antifungal activity of clinically used phenothiazines, we synthesized a fluphenazine derivative (CWHM-974) with 8-fold higher activity against Candida spp. compared to the fluphenazine and with activity against Candida spp. with reduced fluconazole susceptibility due to increased multidrug resistance transporters. Here, we show that the improved C. albicans activity is because fluphenazine induces its own resistance by triggering expression of CDR transporters while CWHM-974 induces expression but does not appear to be a substrate for the transporters or is insensitive to their effects through other mechanisms. We also found that fluphenazine and CWHM-974 are antagonistic with fluconazole in C. albicans but not in C. glabrata , despite inducing CDR1 expression to high levels. Overall, CWHM-974 represents a unique example of a medicinal chemistry-based conversion of chemical scaffold from MDR-sensitive to MDR-resistant and, hence, active against fungi that have developed resistance to clinically used antifungals such as the azoles.

Synthetic derivatives of the antifungal drug ciclopirox are active against herpes simplex virus 2.

We previously showed that the anti-fungal drug ciclopirox olamine effectively inhibits replication of herpes simplex virus (HSV)-1 and HSV-2. Given the rise of HSV strains that are resistant to nucleos(t)ide analog treatment, as well as the incomplete efficacy of nucleos(t)ide analogs, new inhibitory compounds must be explored for potential use in the treatment of HSV infection. In the present study, we analyzed 44 compounds derived from the core structure of ciclopirox olamine for inhibitory activity against HSV. Thirteen of these derivative compounds inhibited HSV-2 replication by > 1000- to ∼100,000-fold at 1 µM and displayed EC50 values lower than that of acyclovir, as well as low cytotoxicity, indicating their strong therapeutic potential. Through structural comparison, we also provide evidence for the importance of various structural motifs to the efficacy of ciclopirox and its derivatives, namely hydrophobic groups at R4 and R6 of the ciclopirox core structure. Like ciclopirox, representative analogs exhibit some oral bioavailability but are rapidly cleared in vivo. Together, these results will guide further development of N-hydroxypyridones as HSV therapeutics.

Repurposing and optimization of drugs for discovery of novel antifungals.

Although fungal diseases are a major and growing public health concern, there are only four major classes of drug to treat primary fungal pathogens. The pipeline of new antifungals in clinical development is relatively thin compared with other disease classes. One approach to rapidly identify and provide novel treatment options is to repurpose existing drugs as antifungals. However, such proposed drug-repurposing candidates often suffer suboptimal efficacy and pharmacokinetics (PK) for fungal diseases. Herein, we briefly review the current antifungal drug pipeline and recent approaches to optimize existing drugs into novel molecules with unique modes of action relative to existing antifungal drug classes.

Mechanism of Action of N-Acyl and N-Alkoxy Fosmidomycin Analogs: Mono- and Bisubstrate Inhibition of IspC from Plasmodium falciparum, a Causative Agent of Malaria.

Malaria is a global health threat that requires immediate attention. Malaria is caused by the protozoan parasite Plasmodium, the most severe form of which is Plasmodium falciparum. The methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis is essential to the survival of many human pathogens, including P. falciparum, but is absent in humans, and thus shows promise as a new antimalarial drug target. The enzyme 1-deoxy-d-xylulose 5-phosphate reductoisomerase (IspC) catalyzes the first committed step in the MEP pathway. In addition to a divalent cation (Mg2+), the enzyme requires the substrates 1-deoxy-D-xylulose 5-phosphate (DXP) and NADPH to catalyze its reaction. We designed N-alkoxy and N-acyl fosmidomycin analogs to inhibit the activity of P. falciparum IspC in a bisubstrate manner. Enzyme assays reveal that the N-alkoxy fosmidomycin analogs have a competitive mode of inhibition relative to both the DXP- and NADPH-binding sites, confirming a bisubstrate mode of inhibition. In contrast, the N-acyl fosmidomycin analogs demonstrate competitive inhibition with respect to DXP but uncompetitive inhibition with respect to NADPH, indicating monosubstrate inhibitory activity. Our results will have a positive impact on the discovery of novel antimalarial drugs.

Efficient Inhibition of Hepatitis B Virus (HBV) Replication and cccDNA Formation by HBV Ribonuclease H Inhibitors during Infection.

The hepatitis B virus (HBV) ribonuclease H (RNase H) is an attractive but unexploited drug target. Here, we addressed three limitations to the current state of RNase H inhibitor development: (a) Efficacy has been assessed only in transfected cell lines. (b) Cytotoxicity data are from transformed cell lines rather than primary cells. (c) It is unknown how the compounds work against nucleos(t)ide analog resistant HBV strains. Three RNase H inhibitors from different chemotypes, 110 (α-hydroxytropolone), 1133 (N-hydroxypyridinedione), and 1073 (N-hydroxynapthyridinone), were tested in HBV-infected HepG2-NTCP cells for inhibition of cccDNA accumulation and HBV product formation. 50% effective concentrations (EC50s) were 0.049-0.078 µM in the infection studies compared to 0.29-1.6 µM in transfected cells. All compounds suppressed cccDNA formation by >98% at 5 µM when added shortly after infection. HBV RNA, intracellular and extracellular DNA, and HBsAg secretion were all robustly suppressed. The greater efficacy of the inhibitors when added shortly after infection is presumably due to blocking amplification of the HBV cccDNA, which suppresses events downstream of cccDNA formation. The compounds had 50% cytotoxic concentrations (CC50s) of 16-100 µM in HepG2-derived cell lines but were nontoxic in primary human hepatocytes, possibly due to the quiescent state of the hepatocytes. The compounds had similar EC50s against replication of wild-type, lamivudine-resistant, and adefovir/lamivudine-resistant HBV, as expected because the RNase H inhibitors do not target the viral reverse transcriptase active site. These studies expand confidence in inhibiting the HBV RNase H as a drug strategy and support inclusion of RNase H inhibitors in novel curative drug combinations for HBV.

Optimization of the Urea Linker of Triazolopyridazine MMV665917 Results in a New Anticryptosporidial Lead with Improved Potency and Predicted hERG Safety Margin.

Cryptosporidiosis is caused by infection of the small intestine by Cryptosporidium parasites, resulting in severe diarrhea, dehydration, malabsorption, and potentially death. The only FDA-approved therapeutic is only partially effective in young children and ineffective for immunocompromised patients. Triazolopyridazine MMV665917 is a previously reported anti-Cryptosporidium screening hit with in vivo efficacy but suffers from modest inhibition of the hERG ion channel, which could portend cardiotoxicity. Herein, we describe our initial development of structure-activity relationships of this novel lead series with a particular focus on optimization of the piperazine-urea linker. We have discovered that piperazine-acetamide is a superior linker resulting in identification of SLU-2633, which has an EC50 of 0.17 µM, an improved projected margin versus hERG, prolonged pharmacokinetic exposure in small intestine, and oral efficacy in vivo with minimal systemic exposure. SLU-2633 represents a significant advancement toward the identification of a new effective and safe treatment for cryptosporidiosis.

Synthetic Derivatives of Ciclopirox are Effective Inhibitors of Cryptococcus neoformans.

Opportunistic fungal infections caused by Cryptococcus neoformans are a significant source of mortality in immunocompromised patients. They are challenging to treat because of a limited number of antifungal drugs, and novel and more effective anticryptococcal therapies are needed. Ciclopirox olamine, a N-hydroxypyridone, has been in use as an approved therapeutic agent for the treatment of topical fungal infections for more than two decades. It is a fungicide, with broad activity across multiple fungal species. We synthesized 10 N-hydroxypyridone derivatives to develop an initial structure-activity understanding relative to efficacy as a starting point for the development of systemic antifungals. We screened the derivatives for antifungal activity against C. neoformans and Cryptococcus gattii and counter-screened for specificity in Candida albicans and two Malassezia species. Eight of the ten show inhibition at 1-3 µM concentration (0.17-0.42 µg per mL) in both Cryptococcus species and in C. albicans, but poor activity in the Malassezia species. In C. neoformans, the N-hydroxypyridones are fungicides, are not antagonistic with either fluconazole or amphotericin B, and are synergistic with multiple inhibitors of the mitochondrial electron transport chain. They appear to function primarily by chelating iron within the active site of iron-dependent enzymes. This preliminary structure-activity relationship points to the need for a lipophilic functional group at position six of the N-hydroxypyridone ring and identifies positions four and six as sites where further substitution may be tolerated. These molecules provide a clear starting point for future optimization for efficacy and target identification.

Identification of 4-Amino-Thieno[2,3-d]Pyrimidines as QcrB Inhibitors in Mycobacterium tuberculosis.

Antibiotic resistance is a global crisis that threatens our ability to treat bacterial infections, such as tuberculosis, caused by Mycobacterium tuberculosis Of the 10 million cases of tuberculosis in 2017, approximately 19% of new cases and 43% of previously treated cases were caused by strains of M. tuberculosis resistant to at least one frontline antibiotic. There is a clear need for new therapies that target these genetically resistant strains. Here, we report the discovery of a new series of antimycobacterial compounds, 4-amino-thieno[2,3-d]pyrimidines, that potently inhibit the growth of M. tuberculosis To elucidate the mechanism by which these compounds inhibit M. tuberculosis, we selected for mutants resistant to a representative 4-amino-thieno[2,3-d]pyrimidine and sequenced these strains to identify the mutations that confer resistance. We isolated a total of 12 resistant mutants, each of which harbored a nonsynonymous mutation in the gene qcrB, which encodes a subunit of the electron transport chain (ETC) enzyme cytochrome bc1 oxidoreductase, leading us to hypothesize that 4-amino-thieno[2,3-d]pyrimidines target this enzyme complex. We found that addition of 4-amino-thieno[2,3-d]pyrimidines to M. tuberculosis cultures resulted in a decrease in ATP levels, supporting our model that these compounds inhibit the M. tuberculosis ETC. Furthermore, 4-amino-thieno[2,3-d]pyrimidines had enhanced activity against a mutant of M. tuberculosis deficient in cytochrome bd oxidase, which is a hallmark of cytochrome bc1 inhibitors. Therefore, 4-amino-thieno[2,3-d]pyrimidines represent a novel series of QcrB inhibitors that build on the growing number of chemical scaffolds that are able to inhibit the mycobacterial cytochrome bc1 complex.IMPORTANCE The global tuberculosis (TB) epidemic has been exacerbated by the rise in drug-resistant TB cases worldwide. To tackle this crisis, it is necessary to identify new vulnerable drug targets in Mycobacterium tuberculosis, the causative agent of TB, and develop compounds that can inhibit the bacterium through novel mechanisms of action. The QcrB subunit of the electron transport chain enzyme cytochrome bc1 has recently been validated to be a potential drug target. In the current work, we report the discovery of a new class of QcrB inhibitors, 4-amino-thieno[2,3-d]pyrimidines, that potently inhibit M. tuberculosis growth in vitro These compounds are chemically distinct from previously reported QcrB inhibitors, and therefore, 4-amino-thieno[2,3-d]pyrimidines represent a new scaffold that can be exploited to inhibit this drug target.

Clinically Advanced p38 Inhibitors Suppress DUX4 Expression in Cellular and Animal Models of Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is characterized by misexpression of the double homeobox 4 (DUX4) developmental transcription factor in mature skeletal muscle, where it is responsible for muscle degeneration. Preventing expression of DUX4 mRNA is a disease-modifying therapeutic strategy with the potential to halt or reverse the course of disease. We previously reported that agonists of the ß-2 adrenergic receptor suppress DUX4 expression by activating adenylate cyclase to increase cAMP levels. Efforts to further explore this signaling pathway led to the identification of p38 mitogen-activated protein kinase as a major regulator of DUX4 expression. In vitro experiments demonstrate that clinically advanced p38 inhibitors suppress DUX4 expression in FSHD type 1 and 2 myoblasts and differentiating myocytes in vitro with exquisite potency. Individual small interfering RNA-mediated knockdown of either p38α or p38ß suppresses DUX4 expression, demonstrating that each kinase isoform plays a distinct requisite role in activating DUX4 Finally, p38 inhibitors effectively suppress DUX4 expression in a mouse xenograft model of human FSHD gene regulation. These data support the repurposing of existing clinical p38 inhibitors as potential therapeutics for FSHD. The surprise finding that p38α and p38ß isoforms each independently contribute to DUX4 expression offers a unique opportunity to explore the utility of p38 isoform-selective inhibitors to balance efficacy and safety in skeletal muscle. We propose p38 inhibition as a disease-modifying therapeutic strategy for FSHD. SIGNIFICANCE STATEMENT: Facioscapulohumeral muscular dystrophy (FSHD) currently has no treatment options. This work provides evidence that repurposing a clinically advanced p38 inhibitor may provide the first disease-modifying drug for FSHD by suppressing toxic DUX4 expression, the root cause of muscle degeneration in this disease.

4-Aryl Pyrrolidines as Novel Orally Efficacious Antimalarial Agents. Part 2: 2-Aryl-N-(4-arylpyrrolidin-3-yl)acetamides.

Malaria is caused by infection from the Plasmodium parasite and kills hundreds of thousands of people every year. Emergence of new drug resistant strains of Plasmodium demands identification of new drugs with novel chemotypes and mechanisms of action. As a follow up to our evaluation of 4-aryl-N-benzylpyrrolidine-3-carboxamides as novel pyrrolidine-based antimalarial agents, we describe herein the structure-activity relationships of the reversed amide homologues 2-aryl-N-(4-arylpyrrolidin-3-yl)acetamides. Unlike their carboxamide homologues, acetamide pyrrolidines do not require a third chiral center to be potent inhibitors of P. falciparum and have good pharmacokinetic properties and improved oral efficacy in a mouse model of malaria. Compound (-)-32a (CWHM-1552) has an in vitro IC50 of 51 nM in the P. falciparum 3D7 assay and an in vivo ED90 of <10 mg/kg/day and ED99 of 30 mg/kg/day in a murine P. chabaudi model. Remarkably, the absolute stereochemical preference for this acetamide series (3S,4R) is opposite of that determined for the homologous carboxamide series. Lead compounds for this class have modest affinities for the hERG channel and inhibit CYP 3A4. Additional optimization is needed in order to eliminate these undesired properties from this otherwise promising series of antimalarial compounds.

Targeting VLA4 integrin and CXCR2 mobilizes serially repopulating hematopoietic stem cells.

Mobilized peripheral blood has become the primary source of hematopoietic stem and progenitor cells (HSPCs) for stem cell transplantation, with a five-day course of granulocyte colony stimulating factor (G-CSF) as the most common regimen used for HSPC mobilization. The CXCR4 inhibitor, plerixafor, is a more rapid mobilizer, yet not potent enough when used as a single agent, thus emphasizing the need for faster acting agents with more predictable mobilization responses and fewer side effects. We sought to improve hematopoietic stem cell transplantation by developing a new mobilization strategy in mice through combined targeting of the chemokine receptor CXCR2 and the very late antigen 4 (VLA4) integrin. Rapid and synergistic mobilization of HSPCs along with an enhanced recruitment of true HSCs was achieved when a CXCR2 agonist was co-administered in conjunction with a VLA4 inhibitor. Mechanistic studies revealed involvement of CXCR2 expressed on BM stroma in addition to stimulation of the receptor on granulocytes in the regulation of HSPC localization and egress. Given the rapid kinetics and potency of HSPC mobilization provided by the VLA4 inhibitor and CXCR2 agonist combination in mice compared to currently approved HSPC mobilization methods, it represents an exciting potential strategy for clinical development in the future.

4-Aryl Pyrrolidines as a Novel Class of Orally Efficacious Antimalarial Agents. Part 1: Evaluation of 4-Aryl- N-benzylpyrrolidine-3-carboxamides.

Identification of novel chemotypes with antimalarial efficacy is imperative to combat the rise of Plasmodium species resistant to current antimalarial drugs. We have used a hybrid target-phenotype approach to identify and evaluate novel chemotypes for malaria. In our search for drug-like aspartic protease inhibitors in publicly available phenotypic antimalarial databases, we identified GNF-Pf-4691, a 4-aryl- N-benzylpyrrolidine-3-carboxamide, as having a structure reminiscent of known inhibitors of aspartic proteases. Extensive profiling of the two terminal aryl rings revealed a structure-activity relationship in which relatively few substituents are tolerated at the benzylic position, but the 3-aryl position tolerates a range of hydrophobic groups and some heterocycles. Out of this effort, we identified (+)-54b (CWHM-1008) as a lead compound. 54b has EC50 values of 46 and 21 nM against drug-sensitive Plasmodium falciparum 3D7 and drug-resistant Dd2 strains, respectively. Furthermore, 54b has a long half-life in mice (4.4 h) and is orally efficacious in a mouse model of malaria (qd; ED99 ∼ 30 mg/kg/day). Thus, the 4-aryl- N-benzylpyrrolidine-3-carboxamide chemotype is a promising novel chemotype for malaria drug discovery.