A Combined Approach Reveals a Regulatory Mechanism Coupling Src's Kinase Activity, Localization, and Phosphotransferase-Independent Functions.

Multiple layers of regulation modulate the activity and localization of protein kinases. However, many details of kinase regulation remain incompletely understood. Here, we apply saturation mutagenesis and a chemical genetic method for allosterically modulating kinase global conformation to Src kinase, providing insight into known regulatory mechanisms and revealing a previously undiscovered interaction between Src's SH4 and catalytic domains. Abrogation of this interaction increased phosphotransferase activity, promoted membrane association, and provoked phosphotransferase-independent alterations in cell morphology. Thus, Src's SH4 domain serves as an intramolecular regulator coupling catalytic activity, global conformation, and localization, as well as mediating a phosphotransferase-independent function. Sequence conservation suggests that the SH4 domain regulatory interaction exists in other Src-family kinases. Our combined approach's ability to reveal a regulatory mechanism in one of the best-studied kinases suggests that it could be applied broadly to provide insight into kinase structure, regulation, and function.

Toxoplasma Calcium-Dependent Protein Kinase 1 Inhibitors: Probing Activity and Resistance Using Cellular Thermal Shift Assays.

In Toxoplasma gondii, calcium-dependent protein kinase 1 (CDPK1) is an essential protein kinase required for invasion of host cells. We have developed several hundred CDPK1 inhibitors, many of which block invasion. Inhibitors with similar 50% inhibitory concentrations (IC50s) were tested in thermal shift assays for their ability to stabilize CDPK1 in cell lysates, in intact cells, or in purified form. Compounds that inhibited parasite growth stabilized CDPK1 in all assays. In contrast, two compounds that showed poor growth inhibition stabilized CDPK1 in lysates but not in cells. Thus, cellular exclusion could explain exceptions in the correlation between the action on the target and cellular activity. We used thermal shift assays to examine CDPK1 in two clones that were independently selected by growth in the CDPK1 inhibitor RM-1-132 and that had increased 50% effective concentrations (EC50s) for the compound. The A and C clones had distinct point mutations in the CDPK1 kinase domain, H201Q and L96P, respectively, residues that lie near one another in the inactive isoform. Purified mutant proteins showed RM-1-132 IC50s and thermal shifts similar to those shown by wild-type CDPK1. Reduced inhibitor stabilization (and a presumed reduced interaction) was observed only in cellular thermal shift assays. This highlights the utility of cellular thermal shift assays in demonstrating that resistance involves reduced on-target engagement (even if biochemical assays suggest otherwise). Indeed, similar EC50s were observed upon overexpression of the mutant proteins, as in the corresponding drug-selected parasites, although high levels of CDPK1(H201Q) only modestly increased resistance compared to that achieved with high levels of wild-type enzyme.

Kinobead and Single-Shot LC-MS Profiling Identifies Selective PKD Inhibitors.

ATP-competitive protein kinase inhibitors are important research tools and therapeutic agents. Because there are >500 human kinases that contain highly conserved active sites, the development of selective inhibitors is extremely challenging. Methods to rapidly and efficiently profile kinase inhibitor targets in cell lysates are urgently needed to discover selective compounds and to elucidate the mechanisms of action for polypharmacological inhibitors. Here, we describe a protocol for microgram-scale chemoproteomic profiling of ATP-competitive kinase inhibitors using kinobeads. We employed a gel-free in situ digestion protocol coupled to nanoflow liquid chromatography-mass spectrometry to profile ∼200 kinases in single analytical runs using as little as 5 µL of kinobeads and 300 µg of protein. With our kinobead reagents, we obtained broad coverage of the kinome, monitoring the relative expression levels of 312 kinases in a diverse panel of 11 cancer cell lines. Further, we profiled a set of pyrrolopyrimidine- and pyrazolopyrimidine-based kinase inhibitors in competition-binding experiments with label-free quantification, leading to the discovery of a novel selective and potent inhibitor of protein kinase D (PKD) 1, 2, and 3. Our protocol is useful for rapid and sensitive profiling of kinase expression levels and ATP-competitive kinase inhibitor selectivity in native proteomes.

5-Aminopyrazole-4-Carboxamide-Based Compounds Prevent the Growth of Cryptosporidium parvum.

Cryptosporidium parvum calcium-dependent protein kinase 1 (CpCDPK1) is a promising target for drug development against cryptosporidiosis. We report a series of low-nanomolar CpCDPK1 5-aminopyrazole-4-carboxamide (AC) scaffold inhibitors that also potently inhibit C. parvum growth in vitro Correlation between anti-CpCDPK1 and C. parvum growth inhibition, as previously reported for pyrazolopyrimidines, was not apparent. Nonetheless, lead AC compounds exhibited a substantial reduction of parasite burden in the neonatal mouse cryptosporidiosis model when dosed at 25 mg/kg.

5-Aminopyrazole-4-carboxamide analogues are selective inhibitors of Plasmodium falciparum microgametocyte exflagellation and potential malaria transmission blocking agents.

Plasmodium falciparum calcium-dependent protein kinase 4 (PfCDPK4) is essential for the exflagellation of male gametocytes. Inhibition of PfCDPK4 is an effective way of blocking the transmission of malaria by mosquitoes. A series of 5-aminopyrazole-4-carboxamide analogues are demonstrated to be potent inhibitors of PfCDPK4. The compounds are also able to block exflagellation of Plasmodium falciparum male gametocytes without observable toxicity to mammalian cells.

Comparative assessment of the effects of bumped kinase inhibitors on early zebrafish embryo development and pregnancy in mice.

Bumped kinase inhibitors (BKIs) are effective against a variety of apicomplexan parasites. Fifteen BKIs with promising in vitro efficacy against Neospora caninum tachyzoites, low cytotoxicity in mammalian cells, and no toxic effects in non-pregnant BALB/c mice were assessed in pregnant mice. Drugs were emulsified in corn oil and were applied by gavage for 5 days. Five BKIs did not affect pregnancy, five BKIs exhibited ~15-35% neonatal mortality and five compounds caused strong effects (infertility, abortion, stillbirth and pup mortality). Additionally, the impact of these compounds on zebrafish (Danio rerio) embryo development was assessed by exposing freshly fertilised eggs to 0.2-50 µM of BKIs and microscopic monitoring of embryo development in a blinded manner for 4 days. We propose an algorithm that includes quantification of malformations and embryo deaths, and established a scoring system that allows the calculation of an impact score (Si) indicating at which concentrations BKIs visibly affect zebrafish embryo development. Comparison of the two models showed that for nine compounds no clear correlation between Si and pregnancy outcome was observed. However, the three BKIs affecting zebrafish embryos only at high concentrations (≥40 µM) did not impair mouse pregnancy at all, and the three compounds that inhibited zebrafish embryo development already at 0.2 µM showed detrimental effects in the pregnancy model. Thus, the zebrafish embryo development test has limited predictive value to foresee pregnancy outcome in BKI-treated mice. We conclude that maternal health-related factors such as cardiovascular, pharmacokinetic and/or bioavailability properties also contribute to BKI-pregnancy effects.

A novel protein kinase is essential in bloodstream Trypanosoma brucei.

Human African trypanosomiasis a fatal disease for which no vaccines exist and treatment regimens are difficult. Here, we evaluate a Trypanosoma brucei protein kinase, AEK1, as a potential drug target. Conditional knockouts confirmed AEK1 essentiality in bloodstream forms. For chemical validation, we overcame the lack of AEK1 inhibitors by creating parasites expressing a single, functional analog-sensitive AEK1 allele. Analog treatment of mice infected with this strain delayed parasitemia and death, with one-third of animals showing no parasitemia. These studies validate AEK1 as a drug target and highlight the need for further understanding of its function.

Bumped kinase inhibitor prohibits egression in Babesia bovis.

Babesiosis is a global zoonotic disease acquired by the bite of a Babesia-infected Ixodes tick or through blood transfusion with clinical relevance affecting humans and animals. In this study, we evaluated a series of small molecule compounds that have previously been shown to target specific apicomplexan enzymes in Plasmodium, Toxoplasma and Cryptosporidium. The compounds, bumped kinase inhibitors (BKIs), have strong therapeutic potential targeting apicomplexa-specific calcium dependent protein kinases (CDPKs). We investigated if BKIs also show inhibitory activities against piroplasms such as Babesia. Using a subset of BKIs that have promising inhibitory activities to Plasmodium and Toxoplasma, we determined that their actions ranged from 100% and no inhibition against Babesia bovis blood stages. One specific BKI, RM-1-152, showed complete inhibition against B. bovis within 48h and was the only BKI that showed noticeable phenotypic changes to the parasites. Focusing our study on this BKI, we further demonstrated that RM-1-152 has Babesia-static activity and involves the prohibition of merozoite egress while replication and re-invasion of host cells are unaffected. The distinct, abnormal phenotype induced by RM-1-152 suggests that this BKI can be used to investigate less studied cellular processes such as egression in piroplasm.

Development of an Orally Available and Central Nervous System (CNS) Penetrant Toxoplasma gondii Calcium-Dependent Protein Kinase 1 (TgCDPK1) Inhibitor with Minimal Human Ether-a-go-go-Related Gene (hERG) Activity for the Treatment of Toxoplasmosis.

New therapies are needed for the treatment of toxoplasmosis, which is a disease caused by the protozoan parasite Toxoplasma gondii. To this end, we previously developed a potent and selective inhibitor (compound 1) of Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) that possesses antitoxoplasmosis activity in vitro and in vivo. Unfortunately, 1 has potent human ether-a-go-go-related gene (hERG) inhibitory activity, associated with long Q-T syndrome, and consequently presents a cardiotoxicity risk. Here, we describe the identification of an optimized TgCDPK1 inhibitor 32, which does not have a hERG liability and possesses a favorable pharmacokinetic profile in small and large animals. 32 is CNS-penetrant and highly effective in acute and latent mouse models of T. gondii infection, significantly reducing the amount of parasite in the brain, spleen, and peritoneal fluid and reducing brain cysts by >85%. These properties make 32 a promising lead for the development of a new antitoxoplasmosis therapy.

SAR Studies of 5-Aminopyrazole-4-carboxamide Analogues as Potent and Selective Inhibitors of Toxoplasma gondii CDPK1.

We previously discovered compounds based on a 5-aminopyrazole-4-carboxamide scaffold to be potent and selective inhibitors of CDPK1 from T. gondii. The current work, through structure-activity relationship studies, led to the discovery of compounds (34 and 35) with improved characteristics over the starting inhibitor 1 in terms of solubility, plasma exposure after oral administration in mice, or efficacy on parasite growth inhibition. Compounds 34 and 35 were further demonstrated to be more effective than 1 in a mouse infection model and markedly reduced the amount of T. gondii in the brain, spleen, and peritoneal fluid, and 35 given at 20 mg/kg eliminated T. gondii from the peritoneal fluid.

Neospora caninum calcium-dependent protein kinase 1 is an effective drug target for neosporosis therapy.

Despite the enormous economic importance of Neospora caninum related veterinary diseases, the number of effective therapeutic agents is relatively small. Development of new therapeutic strategies to combat the economic impact of neosporosis remains an important scientific endeavor. This study demonstrates molecular, structural and phenotypic evidence that N. caninum calcium-dependent protein kinase 1 (NcCDPK1) is a promising molecular target for neosporosis drug development. Recombinant NcCDPK1 was expressed, purified and screened against a select group of bumped kinase inhibitors (BKIs) previously shown to have low IC50s against Toxoplasma gondii CDPK1 and T. gondii tachyzoites. NcCDPK1 was inhibited by low concentrations of BKIs. The three-dimensional structure of NcCDPK1 in complex with BKIs was studied crystallographically. The BKI-NcCDPK1 structures demonstrated the structural basis for potency and selectivity. Calcium-dependent conformational changes in solution as characterized by small-angle X-ray scattering are consistent with previous structures in low Calcium-state but different in the Calcium-bound active state than predicted by X-ray crystallography. BKIs effectively inhibited N. caninum tachyzoite proliferation in vitro. Electron microscopic analysis of N. caninum cells revealed ultra-structural changes in the presence of BKI compound 1294. BKI compound 1294 interfered with an early step in Neospora tachyzoite host cell invasion and egress. Prolonged incubation in the presence of 1294 interfered produced observable interference with viability and replication. Oral dosing of BKI compound 1294 at 50 mg/kg for 5 days in established murine neosporosis resulted in a 10-fold reduced cerebral parasite burden compared to untreated control. Further experiments are needed to determine the PK, optimal dosage, and duration for effective treatment in cattle and dogs, but these data demonstrate proof-of-concept for BKIs, and 1294 specifically, for therapy of bovine and canine neosporosis.

Development of potent and selective Plasmodium falciparum calcium-dependent protein kinase 4 (PfCDPK4) inhibitors that block the transmission of malaria to mosquitoes.

Malaria remains a major health concern for a large percentage of the world's population. While great strides have been made in reducing mortality due to malaria, new strategies and therapies are still needed. Therapies that are capable of blocking the transmission of Plasmodium parasites are particularly attractive, but only primaquine accomplishes this, and toxicity issues hamper its widespread use. In this study, we describe a series of pyrazolopyrimidine- and imidazopyrazine-based compounds that are potent inhibitors of PfCDPK4, which is a calcium-activated Plasmodium protein kinase that is essential for exflagellation of male gametocytes. Thus, PfCDPK4 is essential for the sexual development of Plasmodium parasites and their ability to infect mosquitoes. We demonstrate that two structural features in the ATP-binding site of PfCDPK4 can be exploited in order to obtain potent and selective inhibitors of this enzyme. Furthermore, we demonstrate that pyrazolopyrimidine-based inhibitors that are potent inhibitors of the in vitro activity of PfCDPK4 are also able to block Plasmodium falciparum exflagellation with no observable toxicity to human cells. This medicinal chemistry effort serves as a valuable starting point in the development of safe, transmission-blocking agents for the control of malaria.

Novel C-9, 9'-O-acyl esters of (-)-carinol as free-radical scavengers and xanthine oxidase enzyme inhibitors: synthesis and biological evaluation.

New compounds with hydrophyllic esters of (-)-carinol were synthesized and evaluated as xanthine oxidase enzyme inhibitors and antioxidants. Aliphatic esterfication of C-9,9'-OH groups of (-)-carinol resulted in lowering antioxidant and xanthine oxidase inhibitory activities. However certain aromatic acyl esters considerably improved the xathine oxidase inhibition. Aromatic esterification with electron withdrawing substitutions would preferred for improvement in XOD inhibition while retaining radical scavenging activity, electron withdrawing substitution led to the loss of free radical scavenging property and neutral substituents decrease the enzyme inhibitory potential.