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

Cloning, expression, purification and preliminary X-ray diffraction studies of a putative Mycobacterium smegmatis thiolase

Thiolases are important in fatty-acid degradation and biosynthetic pathways. Analysis of the genomic sequence of Mycobacterium smegmatis suggests the presence of several putative thiolase genes. One of these genes appears to code for an SCP-x protein. Human SCP-x consists of an N-terminal domain (referred to as SCP2 thiolase) and a C-terminal domain (referred as sterol carrier protein 2). Here, the cloning, expression, purification and crystallization of this putative SCP-x protein from M. smegmatis are reported. The crystals diffracted X-rays to 2.5 angstrom resolution and belonged to the triclinic space group P1. Calculation of rotation functions using X-ray diffraction data suggests that the protein is likely to possess a hexameric oligomerization with 32 symmetry which has not been observed in the other six known classes of this enzyme

Structural characterization of a mitochondrial 3-ketoacyl-CoA (T1)-like thiolase from Mycobacterium smegmatis

Thiolases catalyze the degradation and synthesis of 3-ketoacyl-CoA molecules. Here, the crystal structures of a T1-like thiolase (MSM-13 thiolase) from Mycobacterium smegmatis in apo and liganded forms are described. Systematic comparisons of six crystallographically independent unliganded MSM-13 thiolase tetramers (dimers of tight dimers) from three different crystal forms revealed that the two tight dimers are connected to a rigid tetramerization domain via flexible hinge regions, generating an asymmetric tetramer. In the liganded structure, CoA is bound to those subunits that are rotated towards the tip of the tetramerization loop of the opposing dimer, suggesting that this loop is important for substrate binding. The hinge regions responsible for this rotation occur near Val123 and Arg149. The L alpha 1-covering loop-L alpha 2 region, together with the N beta 2-N alpha 2 loop of the adjacent subunit, defines a specificity pocket that is larger and more polar than those of other tetrameric thiolases, suggesting that MSM-13 thiolase has a distinct substrate specificity. Consistent with this finding, only residual activity was detected with acetoacetyl-CoA as the substrate in the degradative direction. No activity was observed with acetyl-CoA in the synthetic direction. Structural comparisons with other well characterized thiolases suggest that MSM-13 thiolase is probably a degradative thiolase that is specific for 3-ketoacyl-CoA molecules with polar, bulky acyl chains

Cloning, expression, purification and preliminary X-ray diffraction studies of a putative Mycobacterium smegmatis thiolase

Thiolases are important in fatty-acid degradation and biosynthetic pathways. Analysis of the genomic sequence of Mycobacterium smegmatis suggests the presence of several putative thiolase genes. One of these genes appears to code for an SCP-x protein. Human SCP-x consists of an N-terminal domain (referred to as SCP2 thiolase) and a C-terminal domain (referred as sterol carrier protein 2). Here, the cloning, expression, purification and crystallization of this putative SCP-x protein from M. smegmatis are reported. The crystals diffracted X-rays to 2.5 Å resolution and belonged to the triclinic space group P1. Calculation of rotation functions using X-ray diffraction data suggests that the protein is likely to possess a hexameric oligomerization with 32 symmetry which has not been observed in the other six known classes of this enzyme

Crystal Structure of a Monomeric Thiolase-Like Protein Type 1 (TLP1) from <em>Mycobacterium smegmatis</em>

<div><p>An analysis of the <em>Mycobacterium smegmatis</em> genome suggests that it codes for several thiolases and thiolase-like proteins. Thiolases are an important family of enzymes that are involved in fatty acid metabolism. They occur as either dimers or tetramers. Thiolases catalyze the Claisen condensation of two acetyl-Coenzyme A molecules in the synthetic direction and the thiolytic cleavage of 3-ketoacyl-Coenzyme A molecules in the degradative direction. Some of the <em>M. smegmatis</em> genes have been annotated as thiolases of the poorly characterized SCP2-thiolase subfamily. The mammalian SCP2-thiolase consists of an N-terminal thiolase domain followed by an additional C-terminal domain called sterol carrier protein-2 or SCP2. The <em>M. smegmatis</em> protein selected in the present study, referred to here as the thiolase-like protein type 1 (<em>Ms</em>TLP1), has been biochemically and structurally characterized. Unlike classical thiolases, <em>Ms</em>TLP1 is a monomer in solution. Its structure has been determined at 2.7 Å resolution by the single wavelength anomalous dispersion method. The structure of the protomer confirms that the N-terminal domain has the thiolase fold. An extra C-terminal domain is indeed observed. Interestingly, it consists of six β-strands forming an anti-parallel β-barrel which is completely different from the expected SCP2-fold. Detailed sequence and structural comparisons with thiolases show that the residues known to be essential for catalysis are not conserved in <em>Ms</em>TLP1. Consistent with this observation, activity measurements show that <em>Ms</em>TLP1 does not catalyze the thiolase reaction. This is the first structural report of a monomeric thiolase-like protein from any organism. These studies show that <em>Ms</em>TLP1 belongs to a new group of thiolase related proteins of unknown function.</p> </div

The overall fold of <i>Ms</i>TLP1.

<p>A) The TLP protomer is divided into two domains. The N-terminal thiolase domain can further be divided into an N-terminal half (green), a thiolase loop region (light blue) and a C-terminal half (dark). The C-terminal extra domain of <i>Ms</i>TLP1 is in dark green. The two domains are connected by a linker region (yellow). B) The N-terminal thiolase domain of <i>Ms</i>TLP1 has the conserved fold of the thiolase superfamily. The two β sheets are sandwiched between three layers of α helices forming the characteristic α/β/α/β/α layered structure found in classical thiolases. The numbering of the strands and helices conforms to the assignment in the classical thiolases.</p

The putative CoA binding groove of <i>Ms</i>TLP1 from the comparison with the <i>Z. ramigera</i> thiolase.

<p>A) The shape of the binding groove. The binding mode of acetyl CoA (red) as obtained by superposition of the complexed <i>Z. ramigera</i> thiolase structure. The N-terminal thiolase domain is shown in pale blue and the C-terminal domain is shown in green. Also highlighted in blue are the positively charged residues Arg227, Arg240, and Arg248 which line the putative CoA binding pocket. B). The disordered loops. Loop regions that are disordered in <i>Ms</i>TLP1 but ordered in <i>Z. ramigera</i> thiolase are highlighted in blue. Also included in the <i>Z. ramigera</i> reference structure is the loop just before the dimer interface Nβ3 strand (residues 73–83) of the adjacent subunit (red). This superposition shows that the linker region of <i>Ms</i>TLP1 clashes with this loop, thereby preventing the formation of <i>Z.ramigera</i> thiolase-like dimers.</p

Mutation update on ACAT1 variants associated with mitochondrial acetoacetyl‐CoA thiolase (T2) deficiency

Abstract Mitochondrial acetoacetyl‐CoA thiolase (T2, encoded by the ACAT1 gene) deficiency is an inherited disorder of ketone body and isoleucine metabolism. It typically manifests with episodic ketoacidosis. The presence of isoleucine‐derived metabolites is the key marker for biochemical diagnosis. To date, 105 ACAT1 variants have been reported in 149 T2‐deficient patients. The 56 disease‐associated missense ACAT1 variants have been mapped onto the crystal structure of T2. Almost all these missense variants concern residues that are completely or partially buried in the T2 structure. Such variants are expected to cause T2 deficiency by having lower in vivo T2 activity because of lower folding efficiency and/or stability. Expression and activity data of 30 disease‐associated missense ACAT1 variants have been measured by expressing them in human SV40‐transformed fibroblasts. Only two variants (p.Cys126Ser and p.Tyr219His) appear to have equal stability as wild‐type. For these variants, which are inactive, the side chains point into the active site. In patients with T2 deficiency, the genotype does not correlate with the clinical phenotype but exerts a considerable effect on the biochemical phenotype. This could be related to variable remaining residual T2 activity in vivo and has important clinical implications concerning disease management and newborn screening

The protomers are assembled with 32 symmetry in the asymmetric unit of <i>Ms</i>TLP1.

<p>The six protomers are arranged as two discs of three protomers each. A) The two discs are related by a non-crystallographic 2-fold symmetry axis. B) A non-crystallographic 3-fold symmetry axis relates the three protomers of each disk.</p

Data collection statistics for <i>Mycobacterium smegmatis</i> TLP1.

<p>Values in parenthesis refer to the highest resolution shell.</p><p><i>I</i> is the integrated intensity and σ (<i>I</i>) is the estimated standard deviation of that intensity.</p><p>R_merge = (Σ_hklΣ_i(|I_i(hkl)−|)/Σ_hklΣI_i(hkl), where I_i(hkl) is the intensity of the i^th measurement of reflection hkl and is its mean.</p