AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

The active ClpP protease from M. tuberculosis is a complex composed of a heptameric ClpP1 and a ClpP2 ring

Mycobacterium tuberculosis (Mtb) contains two clpP genes, both of which are essential for viability. We expressed and purified Mtb ClpP1 and ClpP2 separately. Although each formed a tetradecameric structure and was processed, they lacked proteolytic activity. We could, however, reconstitute an active, mixed ClpP1P2 complex after identifying N-blocked dipeptides that stimulate dramatically (>1000-fold) ClpP1P2 activity against certain peptides and proteins. These activators function cooperatively to induce the dissociation of ClpP1 and ClpP2 tetradecamers into heptameric rings, which then re-associate to form the active ClpP1P2 2-ring mixed complex. No analogous small molecule-induced enzyme activation mechanism involving dissociation and re-association of multimeric rings has been described. ClpP1P2 possesses chymotrypsin and caspase-like activities, and ClpP1 and ClpP2 differ in cleavage preferences. The regulatory ATPase ClpC1 was purified and shown to increase hydrolysis of proteins by ClpP1P2, but not peptides. ClpC1 did not activate ClpP1 or ClpP2 homotetradecamers and stimulated ClpP1P2 only when both ATP and a dipeptide activator were present. ClpP1P2 activity, its unusual activation mechanism and ClpC1 ATPase represent attractive drug targets to combat tuberculosis

Both ClpP1 and ClpP2 are essential for normal growth in mycobacteria.

<p>(A) Schematic representation of mycobacterial recombineering, employed to replace the endogenous promoter of the clpP1P2 operon with a ATc-inducible promoter (Msm strain ptet_clpP1P2). (B) Growth curves of Msm ptet_clpP1P2 in the presence (50 ng/mL) or absence of inducer ATc. Data are represented as mean CFU/mL +/− standard deviation. (C) Growth curves of Msm ptet_clpP1P2 complemented with clpP1, clpP2 or both clpP1 and clpP2 in the absence of inducer ATc. Data are represented as mean CFU/mL +/− standard deviation. (D) Schematic representation of genetic strategy used to create a tetracycline inducible conditional Msm ClpP2 mutant (Msm strain ptet_ClpP2) (E) Growth curves of Msm ptet_clpP2 in the presence (50 ng/mL) or absence of inducer ATc. Msm ptet_clpP2 was also complemented with clpP2 in the absence of ATc. Data are represented as mean OD₆₀₀ +/− standard deviation. Dashed lines represent assumed growth rates until first measured growth point.</p

Clp protease is required for degradation of abnormal proteins and SsrA-tagged proteins in mycobacteria.

<p>(A) Growth curves of Msm ptet_clpP2 in growth medium containing low (1 ng/mL) or high (100 ng/mL) concentrations of inducer ATc, in the presence of either no drug, or amikacin (top left, 0.03 µg/mL), streptomycin (top right, 0.125 µg/mL), and chloramphenicol (bottom, 7.5 µg/mL). Data are represented as mean OD₆₀₀ +/− standard deviation. Dashed lines represent assumed growth rates until first measured growth point. (B) Increase in fluorescence (RFU, 485/520) and initial growth curve (OD₆₀₀) of Msm clpP2_ID expressing the fusion construct GFP-SsrA on a constitutively expressing plasmid, in the presence and absence of inducer, ATc. Data are represented as mean RFU or OD₆₀₀ +/− standard deviation. (C) Depletion of ClpP2 and increase in GFP-SsrA in Msm clpP2_ID expressing the fusion construct GFP-SsrA on a constitutively expressing plasmid was tracked by immunoblot. Blots were probed α-GFP, α-myc, α-FLAG, and α-RpoB (loading control).</p

A catalytically inactive ClpP allele inhibit Mtb growth <i>in vitro and during infection</i>.

<p>(A) Growth curves for Mtb overexpressing wild type ClpP1 or ClpP1 S98A via an ATc-inducible expression vector. Data are represented as mean OD₆₀₀ +/− standard deviation. Dashed lines represent assumed growth rates until first measured growth point. (B) Growth of Mtb containing a doxycycline-inducible plasmid expressing the mutant allele ClpP1 S98A in lungs of C57BL/6 mice 30 days post aerosol infection. Mice were infected via aerosol with a 3∶1 mixture of mutant and wild type bacteria. Mice were fed either with chow containing (dark squares, N = 5 mice) or lacking (gray triangles, N = 5 mice) the inducer doxycycline. As a control, wild type Mtb was co-infected, and representative CFU/organs for the control are represented (right). Each point represents calculated total CFU/organ for each mouse. Not all mice received enough wild type bacteria to quantitate.</p

Inducible protein degradation demonstrates requirement of ClpP2 for normal growth.

<p>(A) Schematic representation of the inducible degradation system used to inducibly deplete ClpP2 (Msm strain clpP2_ID). Induction of HIV-2 protease with ATc leads to cleavage of the HIV-2 protease cutting site and exposure of a SsrA tag on the tagged protein. Cleavage by HIV-2 protease and subsequent degradation can be tracked via the FLAG (square) and c-myc (circle) epitope tags, respectively, included on the inducible degradation tag. (B) Degradation of ClpP2 in clpP2_ID was tracked by Western in the absence or presence of inducer ATc. Blots were probed α-FLAG (loss indicates HIV-2 protease cleavage), α-myc (loss indicates target degradation), and α-RpoB (loading control). (C) Growth curves of Msm clpP2_ID in the absence or presence (50 ng/mL) of inducer ATc. Msm clpP2_ID was also complemented with clpP2 in the presence of ATc. Data are represented as mean CFU/mL +/− standard deviation.</p

Mtb ClpP1 and ClpP2 interact, forming a multi-component protease, and share substantial similarity with ClpP1 and ClpP2 homologs in Msm.

<p>(A) C-terminally myc-tagged Mtb ClpP1 and 6×His-tagged Mtb ClpP2 were expressed in Msm. Lysate (lane 1) was prepared and loaded onto a Ni-column. After washing with PBS (lanes 2,3), Ni-bound material was eluted with 50 mM (lane 4), 100 mM (lane 5), 250 mM (lane 6, 7) of imidazole in PBS, and analyzed by immunoblotting using anti α-myc and α-6×His antibodies. (B) Fraction 6 from (A) was applied to an anti-myc column (lane 1). The flow through (lane 2), and bound material (lane 3) were analyzed by immunoblot with α-myc and α-His antibodies. Bound material was released from the anti-myc agarose beads by boiling in Laemmli buffer after washing with PBS. (C) Bands representing ClpP1 and ClpP2 from (B) were sequenced by MS/MS revealing the presence of both Mtb and Msm homologs. Msm specific peptides are indicated by black lines, those specific to Mtb are indicated by red lines. (D) Cleavage of fluorescent peptide Z-Gly-Gly-Leu-AMC was measured in the presence of 1 µg ClpP1, 1 µg Clp2, and the activating peptide Z-Leu-Leu (see accompanying paper). Addition of 5 µg of catalytically inactive mutants of either ClpP1 (ClpP1S) or ClpP2 (ClpP2S) markedly inhibited cleavage by the ClpP1P2 protease. Results graphed are a representative sample of results obtained.</p

The Cyclic Peptide Ecumicin Targeting ClpC1 Is Active against Mycobacterium tuberculosis In Vivo Downloaded from

M ycobacterium tuberculosis is the cause of one of the most deadly diseases of mankind, and despite the availability of effective treatments, tuberculosis (TB) remains a major public health threat. The difficult challenges in treating multiple-drugresistant (MDR) and extensively drug-resistant (XDR) TB and the importance of shortening the duration of treatment to improve patients&apos; compliance make the discovery of new anti-TB drugs imperative (1-5). Attempts to discover new TB drugs and targets via large-scale screening against intact mycobacteria have largely been confined to synthetic compound libraries and to date have yielded only one new clinical TB drug, the diarylquinoline bedaquiline (6, 7). Although very potent, to be of maximum benefit, bedaquiline, a diarylquinoline, and nitroimidazoles (8) require new companion drugs to be used in a multidrug regimen. While the intensive search for antibiotics from soil microorganisms in the mid-20th century yielded several clinically useful TB drugs, the pathogenic nature of M. tuberculosis and its extremely slow growth rate did not allow classical agar diffusion tests and excluded M. tuberculosis from the initial target panel. The discovery of TB drugs of natural origin at that time therefore relied upon the detection of activity against nonmycobacteria in agar diffusion assays followed by bioassay-guided isolation of the active principle, again using nonmycobacteria. Activity against M. tuberculosis was only assessed once the active principle was purified. Because M. tuberculosis is uniquely susceptible to a number of antimicrobial agents, a high-throughput screening (HTS) of actinomycete extracts directly against the virulent H37Rv strain was conducted, and this campaign revealed selective anti-TB peptides produced by a genetically distinct Nonomuraea species, strain MJM5123. Here, we describe the activity profile of ecumicin, its efficacy in infected mice, the identification of its molecular target, and the elucidation of its unusual mechanism of action. MATERIALS AND METHODS High-throughput screening. Approximately 7,000 actinomycete cultures isolated from Korea, China, Nepal, the Philippines, Vietnam, Antarctica, and the Arctic Circle and maintained at Myongji University, South Korea, were fermented in 20-ml cultures in glucose-soybean starch (GSS) medium (rich medium), Bennett&apos;s medium (normal medium), and dextrinyeast-corn steep liquor (DYC) medium (minimal medium) (se

The Cyclic Peptide Ecumicin Targeting ClpC1 Is Active against Mycobacterium tuberculosis In Vivo

Drug-resistant tuberculosis (TB) has lent urgency to finding new drug leads with novel modes of action. A high-throughput screening campaign of >65,000 actinomycete extracts for inhibition of Mycobacterium tuberculosis viability identified ecumicin, a macrocyclic tridecapeptide that exerts potent, selective bactericidal activity against M. tuberculosis in vitro, including nonreplicating cells. Ecumicin retains activity against isolated multiple-drug-resistant (MDR) and extensively drug-resistant (XDR) strains of M. tuberculosis. The subcutaneous administration to mice of ecumicin in a micellar formulation at 20 mg/kg body weight resulted in plasma and lung exposures exceeding the MIC. Complete inhibition of M. tuberculosis growth in the lungs of mice was achieved following 12 doses at 20 or 32 mg/kg. Genome mining of lab-generated, spontaneous ecumicin-resistant M. tuberculosis strains identified the ClpC1 ATPase complex as the putative target, and this was confirmed by a drug affinity response test. ClpC1 functions in protein breakdown with the ClpP1P2 protease complex. Ecumicin markedly enhanced the ATPase activity of wild-type (WT) ClpC1 but prevented activation of proteolysis by ClpC1. Less stimulation was observed with ClpC1 from ecumicin-resistant mutants. Thus, ClpC1 is a valid drug target against M. tuberculosis, and ecumicin may serve as a lead compound for anti-TB drug development