The squalestatins: synthesis and biological activity of some C3-modified analogues; replacement of a carboxylic acid or methyl ester with an isoelectronic heterocyclic functionality.

A series of squalestatins modified at the C3-position with a heterocyclic functionality was prepared and evaluated in vitro as inhibitors of squalene synthase (SQS). Structure-activity relationships for compounds with the 4,6-dimethyloctenoate at C6(S1 analogues) were different from those for analogues lacking the C6 ester (H1 analogues), with a greater dependence on the nature of the C3-substituent for the H1 series. Potent SQS inhibitory activity equivalent to that of H1 is retained by a C3-(tetrazol-5-yl) analogue, i.e., a carboxylic acid mimetic. The C3-methyl ester derivative is 10-fold less active than H1, and SQS inhibitory activity similar to that of the methyl ester was retained only in those C3-heterocycle-substituted H1 analogues for which electrostatic potential maps of the C3-substituent were closely similar to that of a methyl ester.

The squalestatins: novel inhibitors of squalene synthase. Enzyme inhibitory activities and in vivo evaluation of C1-modified analogues.

Squalestatin analogues modified in the C1 side chain were prepared and evaluated for their ability to inhibit rat liver microsomal and Candida squalene synthase (SQS) in vitro. While maintaining the 4,6-dimethyloctenoate or 4,6-dimethyloctanoate ester groups at C6, a number of modifications to the C1 side chain were well tolerated. However, in the absence of the C6 ester group, similar modifications to the C1 side chain caused substantial loss of activity. Compounds were also evaluated for their ability to inhibit cholesterol biosynthesis in vivo in rats and to reduce serum cholesterol levels in marmosets. These studies revealed that compounds with similar SQS inhibitory activities can possess different in vivo durations of action and lipid-lowering abilities.

The squalestatins: decarboxy and 4-deoxy analogues as potent squalene synthase inhibitors.

Squalestatins without either the hydroxy group at C-4 or the carboxylic acid at C-3 or C-4 were prepared and evaluated for their ability to inhibit rat liver microsomal squalene synthase (SQS) in vitro. These modifications were well tolerated for compounds with the 4,6-dimethyloctenoate ester at C-6 (S1 series). However in analogues without the C-6 ester (H1 series), removal of the C-4 hydroxy group gave compounds with reduced potency, whereas decarboxylation at C-3 resulted in a dramatic loss of SQS inhibitory activity. In comparison with S1 1, C-4 deoxyS1 3 and C-3 decarboxyS1 10 have shorter in vivo durations of action on the inhibition of hepatic cholesterol biosynthesis in rats. C-4 deoxyS1 3 retains good serum cholesterol-lowering ability in marmosets, while C-3 decarboxyS1 10 showed only a marginal effect even at high dose.

Emerging therapies for hepatitis C virus infection.

Hepatitis C virus (HCV) has infected millions of people worldwide and has emerged as a global health crisis. The currently available therapy is interferon (IFN) either alone or in combination with ribavirin. However, the disappointing efficacy of IFN has led to the considerable need for improved treatments and a number of new therapies are under evaluation in clinical trials. These include pegylated IFNs, which have altered physiochemical characteristics allowing once-weekly dosing. Combination of pegylated IFN with ribavirin should further improve sustained response rates. However, not all patients are successfully treated with IFNs, particularly those infected with genotype 1 of the virus, and it is likely that potent, specific drugs will be required. The majority of new approaches currently trying to combat this viral disease are aimed at inhibition of viral targets. Most effort has been directed towards inhibition of the NS3 serine protease, and potent inhibitors have now been described. However, a clinical candidate is yet to emerge against this difficult target. Considerable work by leading researchers has provided crystal structures of the key replicative enzymes, NS3 protease, NS3 helicase, NS5B polymerase and full-length NS3 protease-helicase, and there is much hope that such structural information will bear fruit. More recently, inhibition of host targets, particularly inosine monophosphate dehydrogenase (IMPDH), has become of interest and there are on-going clinical trials with such inhibitors. Research aimed at novel treatments for HCV disease is gathering pace and very recent developments in cell-based assay systems can only hasten the discovery of improved therapies.

Novel approaches to the treatment of hepatitis C virus infection.

Hepatitis C virus (HCV) has infected millions of people worldwide and emerged as a global health crisis. This review reports approaches currently being taken to combat the virus. Viral targets have received the most attention, particularly the NS3 serine protease where potent inhibitors have been described. Crystal structures of key replicative enzymes, NS3 protease, NS3 helicase, NS5B polymerase and now full-length NS3 protease-helicase, are available. More recently, targeting the host system has become of interest, particularly inhibitors of inosine monophosphate dehydrogenase. Research aimed at novel treatments for HCV disease is gathering pace and very recent developments in cell-based assay systems can only hasten the discovery of improved therapies.

TG02, a novel oral multi-kinase inhibitor of CDKs, JAK2 and FLT3 with potent anti-leukemic properties.

TG02 is a novel pyrimidine-based multi-kinase inhibitor that inhibits CDKs 1, 2, 7 and 9 together with JAK2 and FLT3. It dose-dependently inhibits signaling pathways downstream of CDKs, JAK2 and FLT3 in cancer cells with the main targets being CDKs. TG02 is anti-proliferative in a broad range of tumor cell lines, inducing G1 cell cycle arrest and apoptosis. Primary cultures of progenitor cells derived from acute myeloid leukemia (AML) and polycythemia vera patients are very sensitive to TG02. Comparison with reference inhibitors that block only one of the main targets of TG02 demonstrate the benefit of combined CDK and JAK2/FLT3 inhibition in cell lines as well as primary cells. In vivo, TG02 exhibits favorable pharmacokinetics after oral dosing in xenograft models and accumulates in tumor tissues, inducing an effective blockade of both CDK and STAT signaling. TG02 induces tumor regression after oral dosing on both daily and intermittent schedules in a murine model of mutant-FLT3 leukemia (MV4-11) and prolongs survival in a disseminated AML model with wild-type FLT3 and JAK2 (HL-60). These data demonstrate that TG02 is active in various models of leukemia and provide a rationale for the ongoing clinical evaluation of TG02 in patients with advanced leukemias.

SB1518, a novel macrocyclic pyrimidine-based JAK2 inhibitor for the treatment of myeloid and lymphoid malignancies.

SB1518 is an innovative pyrimidine-based macrocycle that shows a unique kinase profile with selective inhibition of Janus Kinase-2 (JAK2; IC50=23 and 19 nM for JAK2(WT) and JAK2(V617F), respectively) within the JAK family (IC50=1280, 520 and 50 nM for JAK1, JK3 and TYK2, respectively) and fms-like tyrosine kinase-3 (FLT3; IC50=22 nM). SB1518 shows potent effects on cellular JAK/STAT pathways, inhibiting tyrosine phosphorylation on JAK2 (Y221) and downstream STATs. As a consequence SB1518 has potent anti-proliferative effects on myeloid and lymphoid cell lines driven by mutant or wild-type JAK2 or FLT3, resulting from cell cycle arrest and induction of apoptosis. SB1518 has favorable pharmacokinetic properties after oral dosing in mice, is well tolerated and significantly reduces splenomegaly and hepatomegaly in a JAK2(V617F)-driven disease model. SB1518 dose-dependently inhibits intra-tumor JAK2/STAT5 signaling, leading to tumor growth inhibition in a subcutaneous model generated with SET-2 cells derived from a JAK2(V617F) patient with megakaryoblastic leukemia. Moreover, SB1518 is active against primary erythroid progenitor cells sampled from patients with myeloproliferative disease. In summary, SB1518 has a unique profile and is efficacious and well tolerated in JAK2-dependent models. These favorable properties are now being confirmed in clinical studies in patients with myelofibrosis and lymphoma.

A fluorescence polarization assay for inhibitors of Hsp90.

Hsp90 encodes a ubiquitous molecular chaperone protein conserved among species which acts on multiple substrates, many of which are important cell-signaling proteins. Inhibition of Hsp90 function has been promoted as a mechanism to degrade client proteins involved in tumorigenesis and disease progression. Several assays to monitor inhibition of Hsp90 function currently exist but are limited in their use for a drug discovery campaign. Using data from the crystal structure of an initial hit compound, we have developed a fluorescence polarization assay to monitor binding of compounds to the ATP-binding site of Hsp90. This assay is very robust (Z' > 0.9) and can detect affinity of compounds with IC50s to 40 nM. We have used this assay in conjunction with cocrystal structures of small molecules to drive a structure-based design program aimed at the discovery and optimization of a novel class of potent Hsp90 inhibitors.