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Cancer cell proliferation requires precise control of E2F1 activity; excess activity promotes apoptosis. Here, we developed cell-permeable and bioavailable macrocycles that selectively kill small cell lung cancer (SCLC) cells with inherent high E2F1 activity by blocking RxL-mediated interactions of cyclin A and cyclin B with select substrates. Genome-wide CRISPR/Cas9 knockout and random mutagenesis screens found that cyclin A/B RxL macrocyclic inhibitors (cyclin A/Bi) induced apoptosis paradoxically by cyclin B- and Cdk2-dependent spindle assembly checkpoint activation (SAC). Mechanistically, cyclin A/Bi hyperactivate E2F1 and cyclin B by blocking their RxL-interactions with cyclin A and Myt1, respectively, ultimately leading to SAC activation and mitotic cell death. Base editor screens identified cyclin B variants that confer cyclin A/Bi resistance including several variants that disrupted cyclin B:Cdk interactions. Unexpectedly but consistent with our base editor and knockout screens, cyclin A/Bi induced the formation of neo-morphic Cdk2-cyclin B complexes that promote SAC activation and apoptosis. Finally, orally-bioavailable cyclin A/Bi robustly inhibited tumor growth in chemotherapy-resistant patient-derived xenograft models of SCLC. This work uncovers gain-of-function mechanisms by which cyclin A/Bi induce apoptosis in cancers with high E2F activity, and suggests cyclin A/Bi as a therapeutic strategy for SCLC and other cancers driven by high E2F activity.

Hyperactivation of mTOR kinase by mutations in the PI3K/mTOR pathway or by crosstalk with other mutant cancer drivers, such as RAS, is a feature of many tumors. Multiple allosteric inhibitors of mTORC1 and orthosteric dual inhibitors of mTORC1 and mTORC2 have been developed as anticancer drugs, but their clinical utility has been limited. To address these limitations, we have developed a novel class of "bi-steric inhibitors" that interact with both the orthosteric and the allosteric binding sites in order to deepen the inhibition of mTORC1 while also preserving selectivity for mTORC1 over mTORC2. In this report, we describe the discovery and preclinical profile of the development candidate RMC-5552 and the in vivo preclinical tool compound RMC-6272. We also present evidence that selective inhibition of mTORC1 in combination with covalent inhibition of KRAS G12C shows increased antitumor activity in a preclinical model of KRAS G12C mutant NSCLC that exhibits resistance to KRAS G12C inhibitor monotherapy.

The clinical benefits of pan-mTOR active-site inhibitors are limited by toxicity and relief of feedback inhibition of receptor expression. To address these limitations, we designed a series of compounds that selectively inhibit mTORC1 and not mTORC2. These 'bi-steric inhibitors' comprise a rapamycin-like core moiety covalently linked to an mTOR active-site inhibitor. Structural modification of these components modulated their affinities for their binding sites on mTOR and the selectivity of the bi-steric compound. mTORC1-selective compounds potently inhibited 4EBP1 phosphorylation and caused regressions of breast cancer xenografts. Inhibition of 4EBP1 phosphorylation was sufficient to block cancer cell growth and was necessary for maximal antitumor activity. At mTORC1-selective doses, these compounds do not alter glucose tolerance, nor do they relieve AKT-dependent feedback inhibition of HER3. Thus, in preclinical models, selective inhibitors of mTORC1 potently inhibit tumor growth while causing less toxicity and receptor reactivation as compared to pan-mTOR inhibitors., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a Zn 2+ deacetylase that is essential for the survival of most pathogenic Gram-negative bacteria. ACHN-975 (N-((S)-3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-(((1R,2R)-2-(hydroxymethyl)cyclopropyl)buta-1,3-diyn-1-yl)benzamide) was the first LpxC inhibitor to reach human clinical testing and was discovered to have a dose-limiting cardiovascular toxicity of transient hypotension without compensatory tachycardia. Herein we report the effort beyond ACHN-975 to discover LpxC inhibitors optimized for enzyme potency, antibacterial activity, pharmacokinetics, and cardiovascular safety. Based on its overall profile, compound 26 (LPXC-516, (S)-N-(2-(hydroxyamino)-1-(3-methoxy-1,1-dioxidothietan-3-yl)-2-oxoethyl)-4-(6-hydroxyhexa-1,3-diyn-1-yl)benzamide) was chosen for further development. A phosphate prodrug of 26 was developed that provided a solubility of >30 mg mL -1 for parenteral administration and conversion into the active drug with a t 1/2 of approximately two minutes. Unexpectedly, and despite our optimization efforts, the prodrug of 26 still possesses a therapeutic window insufficient to support further clinical development., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Previously we reported the results from an effort to improve Gram-negative antibacterial activity in the oxazolidinone class of antibiotics via a systematic medicinal chemistry campaign focused entirely on C-ring modifications. In that series we set about testing if the efflux and permeation barriers intrinsic to the outer membrane of Escherichia coli could be rationally overcome by designing analogs to reside in specific property limits associated with Gram-negative activity: i) low MW (<400), ii) high polarity (clogD 7.4 <1), and iii) zwitterionic character at pH 7.4. Indeed, we observed that only analogs residing within these limits were able to overcome these barriers. Herein we report the results from a parallel effort where we explored structural changes throughout all three rings in the scaffold for the same purpose. Compounds were tested against a diagnostic MIC panel of Escherichia coli and Staphylococcus aureus strains to determine the impact of combining structural modifications in overcoming the OM barriers and in bridging the potency gap between the species. The results demonstrated that distributing the charge-carrying moieties across two rings was also beneficial for avoidance of the outer membrane barriers. Importantly, analysis of the structure-permeation relationship (SPR) obtained from this and the prior study indicated that in addition to MW, polarity, and zwitterionic character, having ≤4 rotatable bonds is also associated with evasion of the OM barriers. These combined results provide the medicinal chemist with a framework and strategy for overcoming the OM barriers in GNB in antibacterial drug discovery efforts., (Copyright © 2017. Published by Elsevier Ltd.)

Novel antibacterials with activity against the Gram-negative bacteria associated with nosocomial infections, including Escherichia coli and other Enterobacteriaceae, are urgently needed due to the increasing prevalence of multidrug-resistant strains. A major obstacle that has stalled progress on nearly all small-molecule classes with potential for activity against these species has been achieving sufficient whole-cell activity, a difficult challenge due to the formidable outer membrane and efflux barriers intrinsic to these species. Using a set of compound design principles derived from available information relating physicochemical properties to Gram-negative entry or activity, we synthesized and evaluated a focused library of oxazolidinone analogues, a currently narrow spectrum class of antibacterials active only against Gram-positive bacteria. In this series, we have explored the effectiveness for improving Gram-negative activity by identifying and combining beneficial structural modifications in the C-ring region. We have found polar and/or charge-carrying modifications that, when combined in hybrid C-ring analogues, appear to largely overcome the efflux and/or permeability barriers, resulting in improved Gram-negative activity. In particular, those analogues least effected by efflux and the permeation barrier had significant zwitterionic character.

Aminoglycoside antibiotics are pseudosaccharides decorated with ammonium groups that are critical for their potent broad-spectrum antibacterial activity. Despite over three decades of speculation whether or not modulation of pKa is a viable strategy to curtail aminoglycoside kidney toxicity, there is a lack of methods to systematically probe amine-RNA interactions and resultant cytotoxicity trends. This study reports the first series of potent aminoglycoside antibiotics harboring fluorinated N1-hydroxyaminobutyryl acyl (HABA) appendages for which fluorine-RNA contacts are revealed through an X-ray cocrystal structure within the RNA A-site. Cytotoxicity in kidney-derived cells was significantly reduced for the derivative featuring our novel β,β-difluoro-HABA group, which masks one net charge by lowering the pKa without compromising antibacterial potency. This novel side-chain assists in evasion of aminoglycoside-modifying enzymes, and it can be easily transferred to impart these properties onto any number of novel analogs.

A series of potent β2-adrenoceptor agonists incorporating a biarylamine secondary binding group was identified. The previously reported milveterol (5), identified by a multivalent approach and containing a typical β2-agonist primary binding group linked via a phenethylamine linker to a hydrophilic secondary binding group, served as an initiation point. A more hydrophobic set of secondary binding groups was explored, prepared rapidly from a common intermediate by Buchwald-Hartwig amination. TD-5471 (25), a potent and selective full agonist of the human β2-adrenoceptor, was identified as the most promising agent. It is potent, with slow onset in an in vitro guinea pig trachea model and shows a dose-dependent and long duration of action in an in vivo guinea pig model of bronchoprotection. TD-5471 is structurally differentiated from milveterol and its long duration of action is consistent with a correlation with hydrophobicity observed in other long-acting β2-agonist discovery programs., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A novel series of CCR1 antagonists based on the 1-(4-phenylpiperazin-1-yl)-2-(1H-pyrazol-1-yl)ethanone scaffold was identified by screening a compound library utilizing CCR1-expressing human THP-1 cells. SAR studies led to the discovery of the highly potent and selective CCR1 antagonist 14 (CCR1 binding IC(50)=4 nM using [(125)I]-CCL3 as the chemokine ligand). Compound 14 displayed promising pharmacokinetic and toxicological profiles in preclinical species., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Deoxygenation of the diol groups in rings A and D of neomycin in combination with the introduction of an N1-(l)-HABA group in the 2-deoxystreptamine subunit (ring B) leads to a novel and potent antibiotic (1) with activity against strains of S. aureus carrying known aminoglycoside resistance determinants, as well as against an extended panel of Methicillin-resistant S. aureus isolates (n = 50). Antibiotic 1 displayed >64 fold improvement in MIC50 and MIC90 against this MRSA collection when compared to the clinically relevant aminoglycosides amikacin and gentamicin. The synthesis was achieved in six steps and 15% overall yield.

Aminoglycosides are broad-spectrum antibiotics with particular clinical utility against life-threatening infections. As resistance to antibiotics, including aminoglycosides, continues to grow, there is a need for new and effective antimicrobial agents. ACHN-490 is a novel aminoglycoside in clinical development with activity against multidrug-resistant Gram-negative and select Gram-positive pathogens. Here we assess the in vivo efficacy of ACHN-490 against a variety of common pathogens in two murine models: the septicemia and neutropenic thigh models. When its activity against a gentamicin-susceptible strain of Escherichia coli was tested in the septicemia model, ACHN-490 improved 7-day survival with a dose-response profile similar to that of gentamicin, with 100% survival seen at doses of 1.6 mg/kg of body weight and above. In animals infected with a gentamicin-susceptible strain of Pseudomonas aeruginosa, treatment with either ACHN-490 or gentamicin led to 100% survival at doses of 16 mg/kg and above in the septicemia model. ACHN-490 was also effective in the neutropenic thigh model, reducing multidrug-resistant Enterobacteriaceae family and methicillin-resistant Staphylococcus aureus strains, as well as broadly susceptible strains, to static levels with dose-dependent activity. Against gentamicin-sensitive Enterobacteriaceae and methicillin-resistant S. aureus, the efficacy of ACHN-490 was comparable to that of gentamicin. However, gentamicin-resistant Enterobacteriaceae strains and those harboring the Klebsiella pneumoniae carbapenemase responded to ACHN-490 but not gentamicin, with static doses ranging from 12 mg/kg to 64 mg/kg for ACHN-490. These results suggest that ACHN-490 has the potential to become a clinically useful agent against drug-resistant pathogens, including Enterobacteriaceae, P. aeruginosa, and methicillin-resistant S. aureus, and support further development of this promising novel aminoglycoside.

ACHN-490 is a neoglycoside, or "next-generation" aminoglycoside (AG), that has been identified as a potentially useful agent to combat drug-resistant bacteria emerging in hospitals and health care facilities around the world. A focused medicinal chemistry campaign produced a collection of over 400 sisomicin analogs from which ACHN-490 was selected. We tested ACHN-490 against two panels of Gram-negative and Gram-positive pathogens, many of which harbored AG resistance mechanisms. Unlike legacy AGs, ACHN-490 was active against strains expressing known AG-modifying enzymes, including the three most common such enzymes found in Enterobacteriaceae. ACHN-490 inhibited the growth of AG-resistant Enterobacteriaceae (MIC(90), ≤4 μg/ml), with the exception of Proteus mirabilis and indole-positive Proteae (MIC(90), 8 μg/ml and 16 μg/ml, respectively). ACHN-490 was more active alone in vitro against Pseudomonas aeruginosa and Acinetobacter baumannii isolates with AG-modifying enzymes than against those with altered permeability/efflux. The MIC(90) of ACHN-490 against AG-resistant staphylococci was 2 μg/ml. Due to its promising in vitro and in vivo profiles, ACHN-490 has been advanced into clinical development as a new antibacterial agent.

The in vitro activity of ACHN-490, a novel aminoglycoside ("neoglycoside"), was evaluated against 102 multidrug-resistant (MDR) Klebsiella pneumoniae strains, including a subset of 25 strains producing the KPC carbapenemase. MIC50 values for gentamicin, tobramycin, and amikacin were 8 microg/ml, 32 microg/ml, and 2 microg/ml, respectively; MIC90 values for the same antimicrobials were > or = 64 microg/ml, > or = 64 microg/ml, and 32 microg/ml, respectively. ACHN-490 showed an MIC50 of 0.5 microg/ml and an MIC90 of 1 microg/ml, which are significantly lower than those of comparator aminoglycosides. ACHN-490 represents a promising aminoglycoside for the treatment of MDR K. pneumoniae isolates, including those producing KPC beta-lactamase.

Further investigations towards novel glycopeptide/beta-lactam heterodimers are reported. Employing a multivalent approach to drug discovery, vancomycin and cephalosporin synthons, 4, 2, 5 and 10, 18, 25 respectively, were chemically linked to yield heterodimer antibiotics. These novel compounds were designed to inhibit Gram-positive bacterial cell wall biosynthesis by simultaneously targeting the principal cellular targets of both glycopeptides and beta-lactams. The positional attachment of both the vancomycin and the cephalosporin central cores has been explored and the SAR is reported. This novel class of bifunctional antibiotics 28-36 all displayed remarkable potency against a wide range of Gram-positive organisms, including methicillin-resistant Staphylococcus aureus (MRSA). A subset of compounds, 29, 31 and 35 demonstrated excellent bactericidal activity against MRSA (ATCC 33591) and 31 and 35 also exhibited superb in vivo efficacy in a mouse model of MRSA infection. As a result of this work compound 35 was selected as a clinical candidate, TD-1792.

The design, synthesis and antibacterial activity of novel glycopeptide/beta-lactam heterodimers is reported. Employing a multivalent approach to drug discovery, vancomycin and cephalosporin synthons, A and B respectively, were chemically linked to yield heterodimer antibiotics. These novel compounds were designed to inhibit Gram-positive bacterial cell wall biosynthesis by simultaneously targeting the principal cellular targets of both glycopeptides and beta-lactams. The antibiotics 8a-f displayed remarkable potency against a wide range of Gram-positive organisms including methicillin-resistant Staphylococcus aureus (MRSA). Compound 8e demonstrated excellent bactericidal activity against MRSA (ATCC 33591) and initial evidence supports a multivalent mechanism of action for this important new class of antibiotic.

A series of greatly simplified microcystin analogues comprised only of Adda (the beta-amino acid common to the microcystins, nodularins, and motuporin,) and a single additional amino acid residue was synthesized and screened for inhibition of the protein phosphatases 1 and 2A. Several of the analogues were shown to be mid-nanomolar inhibitors of the enzymes.

A series of acyclic, truncated microcystin analogues, comprised of the dienic beta-amino acid (Adda) and up to four additional amino acids characteristic of the parent toxin, was synthesized and screened for activity as inhibitors of PP1 and PP2A. Despite a recent report to the contrary for a microcystin-derived tetrapeptide degradation product, none approaches the potency of microcystin itself.

Cyanobacteria (blue-green algae) (e.g., Microcystis and Nodularia spp.) capable of producing toxic peptides are found in fresh and brackish water worldwide. These toxins include the microcystin (MC) heptapeptides (>60 congeners) and the nodularin pentapeptides (ca. 5 congeners). Cyanobacterial cyclic peptide toxins are harmful to man, other mammals, birds, and fish. Acute exposure to high concentrations of these toxins causes liver damage, while subchronic or chronic exposure may promote liver tumor formation. The detection of cyclic peptide cyanobacterial toxins in surface and drinking waters has been hampered by the low limits of detection required and that the present routine detection is restricted to a few of the congeners. The unusual beta-amino acid ADDA (4E,6E-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid) is present in most (>80%) of the known toxic penta- and heptapeptide toxin congeners. Here, we report the synthesis of two ADDA-haptens, the raising of antibodies to ADDA, and the development of a competitive indirect ELISA for the detection of microcystins and nodularins utilizing these antibodies. The assay has a limit of quantitation of 0.02-0.07 ng/mL (depending on which congeners are present), lower than the WHO-proposed guideline (1 ng/mL) for drinking water, irrespective of the sample matrix (raw water, drinking water, or pure toxin in PBS). This new ELISA is robust, can be performed without sample preconcentration, detects toxins in freshwater samples at lower concentrations than does the protein phosphatase inhibition assay, and shows very good cross-reactivity with all cyanobacterial cyclic peptide toxin congeners tested to date (MC-LR, -RR, -YR, -LW, -LF, 3-desmethyl-MC-LR, 3-desmethyl-MC-RR, and nodularin).

Reversible protein phosphorylation is a major regulatory mechanism of intracellular signal transduction. Protein phosphatase 1 (PP1) is one of four major types of serine-threonine phosphatases mediating signaling pathways, but the means by which its activity is modulated has only recently begun to come into focus.

Based on the results from previously reported molecular modeling analyses of the interactions between the inhibitor microcystin and the serine-threonine protein phosphatases 1 and 2A, we have designed analogues of microcystin LA with structural modifications intended to impart PP1 selectivity. The synthesis of several first generation analogues followed by inhibition assays revealed that all three are PP1-selective, as predicted. Although the observed selectivities are modest, one of the designed analogues is more selective for PP1 than any known small molecule inhibitor.