Structure-based discovery of LpxC inhibitors.

The emergence and spread of multidrug-resistant (MDR) Gram negative bacteria presents a serious threat for public health. Novel antimicrobials that could overcome the resistance problems are urgently needed. UDP-3-O-(R-3-hydroxymyristol)-N-acetylglucosamine deacetylase (LpxC) is a cytosolic zinc-based deacetylase that catalyzes the first committed step in the biosynthesis of lipid A, which is essential for the survival of Gram-negative bacteria. Our efforts toward the discovery of novel LpxC inhibitors are presented herein.

Reduced pulmonary surfactant interaction of daptomycin analogs via tryptophan replacement with alternative amino acids.

Daptomycin was shown to interact in vitro with pulmonary surfactant leading to reduction of its antibacterial activity. We report herein the preparation and anti-staphylococcal activity of a series of daptomycin analogs with reduced pulmonary surfactant interaction by replacing tryptophan with various amino acids.

Discovery of 5-(arenethynyl) hetero-monocyclic derivatives as potent inhibitors of BCR-ABL including the T315I gatekeeper mutant.

Ponatinib (AP24534) was previously identified as a pan-BCR-ABL inhibitor that potently inhibits the T315I gatekeeper mutant, and has advanced into clinical development for the treatment of refractory or resistant CML. In this study, we explored a novel series of five and six membered monocycles as alternate hinge-binding templates to replace the 6,5-fused imidazopyridazine core of ponatinib. Like ponatinib, these monocycles are tethered to pendant toluanilides via an ethynyl linker. Several compounds in this series displayed excellent in vitro potency against both native BCR-ABL and the T315I mutant. Notably, a subset of inhibitors exhibited desirable PK and were orally active in a mouse model of T315I-driven CML.

Discovery of a Novel Cabazitaxel Nanoparticle-Drug Conjugate (CRLX522) with Improved Pharmacokinetic Properties and Anticancer Effects Using a ß-Cyclodextrin-PEG Copolymer Based Delivery Platform.

Novel nanoparticle-drug conjugates (NDCs) containing diverse, clinically relevant anticancer drug payloads (docetaxel, cabazitaxel, and gemcitabine) were successfully generated and tested in drug discovery studies. The NDCs utilized structurally varied linkers that attached the drug payloads to a ß-cyclodextrin-PEG copolymer to form self-assembled nanoparticles. In vitro release studies revealed a diversity of release rates driven by linker structure-activity relationships (SARs). Improved in vivo pharmacokinetics (PK) for the cabazitaxel (CBTX) NDCs with glycinate-containing (1c) and hexanoate-containing linkers (2c) were demonstrated, along with high and sustained tumor levels (>168 h of released drug in tumor tissues). This led to potent efficacy and survival in both taxane- and docetaxel-resistant in vivo anticancer mouse efficacy models. Overall, the CBTX-hexanoate NDC 2c (CRLX522), demonstrated optimal and improved in vivo PK (plasma and tumor) and efficacy profile versus those of the parent drug, and the results support the potential therapeutic use of CRLX522 as a new anticancer agent.

SRC inhibitors in metastatic bone disease.

Src tyrosine kinase was the first gene product shown to have an essential function in bone using recombinant DNA technology after its expression was knocked out in mice approximately 15 years ago. Since then, our understanding of the regulation of bone catabolism has advanced significantly with the identification of other key enzymes that regulate osteoclast formation, activation, and survival after their knockout in mice or recognition of mutations in them in humans. This led to the discovery or development of specific inhibitors of some of these key enzymes, including Src, as proof-of-concept lead compounds or potential clinical candidates for the prevention of diseases associated with increased bone resorption, such as osteoporosis and metastatic bone disease. Although bisphosphonates have been prescribed with proven and improving efficacy for the prevention of bone loss for >30 years, adverse effects, such as upper gastrointestinal tract symptoms, and the requirement to take them at least 2 hours before food have limited patient compliance. Thus, with growing knowledge of the pathways regulating osteoclast function and the appreciation that some of these are active also in tumor cells, drug companies have made efforts to identify small-molecular lead compounds for development into new therapeutic agents for the prevention of bone loss with efficacy that matches or supersedes that of bisphosphonates. In this article, we review our current understanding of the signaling pathways that regulate osteoclast formation, activation, and survival with specific reference to the role of Src tyrosine kinase and downstream signaling and highlight in a variety of models of increased bone resorption the effects of Src kinase inhibitors that have been targeted to bone to limit potential adverse effects on other cells.

Identification of Src-specific phosphorylation site on focal adhesion kinase: dissection of the role of Src SH2 and catalytic functions and their consequences for tumor cell behavior.

Src tyrosine kinase expression and activity are elevated during colon cancer progression. How this contributes to the malignant phenotype is not fully understood. We show that in KM12C colon carcinoma cells, expression of kinase-deficient Src proteins (SrcMF and Src251) does not alter cell growth. Src kinase activity is required for turnover of cell-matrix adhesions and, in particular, the Src-dependent phosphorylation of focal adhesion kinase (FAK) is required for their disassembly. Surprisingly, we found that expression of SrcMF or Src251 resulted in increased tyrosine phosphorylation of FAK on Tyr(407), Tyr(576), Tyr(577), and Tyr(861), which are considered to be Src kinase substrates. This Src kinase-independent phosphorylation of FAK required an intact Src SH2 domain that mediates association of Src and FAK at peripheral adhesions. Use of a novel highly potent and selective Src kinase inhibitor AP23464 combined with experiments in Src/Fyn/Yes-deficient fibroblasts showed that increased phosphorylation of FAK in cells expressing SrcMF did not require Src-like kinases. However, specific phosphorylation on Tyr(925) of FAK was not evident in SrcMF- or Src251-expressing cells, and lack of Src kinase-dependent phosphorylation on this site was associated with impaired adhesion turnover. Our data show that Src kinase activity is required for adhesion turnover associated with cell migration in cancer cells and that, in addition to the catalytic activity, Src also acts as an adaptor to recruit other kinases that can phosphorylate key substrates including FAK. These studies have implications for tumor progression with respect to the use of Src kinase inhibitors.

Future anti-catabolic therapeutic targets in bone disease.

Understanding of the regulation of bone catabolism has advanced significantly over the past two decades with the identification of key enzymes that regulate osteoclast formation, activation, and survival following their knockout in mice or recognition of mutations in humans. This led to the discovery of specific inhibitors of some of these key enzymes as proof-of-concept lead compounds or potential clinical candidates for the prevention of osteoporosis and other diseases associated with increased bone resorption. Bisphosphonates have been the major therapeutic agents prescribed for the prevention of bone loss in a variety of pathologic conditions for over 30 years. More potent amino bisphosphonates have increased efficacy than earlier drugs, but side effects such as upper gastrointestinal symptoms and the requirement to take them at least 2 h before food have limited patient compliance. This, coupled with the growing knowledge of the pathways regulating osteoclast function, has driven efforts to identify small molecular lead compounds that could be developed into new therapeutic agents with efficacy that matches or supersedes that of bisphosphonates for the prevention of bone loss. In this article, we review briefly the effects of specific inhibitors of bone resorption that have been developed to date and highlight in a variety of models of increased bone resorption the effects of Src kinase inhibitors that have been targeted to bone to limit potential unwanted side effects on other cells.

AP23846, a novel and highly potent Src family kinase inhibitor, reduces vascular endothelial growth factor and interleukin-8 expression in human solid tumor cell lines and abrogates downstream angiogenic processes.

c-Src is frequently activated in human malignancies, including colon, breast, and pancreatic carcinomas. Several recent studies have shown that activation of Src family kinases leads to tumor progression and metastasis by increasing cellular migration and invasion, promoting cell growth and survival, and deregulating expression of proangiogenic molecules. Therefore, selective inhibitors of Src are being developed for cancer therapy. In this study, we characterize the biological effects of the novel ATP-based Src family kinase inhibitor, AP23846, in tumor cells with high Src activity. As a lead compound, AP23846 is a potent c-Src kinase inhibitor (IC50 approximately 0.5 nmol/L in vitro, approximately 10-fold more potent than PP2, the most widely used commercially available Src family kinase inhibitor). At concentrations of 1 micromol/L, AP23846 led to complete Src inhibition for 48 hours in cells. No cytotoxicity was observed under these conditions, although proliferation rates were slower. Therefore, this was an excellent inhibitor to examine Src-regulated signaling pathways in tumor cells. AP23846 reduced cellular migration, vascular endothelial growth factor, and interleukin-8 in a dose-dependent fashion in pancreatic adenocarcinoma cells grown in vitro. Correspondingly, cell culture supernatants from L3.6pl pancreatic adenocarcinoma cells pretreated with AP23846 failed to promote migration of hepatic endothelial cells in vitro and failed to support angiogenesis into gel foams implanted s.c. in mice in vivo. These results suggest that Src inhibitors affect biological properties of tumor progression and may be useful as cancer therapeutic agents in more advanced disease.

Discovery of Pyrazolopyridones as a Novel Class of Gyrase B Inhibitors Using Structure Guided Design.

The ATPase subunit of DNA gyrase B is an attractive antibacterial target due to high conservation across bacteria and the essential role it plays in DNA replication. A novel class of pyrazolopyridone inhibitors was discovered by optimizing a fragment screening hit scaffold using structure guided design. These inhibitors show potent Gram-positive antibacterial activity and low resistance incidence against clinically important pathogens.

Structure-Activity Relationship Studies of a Series of Semisynthetic Lipopeptides Leading to the Discovery of Surotomycin, a Novel Cyclic Lipopeptide Being Developed for the Treatment of Clostridium difficile-Associated Diarrhea.

Novel cyclic lipopeptides with different acyl tails were synthesized via a semisynthetic approach. Structure-activity relationship studies revealed that lipophilicity, chain length, and the location of key aromatic functionalities of the tail modulated activity. The lead compound surotomycin exhibited significantly improved in vitro activity compared with daptomycin (MIC90 0.5 vs 2 µg/mL) against Clostridium difficile including NAP1 epidemic strains. In hamster efficacy studies, surotomycin protected animals at a dose of 0.5 mg/kg, PO.

Discovery of Indazole Derivatives as a Novel Class of Bacterial Gyrase B Inhibitors.

Antibacterials with a novel mechanism of action offer a great opportunity to combat widespread antimicrobial resistance. Bacterial DNA Gyrase is a clinically validated target. Through physiochemical property optimization of a pyrazolopyridone hit, a novel class of GyrB inhibitors were discovered. Guided by structure-based drug design, indazole derivatives with excellent enzymatic and antibacterial activity as well as great animal efficacy were discovered.

Discovery of Azaindole Ureas as a Novel Class of Bacterial Gyrase B Inhibitors.

The emergence and spread of multidrug resistant bacteria are widely believed to endanger human health. New drug targets and lead compounds exempt from cross-resistance with existing drugs are urgently needed. We report on the discovery of azaindole ureas as a novel class of bacterial gyrase B inhibitors and detail the story of their evolution from a de novo design hit based on structure-based drug design. These inhibitors show potent minimum inhibitory concentrations against fluoroquinolone resistant MRSA and other Gram-positive bacteria.

Targeting protein kinases for bone disease: discovery and development of Src inhibitors.

The dynamic and highly regulated processes of bone remodeling involve two major cells, osteoclasts and osteoblasts, both of which command a multitude of cellular signaling pathways involving protein kinases. Of the possible kinases in these cells, Src tyrosine kinase stands out as a promising therapeutic target for bone disease as validated by Src knockout mouse studies and in vitro cellular experiments, suggesting a regulatory role for Src in both osteoclasts (positive) and osteoblasts (negative). Advances in structural studies involving both Src and non-Src family kinases, in activated and unactivated protein states, have uncovered key binding site interactions that have led to the design of potent Src inhibitors. The lead compounds originate from a variety of synthetic templates and have demonstrated nM potency in enzymatic/binding assays and efficacy in animal models of bone disease. This review will provide a current understanding of critical Src signalling pathways in osteoclasts and osteoblasts, while detailing the structure-based design and screening-based lead discovery of Src inhibitors to be developed as therapeutic agents for bone disease.

Novel bone-targeted Src tyrosine kinase inhibitor drug discovery.

Bone-targeted Src tyrosine kinase (STK) inhibitors have recently been developed for the treatment of osteoporosis and cancer-related bone diseases. The concept of bone targeting derives from bisphosphonates, and from the evolution of such molecules in terms of therapeutic efficacy for the treatment of bone disorders. Interestingly, some of the earliest bisphosphonates were recognized for their ability to inhibit calcium carbonate precipitation (scaling) by virtue of their affinity to chelate calcium. This chelating property was subsequently exploited in the development of bisphosphonate analogs as inhibitors of the bone-resorbing cells known as osteoclasts, giving rise to breakthrough medicines, such as Fosamax (for the treatment of osteoporosis) and Zometa (for the treatment of osteoporosis and bone metastases). Relative to these milestone achievements, there is a tremendous opportunity to explore beyond the limited chemical space (functional group diversity) of such bisphosphonates to design novel bone-targeting moieties, which may be used to develop other classes of promising small-molecule drugs affecting different biological pathways. Here, we review studies focused on bone-targeted inhibitors of STK, a key enzyme in osteoclast-dependent bone resorption. Two strategies are described relative to bone-targeted STK inhibitor drug discovery: (i) the development of novel Src homology (SH)-2 inhibitors incorporating non-hydrolyzable phosphotyrosine mimics and exhibiting molecular recognition and bone-targeting properties, leading to the in vivo-effective lead compound AP-22408; and (ii) the development of novel ATP-based Src kinase inhibitors incorporating bone-targeting moieties, leading to the in vivo-effective lead compound AP-23236. In summary, AP-22408 and AP-23236, which differ mechanistically by virtue of blocking Src-dependent non-catalytic or catalytic activities in osteoclasts, exemplify ARIAD Pharmaceuticals' structure-based design of novel bone-targeted lead compounds, successfully achieving in vivo proof-of-concept and providing the framework for the next-generation molecules that have further advanced, in terms of preclinical studies, for the treatment of osteoporosis and related bone diseases, including osteolytic bone metastases.

SRC homology-2 inhibitors: peptidomimetic and nonpeptide.

The structural and functional characterization of Src homology-2 (SH2) domains and their relationship to catalytic proteins (e.g., kinases, phosphatases, and lipases) or non-catalytic proteins (e.g., upstream adapters, and downstream transcription factors) has significantly impacted our understanding of signal transduction pathways and the identification of promising therapeutic targets for drug discovery. Such SH2-containing proteins are known to be intimately involved in the regulation of a number of cellular processes, including growth, mitogenesis, motility, metabolism, and gene transcription. Molecular recognition and biochemical selectivity exists for various SH2 domains based on their binding to phosphotyrosine (pTyr) and contiguous C-terminal amino acids of cognate protein 'partners' in a sequence-dependent manner (i.e., -pTyr-AA(1)-AA(2)-AA(3)-) which result in the formation of signal transduction protein complexes in cells. In recent years, drug discovery efforts have advanced peptidomimetic and nonpeptide inhibitors of such protein-protein interactions based on mimicking pTyr-containing peptide ligands as well as SH2 structure-based de novo design of nonpeptide templates that can capture key binding sites on the target protein. Noteworthy are peptidomimetic and nonpeptide inhibitors of Src, Lck, Grb2, PI-3K, and Zap70 from pioneering efforts that led to the first examples of cellularly and in vivo active SH2 inhibitors. This mini-review highlights key achievements in SH2 inhibitor drug discovery with an emphasis on peptidomimetic and nonpeptide lead compounds in terms of structure-based design, key chemical and biological properties, and proof-of-concept studies relative to further defining the role(s) of SH2 domains in signal transduction processes, cellular functions, and in vivo disease models.

Discovery of 3-[2-(imidazo[1,2-b]pyridazin-3-yl)ethynyl]-4-methyl-N-{4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl}benzamide (AP24534), a potent, orally active pan-inhibitor of breakpoint cluster region-abelson (BCR-ABL) kinase including the T315I gatekeeper mutant.

In the treatment of chronic myeloid leukemia (CML) with BCR-ABL kinase inhibitors, the T315I gatekeeper mutant has emerged as resistant to all currently approved agents. This report describes the structure-guided design of a novel series of potent pan-inhibitors of BCR-ABL, including the T315I mutation. A key structural feature is the carbon-carbon triple bond linker which skirts the increased bulk of Ile315 side chain. Extensive SAR studies led to the discovery of development candidate 20g (AP24534), which inhibited the kinase activity of both native BCR-ABL and the T315I mutant with low nM IC(50)s, and potently inhibited proliferation of corresponding Ba/F3-derived cell lines. Daily oral administration of 20g significantly prolonged survival of mice injected intravenously with BCR-ABL(T315I) expressing Ba/F3 cells. These data, coupled with a favorable ADME profile, support the potential of 20g to be an effective treatment for CML, including patients refractory to all currently approved therapies.

AP24534, a pan-BCR-ABL inhibitor for chronic myeloid leukemia, potently inhibits the T315I mutant and overcomes mutation-based resistance.

Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABL(T315I) mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and preclinical evaluation of AP24534, a potent, orally available multitargeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABL(T315I)-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML.

Activity of dual SRC-ABL inhibitors highlights the role of BCR/ABL kinase dynamics in drug resistance.

Mutation in the ABL kinase domain is the principal mechanism of imatinib resistance in patients with chronic myelogenous leukemia. Many mutations favor active kinase conformations that preclude imatinib binding. Because the active forms of ABL and SRC resemble one another, we tested two dual SRC-ABL kinase inhibitors, AP23464 and PD166326, against 58 imatinib-resistant (IM(R)) BCR/ABL kinase variants. Both compounds potently inhibit most IM(R) variants, and in vitro drug selection demonstrates that active (AP23464) and open (PD166326) conformation-specific compounds are less susceptible to resistance than imatinib. Combinations of inhibitors suppressed essentially all resistance mutations, with the notable exception of T315I. Guided by mutagenesis studies and molecular modeling, we designed a series of AP23464 analogues to target T315I. The analogue AP23846 inhibited both native and T315I variants of BCR/ABL with submicromolar potency but showed nonspecific cellular toxicity. Our data illustrate how conformational dynamics of the ABL kinase accounts for the activity of dual SRC-ABL inhibitors against IM(R)-mutants and provides a rationale for combining conformation specific inhibitors to suppress resistance.