Search

While epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have changed the treatment landscape for EGFR mutant (L858R and ex19del)-driven non-small-cell lung cancer (NSCLC), most patients will eventually develop resistance to TKIs. In the case of first- and second-generation TKIs, up to 60% of patients will develop an EGFR T790M mutation, while third-generation irreversible TKIs, like osimertinib, lead to C797S as the primary on-target resistance mutation. The development of reversible inhibitors of these resistance mutants is often hampered by poor selectivity against wild-type EGFR, resulting in potentially dose-limiting toxicities and a sub-optimal profile for use in combinations. BLU-945 (compound

Background: Cyclin-dependent kinases (CDK) are a class of enzymes that, along with their regulatory cyclin binding partners, drive cell cycle progression. Cell cycle dysregulation is a hallmark of cancer and targeting its genetic drivers can confer therapeutic benefits. Cyclin E1 (CCNE1) gene alterations are common in patients with ovarian cancer and can be found in ~20% of high-grade serous ovarian cancer cases, which tend to be platinum therapy resistant and therefore represent a high medical need. Cyclin E1 is the canonical binding partner of CDK2, which becomes constitutively active when CCNE1 is amplified and overexpressed. Selectively inhibiting CDK2 is an attractive therapeutic option for CCNE1-amplified tumors and may limit off-target CDK-driven toxicities. Here we report preclinical validation studies leading to the development of an orally available CDK2 inhibitor, BLU-222, for the treatment of ovarian cancer harboring a CCNE1 amplification. Methods: BLU-222 selectivity was measured by enzyme assays, cellular target engagement assays (NanoBRET), and proliferation assays in a panel of ovarian cancer cell lines. In vitro cellular potency was assessed by phospho-Rb levels. In vivo antitumor activity of BLU-222 as a single agent or in combination with carboplatin was measured in an OVCAR-3 cell line-derived xenograft (CDX) tumor model harboring a CCNE1 amplification. Results: BLU-222 demonstrated selectivity, both biochemically and in cells, with low nanomolar potency for CDK2 vs other CDK family members (CDK1, -4, -6, -7, and -9). In a panel of ovarian cancer cell lines, those with CCNE1 amplifications were highly sensitive to BLU-222. Consistent with the in vitro profiling, BLU-222 exhibited significant antitumor activity in the OVCAR-3 CDX model. While administration of single-agent carboplatin led to stasis in vivo, the combination of BLU-222 + carboplatin induced durable tumor regression even after treatment cessation. Conclusions: These data provide a strong rationale for advancing BLU-222 towards clinical development in patients with CCNE1-amplified ovarian cancer. Citation Format: Victoria Brown, Phil Ramsden, Nealia House, Richard Vargas, Jian Guo, Ruduan Wang, Riadh Lobbardi, Maxine Chen, Douglas Wilson, Joseph Kim, Neil Bifulco, Michelle Maynard, Emanuele Perola, Dean Zhang, Steve Wenglowsky, Yoon Jong Choi. BLU-222, an investigational, potent, and selective CDK2 inhibitor, demonstrated robust antitumor activity in CCNE1-amplified ovarian cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2306.

BACKGROUND: Non-small cell lung cancer (NSCLC) represents 80% of lung cancers, the second leading cause of cancer deaths globally. Mutations in the kinase domain of the epidermal growth factor receptor (EGFR) are genomic drivers in a molecular sub-type of NSCLC that is sensitive to tyrosine kinase inhibitors (TKIs). Third-generation, irreversible TKIs, such as osimertinib and lazertinib, have become standard of care and are now widely used as first-line therapy in EGFR-driven metastatic NSCLC. However, resistance invariably arises during treatment. The C797S mutation is the most frequent on-target resistance mechanism in response to third-generation TKIs when used in the first-line setting. There are currently no approved therapies for patients who progress with a C797S mutation. Although first-generation TKIs inhibit EGFR with a C797S mutation, they carry liabilities of 1) insufficient selectivity over wild-type (WT) EGFR, which precludes maximal target coverage and results in dose-limiting adverse events; and 2) limited brain penetration. METHODS: BLU-701 activity was tested in biochemical assays for EGFR mutants and EGFR WT. Cellular activity was evaluated by a phosphorylation specific EGFR AlphaLisa assay in WT cell lines or those expressing EGFR mutations. The in vivo antitumor activity of BLU-701 was assessed in a PC9 ex19del cell line-derived tumor xenograft (CDX) model. RESULTS: We have developed an orally available, single-digit nanomolar, brain-penetrant, WT-sparing, reversible EGFR inhibitor that is active against the C797S resistance mutation. BLU-701 is equally potent against both the sensitizing mutations ex19del and L858R, and the resistance double mutants ex19del/C797S and L858R/C797S. BLU-701 is brain penetrant (Kpu,u >0.9) and demonstrates a selectivity over WT EGFR that compares favorably with osimertinib. Oral administration of BLU-701 to PC9 ex19del tumor-bearing mice resulted in strong and prolonged pathway suppression and tumor regression at tolerated doses. CONCLUSION: Due to its potent pharmacological activity and selectivity for mutant EGFR, BLU-701 has the potential to demonstrate activity in first-line and resistance settings as a single agent and in combination therapy, addressing potential tumor heterogeneity. The pre-clinical data described here supports the clinical development of BLU-701 in EGFR-driven NSCLC. Citation Format: Chiara Conti, John Campbell, Rich Woessner, Jian Guo, Yoav Timsit, Maria Iliou, Scott Wardwell, Alison Davis, Sharon Chicklas, John Hsieh, Meredith Eno, Omar Ahmad, Dilinie Fernando, Kevin Barvian, Joseph Kim, Steven Kazmirski, Emanuele Perola, Tom Dineen, Victoria Brown, Timothy Guzi, Ayşegül Özen, Faith Stevison, Caitlin Utt, Clare Medendorp, Robert Meissner, Marion Dorsch, Klaus Hoeflich. BLU-701 is a highly potent, brain-penetrant and WT-sparing next-generation EGFR TKI for the treatment of sensitizing (ex19del, L858R) and C797S resistance mutations in metastatic NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1262.

Background: Cyclin dependent kinases (CDK) comprise a family of proteins which are activated upon cyclin binding to promote cell cycle progression. Historically, CDK family inhibitors have had limited utility in the clinic due to 1) toxicities associated with broad spectrum CDK inhibition; and 2) varied and unpredictable efficacy due to lack of a patient selection strategy. The recent approval of three selective CDK4/6 drugs (palbociclib, ribociclib and abemaciclib) has revived the pursuit of therapies against a related interphase kinase, CDK2. Cyclin E gene (CCNE) alterations are prevalent in cancers with high unmet medical need and are emerging as a potential mechanism of clinical resistance to targeted therapies (e.g. CDK4/6 therapies). Methods: Here we report preclinical validation leading to the development of a selective CDK2 inhibitor for the treatment of cancers harboring CCNE alterations. We highlight advanced, orally bioavailable compounds exhibiting single-digit low nanomolar biochemical and cellular potency (pRB), exquisite CDK family selectivity (CDK1, 4, 6, 7 and 9), and whole kinome selectivity. We have characterized the phenotypic and mechanistic consequences of targeting CDK2, employing both genetic and pharmacologic methods across cell lines and xenograft models (e.g. OVCAR-3, MKN-1, HCC1569). Results: In a panel of cell lines, CCNE1-amplified lines exhibited profound sensitivity to selective CDK2 inhibition by halting cells at the G1/S phase of the cell cycle. Upon further inspection, CDK2 inhibition induced multiple markers of senescence, e.g. SA-b-gal and IL-6, in CCNE1-amplified but not in CCNE non-amplified cell lines. Consistent with this, these compounds exhibit robust anti-tumor activity in multiple CCNE1-amplified xenograft models, which was sustained after removal of treatment with these compounds. Conclusions: These data provide a strong rationale for advancing this class of compounds toward clinical development in CCNE-altered cancers. Citation Format: Yoon J. Choi, Steve Wenglowsky, Victoria Brown, Neil Bifulco, Yeon S. Choi, Jian Guo, Megan Hatlen, Joseph Kim, Tim LaBranche, Riadh Lobbardi, Emanuele Perola, Emily Rozsahegyi, Michelle Maynard, Phil Ramsden, Grace Silva, Faith Stevison, Richard Vargas, Ruduan Wang, Doug Wilson, Rich Woessner, Dean Zhang, Rob Meissner, Klaus Hoeflich, Marion Dorsch. Development of a selective CDK2-E inhibitor in CCNE driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1279.

Hematopoietic progenitor kinase 1 (HPK1, MAP4K1) is a serine/threonine kinase that has been demonstrated to have suppressive effects across a range of immune cells, including T cells and dendritic cells. Loss of MAP4K1 kinase activity is sufficient to enhance T cell receptor (TCR) signaling resulting in robust anti-tumor immunity alone and in combination with checkpoint inhibition. These data support that MAP4K1 is a novel and attractive target for cancer immunotherapy. We have designed a series of potent, selective, and orally bioavailable inhibitors of MAP4K1. Treatment of primary human T cells or peripheral blood with either BLU2069 or BLU6348 was able to inhibit phosphorylation of pSLP76, a scaffolding protein that regulates MAPK downstream of the TCR. In addition, we show that compound treatment can enhance cytokine secretion and proliferation in human T cells in response to TCR crosslinking. The therapeutic benefit of MAP4K1 inhibition alone and in combination with anti-PD-L1 was evaluated in multiple syngeneic mouse tumor models including MCA205, MC38 and EMT-6. Treatment with either compound alone led to a reduction in tumor growth that was further enhanced when combined with anti-PD-L1 therapy. When tumors were grown in immunocompromised mice (MCA-205) or in the setting of CD8+ T cell depletion (MC-38), the anti-tumor effect of BLU2069 and BLU6348 respectively was lost, confirming the importance of immune cells in compound mediated antitumor effects. We further show that MCA205 tumors harvested from mice treated with BLU2069 had increased intratumoral CD8+ T cell infiltration, resulting in enhanced CD8/Treg ratios. In addition, transcriptional analysis of tumor lysates showed that BLU2069 significantly increased genes associated with an effector phenotype. These data support that pharmacological inhibition of MAP4K1 reduced tumor burden and enhanced antitumor immunity in preclinical tumor models. Finally, we show that MAP4K1 inhibition can enhance CD3/CD28-induced IL2 and IFNγ in human tumor infiltrating lymphocytes (TILs) generated from melanoma or non-small cell lung cancer (NSCLC) primary tumors. This work describes the identification of potent small molecule inhibitors of MAP4K1 which could be novel therapeutic agents and induce an effective immune response either alone or in combination with approved checkpoint inhibitors. Citation Format: Kerrie Faia, Alberto Toso, Kristina Fetalvero, Marly Roche, Steven Bench, Erin O'Hearn, Qiongfang Cao, Kerry-Ann Bright, Debora Paduraru, Andrea Romagnani, Weifan Weng, Tina Zimmermann, Michael Burke, Joshua Close, Luke Green, Joseph Kim, Chandra Miduturu, Alison Ribeiro, Marina Bacac, Sylvia Herter, Emanuele Perola, Michael Sheets, Jan Eckmann, Gordon Heidkamp, Tary Traore, Erik Gerson, Rich Woessner, Carsten Wolter, Felix Scheuplein, Nisha Perez, Timothy LaBranche, Grace Silva, Chaoyang Ye, Caitlin Utt, Stefan Gross, James R. Bischoff, Marion Dorsch, Tim Guzi, Klaus Hoeflich, Jason Brubaker. MAP4K1 inhibition enhances immune cell activation and anti-tumor immunity in preclinical tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1717.

JNJ-63623872 (2) is a first-in-class, orally bioavailable compound that offers significant potential for the treatment of pandemic and seasonal influenza. Early lead optimization efforts in our 7-azaindole series focused on 1,3-diaminocyclohexyl amide and urea substitutions on the pyrimidine-7-azaindole motif. In this work, we explored two strategies to eliminate observed aldehyde oxidase (AO)-mediated metabolism at the 2-position of these 7-azaindole analogues. Substitution at the 2-position of the azaindole ring generated somewhat less potent analogues, but reduced AO-mediated metabolism. Incorporation of a ring nitrogen generated 7-azaindazole analogues that were equipotent to the parent 2-H-7-azaindole, but surprisingly, did not appear to improve AO-mediated metabolism. Overall, we identified multiple 2-substituted 7-azaindole analogues with enhanced AO stability and we present data for one such compound (12) that demonstrate a favorable oral pharmacokinetic profile in rodents. These analogues have th...

There are currently no treatments for life-threatening infections caused by human polyomaviruses JCV and BKV. We therefore report herein the first crystal structure of the hexameric helicase of JCV large T antigen (apo) and its use to drive the structure-based design of dual JCV and BKV ATP-competitive inhibitors. The crystal structures obtained by soaking our early inhibitors into the JCV helicase allowed us to rapidly improve the biochemical activity of our inhibitors from 18 μM for the early 6-(2-methoxyphenyl)- and the 6-(2-ethoxyphenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole hits 1a and 1b to 0.6 μM for triazolopyridine 12i. In addition, we were able to demonstrate measurable antiviral activity in Vero cells for our thiazolopyridine series in the absence of marked cytotoxicity, thus confirming the usefulness of this approach.

VX-787 is a first in class, orally bioavailable compound that offers unparalleled potential for the treatment of pandemic and seasonal influenza. As a part of our routine SAR exploration, carboxylic acid isosteres of VX-787 were prepared and tested against influenza A. It was found that the negative charge is important for maintaining potency and selectivity relative to kinase targets. Neutral carboxylic acid replacements generally resulted in compounds that were significantly less potent and less selective relative to the charged species.

New drugs to treat drug-resistant tuberculosis are urgently needed. Extensively drug-resistant and probably the totally drug-resistant tuberculosis strains are resistant to fluoroquinolones like moxifloxacin, which target gyrase A, and most people infected with these strains die within a year. In this study, we found that a novel aminobenzimidazole, VXc-486, which targets gyrase B, potently inhibits multiple drug-sensitive isolates and drug-resistant isolates of Mycobacterium tuberculosis in vitro (MICs of 0.03 to 0.30 μg/ml and 0.08 to 5.48 μg/ml, respectively) and reduces mycobacterial burdens in lungs of infected mice in vivo . VXc-486 is active against drug-resistant isolates, has bactericidal activity, and kills intracellular and dormant M. tuberculosis bacteria in a low-oxygen environment. Furthermore, we found that VXc-486 inhibits the growth of multiple strains of Mycobacterium abscessus , Mycobacterium avium complex, and Mycobacterium kansasii (MICs of 0.1 to 2.0 μg/ml), as well as that of several strains of Nocardia spp. (MICs of 0.1 to 1.0 μg/ml). We made a direct comparison of the parent compound VXc-486 and a phosphate prodrug of VXc-486 and showed that the prodrug of VXc-486 had more potent killing of M. tuberculosis than did VXc-486 in vivo . In combination with other antimycobacterial drugs, the prodrug of VXc-486 sterilized M. tuberculosis infection when combined with rifapentine-pyrazinamide and bedaquiline-pyrazinamide in a relapse infection study in mice. Furthermore, the prodrug of VXc-486 appeared to perform at least as well as the gyrase A inhibitor moxifloxacin. These findings warrant further development of the prodrug of VXc-486 for the treatment of tuberculosis and nontuberculosis mycobacterial infections.

To develop agents for the treatment of infections caused by Mycobacterium tuberculosis , a novel phenotypic screen was undertaken that identified a series of 2- N -aryl thiazole-based inhibitors of intracellular Mycobacterium tuberculosis . Analogs were optimized to improve potency against an attenuated BSL2 H37Ra laboratory strain cultivated in human macrophage cells in vitro . The insertion of a carboxylic acid functionality resulted in compounds that retained potency and greatly improved microsomal stability. However, the strong potency trends we observed in the attenuated H37Ra strain were inconsistent with the potency observed for virulent strains in vitro and in vivo .

In our efforts to develop novel small-molecule inhibitors for the treatment of influenza, we utilized molecular modeling and the X-ray crystal structure of the PB2 subunit of the influenza polymerase to optimize a series of acyclic β-amino acid inhibitors, highlighted by compound 4. Compound 4 showed good oral exposure in both rat and mouse. More importantly, it showed strong potency versus multiple influenza-A strains, including pandemic 2009 H1N1 and avian H5N1 strains and showed a strong efficacy profile in a mouse influenza model even when treatment was initiated 48 h after infection. Compound 4 offers good oral bioavailability with great potential for the treatment of both pandemic and seasonal influenza.

A series of 2,6-disubstituted aminoalkoxypyrimidine carboxamides (AAPCs) with potent inhibition of bacterial NAD(+)-dependent DNA ligase was discovered through the use of structure-guided design. Two subsites in the NAD(+)-binding pocket were explored to modulate enzyme inhibitory potency: a hydrophobic selectivity region was explored through a series of 2-alkoxy substituents while the sugar (ribose) binding region of NAD(+) was explored via 6-alkoxy substituents.

A series of potent thiol-containing aryl sulfone TACE inhibitors were designed and synthesized. The SAR and MMP selectivity of the series were investigated. In particular, compound 8b showed excellent in vitro potency against the isolated enzyme and good selectivity over MMP-2, -7, -8, -9, and -13. The X-ray structure of 8b in complex with TACE was also obtained.

Mycobacterium tuberculosis (Mtb) relies on a specialized set of metabolic pathways to support growth in macrophages. By conducting an extensive, unbiased chemical screen to identify small molecules that inhibit Mtb metabolism within macrophages, we identified a significant number of novel compounds that limit Mtb growth in macrophages and in medium containing cholesterol as the principle carbon source. Based on this observation, we developed a chemical-rescue strategy to identify compounds that target metabolic enzymes involved in cholesterol metabolism. This approach identified two compounds that inhibit the HsaAB enzyme complex, which is required for complete degradation of the cholesterol A/B rings. The strategy also identified an inhibitor of PrpC, the 2-methylcitrate synthase, which is required for assimilation of cholesterol-derived propionyl-CoA into the TCA cycle. These chemical probes represent new classes of inhibitors with novel modes of action, and target metabolic pathways required to support growth of Mtb in its host cell. The screen also revealed a structurally-diverse set of compounds that target additional stage(s) of cholesterol utilization. Mutants resistant to this class of compounds are defective in the bacterial adenylate cyclase Rv1625/Cya. These data implicate cyclic-AMP (cAMP) in regulating cholesterol utilization in Mtb, and are consistent with published reports indicating that propionate metabolism is regulated by cAMP levels. Intriguingly, reversal of the cholesterol-dependent growth inhibition caused by this subset of compounds could be achieved by supplementing the media with acetate, but not with glucose, indicating that Mtb is subject to a unique form of metabolic constraint induced by the presence of cholesterol., Author Summary Human beings are the sole ecological niche for M. tuberculosis (Mtb), and it is estimated that 1.8 billion people are currently infected with Mtb. An important aspect of this infection is Mtb’s ability to maintain infection by replicating within macrophages. Within macrophages, Mtb exploits a specialized set of metabolic pathways to utilize host-derived nutrients, such as fatty acids and/or cholesterol, for energy production. Many details regarding Mtb metabolism during infection remain unknown. Here we took a chemical approach to identify small molecule probes, which target Mtb metabolism during infection in macrophages. We found that many of the small molecule inhibitors that we identified require cholesterol for activity. Here we report a novel chemical rescue approach to identify the metabolic targets of three novel inhibitors, and discovered that cAMP signaling is linked to cholesterol utilization in Mtb. Together, these data demonstrate that cholesterol exerts a dominant effect on Mtb metabolism within macrophages. Additionally, the novel inhibitors identified in this study will facilitate evaluation of cholesterol metabolism as a target for chemotherapeutic intervention.

VX-787 is a novel inhibitor of influenza virus replication that blocks the PB2 cap-snatching activity of the influenza viral polymerase complex. Viral genetics and X-ray crystallography studies provide support for the idea that VX-787 occupies the 7-methyl GTP (m 7 GTP) cap-binding site of PB2. VX-787 binds the cap-binding domain of the PB2 subunit with a K D (dissociation constant) of 24 nM as determined by isothermal titration calorimetry (ITC). The cell-based EC 50 (the concentration of compound that ensures 50% cell viability of an uninfected control) for VX-787 is 1.6 nM in a cytopathic effect (CPE) assay, with a similar EC 50 in a viral RNA replication assay. VX-787 is active against a diverse panel of influenza A virus strains, including H1N1pdm09 and H5N1 strains, as well as strains with reduced susceptibility to neuraminidase inhibitors (NAIs). VX-787 was highly efficacious in both prophylaxis and treatment models of mouse influenza and was superior to the neuraminidase inhibitor, oseltamivir, including in delayed-start-to-treat experiments, with 100% survival at up to 96 h postinfection and partial survival in groups where the initiation of therapy was delayed up to 120 h postinfection. At different doses, VX-787 showed a 1-log to >5-log reduction in viral load (relative to vehicle controls) in mouse lungs. Overall, these favorable findings validate the PB2 subunit of the viral polymerase as a drug target for influenza therapy and support the continued development of VX-787 as a novel antiviral agent for the treatment of influenza infection.

A thorough evaluation of some of the most advanced docking and scoring methods currently available is described, and guidelines for the choice of an appropriate protocol for docking and virtual screening are defined. The generation of a large and highly curated test set of pharmaceutically relevant protein-ligand complexes with known binding affinities is described, and three highly regarded docking programs (Glide, GOLD, and ICM) are evaluated on the same set with respect to their ability to reproduce crystallographic binding orientations. Glide correctly identified the crystallographic pose within 2.0 A in 61% of the cases, versus 48% for GOLD and 45% for ICM. In general Glide appears to perform most consistently with respect to diversity of binding sites and ligand flexibility, while the performance of ICM and GOLD is more binding site-dependent and it is significantly poorer when binding is predominantly driven by hydrophobic interactions. The results also show that energy minimization and reranking of the top N poses can be an effective means to overcome some of the limitations of a given docking function. The same docking programs are evaluated in conjunction with three different scoring functions for their ability to discriminate actives from inactives in virtual screening. The evaluation, performed on three different systems (HIV-1 protease, IMPDH, and p38 MAP kinase), confirms that the relative performance of different docking and scoring methods is to some extent binding site-dependent. GlideScore appears to be an effective scoring function for database screening, with consistent performance across several types of binding sites, while ChemScore appears to be most useful in sterically demanding sites since it is more forgiving of repulsive interactions. Energy minimization of docked poses can significantly improve the enrichments in systems with sterically demanding binding sites. Overall Glide appears to be a safe general choice for docking, while the choice of the best scoring tool remains to a larger extent system-dependent and should be evaluated on a case-by-case basis.

Compound 3 is a potent aminobenzimidazole urea with broad-spectrum Gram-positive antibacterial activity resulting from dual inhibition of bacterial gyrase (GyrB) and topoisomerase IV (ParE), and it demonstrates efficacy in rodent models of bacterial infection. Preclinical in vitro and in vivo studies showed that compound 3 covalently labels liver proteins, presumably via formation of a reactive metabolite, and hence presented a potential safety liability. The urea moiety in compound 3 was identified as being potentially responsible for reactive metabolite formation, but its replacement resulted in loss of antibacterial activity and/or oral exposure due to poor physicochemical parameters. To identify second-generation aminobenzimidazole ureas devoid of reactive metabolite formation potential, we implemented a metabolic shift strategy, which focused on shifting metabolism away from the urea moiety by introducing metabolic soft spots elsewhere in the molecule. Aminobenzimidazole urea 34, identified through this strategy, exhibits similar antibacterial activity as that of 3 and did not label liver proteins in vivo, indicating reduced/no potential for reactive metabolite formation.

In our effort to develop agents for the treatment of influenza, a phenotypic screening approach utilizing a cell protection assay identified a series of azaindole based inhibitors of the cap-snatching function of the PB2 subunit of the influenza A viral polymerase complex. Using a bDNA viral replication assay (Wagaman, P. C., Leong, M. A., and Simmen, K. A. Development of a novel influenza A antiviral assay. J. Virol. Methods 2002, 105, 105-114) in cells as a direct measure of antiviral activity, we discovered a set of cyclohexyl carboxylic acid analogues, highlighted by VX-787 (2). Compound 2 shows strong potency versus multiple influenza A strains, including pandemic 2009 H1N1 and avian H5N1 flu strains, and shows an efficacy profile in a mouse influenza model even when treatment was administered 48 h after infection. Compound 2 represents a first-in-class, orally bioavailable, novel compound that offers potential for the treatment of both pandemic and seasonal influenza and has a distinct advantage over the current standard of care treatments including potency, efficacy, and extended treatment window.

A series of dual targeting inhibitors of bacterial gyrase B and topoisomerase IV were identified and optimized to mid-to-low nanomolar potency against a variety of bacteria. However, in spite of seemingly adequate exposure achieved upon IV administration, the in vivo efficacy of the early lead compounds was limited by high levels of binding to serum proteins. To overcome this limitation, targeted serum shift prediction models were generated for each subclass of interest and were applied to the design of prospective analogs. As a result, numerous compounds with comparable antibacterial potency and reduced protein binding were generated. These efforts culminated in the synthesis of compound 10, a potent inhibitor with low serum shift that demonstrated greatly improved in vivo efficacy in two distinct rat infection models.

The crystal structure of Torpedo californica (Tc) acetylcholinesterase (AChE) carbamoylated by the physostigmine analogue 8-(cis-2,6-dimethylmorpholino)octylcarbamoyleseroline (MF268) is reported at 2.7 A resolution. In the X-ray structure, the dimethylmorpholinooctylcarbamic moiety of MF268 is covalently bound to the catalytic serine, which is located at the bottom of a long and narrow gorge. The alkyl chain of the inhibitor fills the upper part of the gorge, blocking the entrance of the active site. This prevents eseroline, the leaving group of the carbamoylation process, from exiting through this path. Surprisingly, the relatively bulky eseroline is not found in the crystal structure, thus implying the existence of an alternative route for its clearance. This represents indirect evidence that a "back door" opening may occur and shows that the release of products via a "back door" is a likely alternative for this enzyme. However, its relevance as far as the mechanism of substrate hydrolysis is concerned needs to be established. This study suggests that the use of properly designed acylating inhibitors, which can block the entrance of catalytic sites, may be exploited as a general approach for investigating the existence of "back doors" for the clearance of products.

A high-quality NMR solution structure of the chimeric hybrid duplex r(gcaguggc).r(gcca)d(CTGC) was determined using the program DYANA with its recently implemented new module FOUND, which performs exhaustive conformational grid searches for dinucleotides. To ensure conservative data interpretation, the use of 1H-1H lower distance limit constraints was avoided. The duplex comprises the tRNA-DNA junction formed during the initiation of HIV-1 reverse transcription. It forms an A-type double helix that exhibits distinct structural deviations from a standard A-conformation. In particular, the minor groove is remarkably narrow, and its width decreases from about 7.5 A in the RNA/RNA stem to about 4.5 A in the RNA/DNA segment. This is unexpected, since minor groove widths for A-RNA and RNA/DNA hybrid duplexes of approximately 11 A and approximately 8.5 A, respectively, were previously reported. The present, new structure supports that reverse transcriptase-associated RNaseH specificity is related primarily to conformational adaptability of the nucleic acid in 'induced-fit'-type interactions, rather than the minor groove width of a predominantly static nucleic acid duplex.

The recent article “On Evaluating Molecular-Docking Methods for Pose Prediction and Enrichment Factors” (Chen H. et al. J. Chem. Inf. Model. 2006, 46, 401−415) contains a series of comments on a similar study we published in Proteins in 2004 (Perola et. al. Proteins 2004, 56, 235−249). We believe that some of those comments are misleading, and we feel that an adequate response is in order.

Several samples of oversulfated chondroitin and dermatan were obtained by chemical sulfation and by SAX-HPLC enrichment. The starting products and oversulfated products were tested as potential inhibitors of human leukocyte elastase, an enzyme hypothesized to be involved in the etiology of diseases such as emphysema, atherosclerosis, and rheumatoid arthritis. Chemical oversulfation (SO3H/COOH 1.6-3.2), preferentially occurring at C-6 of galactosamine residues, was found generally to increase the inhibitory power on elastase. Chemically oversulfated galactosaminoglycans thus have potential as therapeutic agents, considering that they produce non-significant effects on the hemocoagulative system. Two naturally oversulfated dermatans sulfate (SO3H/COOH ca. 1.2), mainly oversulfated at C-2 of iduronic acid residues, showed comparatively higher anticoagulant activity (in the HC-II mediated thrombin inhibition test).

A series of 4-amino-pyrido[2,3-d]pyrimidin-5(8H)-ones were designed and synthesized as a novel class of inhibitors of NAD(+)-dependent DNA ligase that possess potency against Gram-positive bacteria.

A series of N-[ω-N′-(adamant-1′-yl)aminoalkyl]-2-(4′-dimethylaminophenyl)acetamides were synthesized and tested as acetylcholinesterase inhibitors. A significant selectivity toward acetylcholinesterases from various natural sources, mainly differing in their quaternary structure and solubility, was pointed out. The interest of this kind of molecules as potential therapeutic agents for Alzheimer's disease is discussed.

Virtual screening of chemical databases is an emerging approach in drug discovery that uses computers to dock chemicals into the active site of a drug target to identify leads through evaluation of binding affinities of the chemicals. However, there are concerns about the validity and scope of the reported virtual screens due to lack of studies to show that randomly selected chemicals are not equally active and due to the fact that metalloproteins were rarely used as drug targets. We have performed a virtual screening of a chemical database to identify prototypic inhibitors of farnesyltransferase (FT) with zinc present in the active site. Among the 21 compounds identified by computers, four inhibited FT in vitro with IC50 values in the range from 25 to 100 μM. The most potent inhibitor also inhibited FT in human lung cancer cells. In contrast, none of 21 randomly selected compounds have an IC50 lower than 100 μM. The results demonstrate the validity of virtual screening and the feasibility of applications...

In order to investigate the evolution of binding efficiency in successful drug discovery programs, a data set of 60 lead/drug pairs with known binding affinities has been compiled and analyzed. Low-end thresholds for the binding efficiencies of viable leads and drugs have been derived. On average, the drugs in the set are significantly larger and more potent but have similar lipophilicity relative to their originating leads, suggesting that the ability to maintain low levels of lipophilicity while increasing molecular weight is one of the keys to a successful drug discovery program. A number of examples demonstrate that large increases in binding efficiency from leads to more elaborate drugs sharing the same scaffold can be achieved. The importance of dissecting a lead structure to identify the most efficient fragments and the option of sacrificing binding efficiency to optimize other properties are discussed, and relevant examples are highlighted.

The discovery of new antibacterial agents with novel mechanisms of action is necessary to overcome the problem of bacterial resistance that affects all currently used classes of antibiotics. Bacterial DNA gyrase and topoisomerase IV are well-characterized clinically validated targets of the fluoroquinolone antibiotics which exert their antibacterial activity through inhibition of the catalytic subunits. Inhibition of these targets through interaction with their ATP sites has been less clinically successful. The discovery and characterization of a new class of low molecular weight, synthetic inhibitors of gyrase and topoisomerase IV that bind to the ATP sites are presented. The benzimidazole ureas are dual targeting inhibitors of both enzymes and possess potent antibacterial activity against a wide spectrum of relevant pathogens responsible for hospital- and community-acquired infections. The discovery and optimization of this novel class of antibacterials by the use of structure-guided design, modeling, and structure-activity relationships are described. Data are presented for enzyme inhibition, antibacterial activity, and in vivo efficacy by oral and intravenous administration in two rodent infection models.

In spite of recent improvements in docking and scoring methods, high false-positive rates remain a common issue in structure-based virtual screening. In this study, the distinctive features of false positives in kinase virtual screens were investigated. A series of retrospective virtual screens on kinase targets was performed on specifically designed test sets, each combining true ligands and experimentally confirmed inactive compounds. A systematic analysis of the docking poses generated for the top-ranking compounds highlighted key aspects differentiating true hits from false positives. The most recurring feature in the poses of false positives was the absence of certain key interactions known to be required for kinase binding. A systematic analysis of 444 crystal structures of ligand-bound kinases showed that at least two hydrogen bonds between the ligand and the backbone protein atoms in the kinase hinge region are present in 90% of the complexes, with very little variability across targets. Closer inspection showed that when the two hydrogen bonds are present, one of three preferred hinge-binding motifs is involved in 96.5% of the cases. Less than 10% of the false positives satisfied these two criteria in the minimized docking poses generated by our standard protocol. Ligand conformational artifacts were also shown to contribute to the occurrence of false positives in a number of cases. Application of this knowledge in the form of docking constraints and post-processing filters provided consistent improvements in virtual screening performance on all systems. The false-positive rates were significantly reduced and the enrichment factors increased by an average of twofold. On the basis of these results, a generalized two-step protocol for virtual screening on kinase targets is suggested.

This paper describes a large-scale study on the nature and the energetics of the conformational changes drug-like molecules experience upon binding. Ligand strain energies and conformational reorganization were analyzed with different computational methods on 150 crystal structures of pharmaceutically relevant protein-ligand complexes. The common knowledge that ligands rarely bind in their lowest calculated energy conformation was confirmed. Additionally, we found that over 60% of the ligands do not bind in a local minimum conformation. While approximately 60% of the ligands were calculated to bind with strain energies lower than 5 kcal/mol, strain energies over 9 kcal/mol were calculated in at least 10% of the cases regardless of the method used. A clear correlation was found between acceptable strain energy and ligand flexibility, while there was no correlation between strain energy and binding affinity, thus indicating that expensive conformational rearrangements can be tolerated in some cases without overly penalizing the tightness of binding. On the basis of the trends observed, thresholds for the acceptable strain energies of bioactive conformations were defined with consideration of the impact of ligand flexibility. An analysis of the degree of folding of the bound ligands confirmed the general tendency of small molecules to bind in an extended conformation. The results suggest that the unfolding of hydrophobic ligands during binding, which exposes hydrophobic surfaces to contact with protein residues, could be one of the factors accounting for high reorganization energies. Finally, different methods for conformational analysis were evaluated, and guidelines were defined to maximize the prevalence of bioactive conformations in computationally generated ensembles.

The 3D structure of a complex of the anti-Alzheimer drug galanthamine with Torpedo californica acetylcholinesterase is reported. Galanthamine, a tertiary alkaloid extracted from several species of Amarylidacae, is so far the only drug that shows a dual activity, being both an acetylcholinesterase inhibitor and an allosteric potentiator of the nicotinic response induced by acetylcholine and competitive agonists. The X-ray structure, at 2.5A resolution, shows an unexpected orientation of the ligand within the active site, as well as unusual protein-ligand interactions. The inhibitor binds at the base of the active site gorge, interacting with both the acyl-binding pocket and the principal quaternary ammonium-binding site. However, the tertiary amine group of galanthamine does not directly interact with Trp84. A docking study using the program AUTODOCK correctly predicts the orientation of galanthamine in the active site. The docked lowest-energy structure has a root mean square deviation of 0.5A with respect to the corresponding crystal structure of the complex. The observed binding mode explains the affinities of a series of structural analogs of galanthamine and provides a rational basis for structure-based drug design of synthetic derivatives with improved pharmacological properties. Proteins 2001;42:182-191.

Virtual screening of chemical databases is an emerging approach in drug discovery that uses computers to dock chemicals into the active site of a drug target to identify leads through evaluation of binding affinities of the chemicals. However, there are concerns about the validity and scope of the reported virtual screens due to lack of studies to show that randomly selected chemicals are not equally active and due to the fact that metalloproteins were rarely used as drug targets. We have performed a virtual screening of a chemical database to identify prototypic inhibitors of farnesyltransferase (FT) with zinc present in the active site. Among the 21 compounds identified by computers, four inhibited FT in vitro with IC(50) values in the range from 25 to 100 microM. The most potent inhibitor also inhibited FT in human lung cancer cells. In contrast, none of 21 randomly selected compounds have an IC(50) lower than 100 microM. The results demonstrate the validity of virtual screening and the feasibility of applications of this approach to metalloprotein drug targets, such as matrix metalloproteinases, farnesyltransferase, and HIV-1 integrase, for the treatments of cardiovascular diseases, cancers, and AIDS.

A series of N-[ω-N′-(adamant-1′-yl)aminoalkyl]-2-(4′-dimethylaminophenyl)acetamides were synthesized and tested as acetylcholinesterase inhibitors. A significant selectivity toward acetylcholinesterases from various natural sources, mainly differing in their quaternary structure and solubility, was pointed out. The interest of this kind of molecules as potential therapeutic agents for Alzheimer's disease is discussed.

Heptylphysostigmine is in advanced clinical trial as a drug for Alzheimer's disease. 8-Morpholinooctylphysostigmine and 8-(cis-2,6-dimethylmorpholino)octylphysostigmine are currently undergoing pre-clinical evaluation. The mechanism of action of these compounds in the inhibition of acetylcholinesterase has been investigated. All the examined compounds display non competitive-like kinetics of inhibition. There are no reversible components in the observed inhibition: the whole inhibitory effect is due to the time-dependent pseudo-irreversible carbamylation of the active site. Yet the observed time course of the inhibition does not match a simple second order kinetics. An influence of the quaternary structure of the enzyme on the more complex kinetics of carbamylation is hypothesized. Reactivation experiments on the inhibited enzyme show long lasting inhibitory effects for these compounds. The higher duration of the anticholinesterase effect of the morpholino derivatives compared to heptylphysostigmine should provide the basis for their higher therapeutic potential.